MHC II Hek 2021

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Curr Protoc. Author manuscript; available in PMC 2022 February 01.
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Published in final edited form as:

Curr Protoc. 2021 February ; 1(2): e36. doi:10.1002/cpz1.36.
Production of Class II MHC Proteins in Lentiviral Vector-

Transduced HEK-293T Cells for Tetramer Staining Reagents
Richard A. Willis1,2 [Co-equal], Vasanthi Ramachandiran1,2 [Co-equal], John C. Shires1,2
[Co-equal], Ge Bai1,2, Kelly Jeter1,2, Donielle L. Bell1,2, Lixia Han1,2, Tamara Kazarian1,2,
Kyla C. Ugwu1,2, Oskar Laur3,5, Susana Contreras-Alcantara1,2, Dale L. Long1,2, John D.
Altman1,2,3,4
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1Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30329, USA
2Yerkes National Primate Research Center, Atlanta, GA 30329, USA
3Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA
30322, USA
4Center for AIDS Research, Emory University, Atlanta, GA 30322, USA
5Emory Custom Cloning Core Facility, Emory University School of Medicine, Atlanta, GA 30322,
USA
Abstract
Class II major histocompatibility complex peptide (MHC-IIp) multimers are precisely engineered
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reagents used to detect T cells specific for antigens from pathogens, tumors, and self-proteins.
While the related class I MHC/peptide (MHC-Ip) multimers are usually produced from subunits
expressed in E. coli, most class II MHC alleles cannot be produced in bacteria, and this has
contributed to the reputation that MHC-IIp reagents are harder to produce. Herein, we present a
robust constitutive expression system for soluble biotinylated MHC-IIp proteins that uses stable
lentiviral vector transduced derivatives of HEK-293T cells. The expression design includes allele-
specific peptide ligands tethered to the amino-terminus of the MHC-II β chain via a protease
cleavable linker. Following cleavage of the linker, HLA-DM is used to catalyze efficient peptide
exchange, enabling high throughput production of many distinct MHC-IIp complexes from a
single production cell line. Peptide exchange is monitored using either of two label-free methods,
native isoelectric focusing gel electrophoresis or matrix-assisted laser desorption/ionization time-
of-flight (MALDI-TOF) mass spectrometry of eluted peptides. Together, these methods produce
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MHC-IIp complexes that are highly homogeneous and that form the basis for excellent MHC-IIp
multimer reagents.
Keywords
Class II MHC tetramers; Class II MHC multimers; Antigen-specific T cells; Flow cytometry;
Protein expression; Protein engineering; Protein purification; Lentiviral transduction
Corresponding 404-727-5981, jaltman@emory.edu.

CONFLICTS OF INTEREST
None of the authors have any conflicts of interest to declare.
Willis et al. Page 2
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INTRODUCTION
Identification of antigen-specific T cells by staining with MHC/peptide (MHCp) multimers
was introduced in 1996 (Altman et al., 1996) and remains a widely used and essential
immunological tool for studying T cell immune responses to pathogens, tumors, and self-
antigens. The early days of applications of this technology were dominated by the
development of class I MHCp multimers (MHC-Ip), in large part driven by availability of
robust protein expression methods for production of high-quality, homogeneous MHC-Ip
complexes and the relatively greater difficulty of producing MHC-IIp complexes of similar
quality across a wide range of MHC-II alleles. Here, we describe highly evolved methods
we have developed in the NIH Tetramer Core Facility (NTCF) for the production of a wide
range of MHC-IIp multimers of extraordinary quality and provide guides to their use.
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The methods in this chapter are organized into a number of Basic Protocols, Support
Protocols, and Alternate Protocols. Support Protocols 1 and 2 describe methods for
measuring expression yields from established production cell lines. Basic Protocol 1
describes production of lentiviral vectors that are used to produce stable expression cell lines
in a derivative of HEK293T cells. Basic Protocol 2 describes production culture methods,
while Basic Protocol 3, Alternate Protocol 1, and Support Protocols 3-5 describe purification
and quality control procedures. Basic Protocol 4 describes a robust method for peptide
exchange, while Basic Protocol 5 and Alternate Protocol 6 describe methods for validating
peptide exchange. Finally, Basic Protocol 6 describes how to multimerize biotinylated
MHC-IIp with streptavidin, and Basic Protocol 7 describes use of these reagents to stain T
cells and analyze them by flow cytometry.
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STRATEGIC PLANNING
BIOSAFETY
All of the protocols in this unit require a BSL-2 laboratory and approval by an institutional
biosafety committee.
CELL LINES
There are numerous derivatives of HEK293 cells available, and at the outset, we chose the
HEK293T/17 line (ATCC CRL-11268). We have modified this line over the years in a
number of ways. First, we prepared a subclone that was transduced with a lentiviral vector
coding for a secreted version of the E. coli BirA biotin ligase, providing biotinylation of the
expressed protein in culture (the marker gene in our BirA vector is eGFP); we call this line
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293TB15. Second, using Cas9/CRISPR technology, we prepared a second generation

subclone that lacks expression of the TAP2 peptide transporter (we now also use these cells
for expression of class I MHC, but that is beyond the scope of this chapter). Finally, again
using Cas9/CRISPR technology, we knocked out the MGAT1 gene, recapitulating the
mutation in the popular 293S.GNTI- cell line (Reeves et al., 2002) that reduces
heterogeneity of N-linked carbohydrates, but on the background of cells expressing BirA
and lacking TAP2. We refer to this line as 293TPM or simply TPM, and it is now the
standard line used in the TCF for all new expression constructs.

CULTURE SYSTEMS
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We have explored many production culture systems over the years, including: (1) BelloCell
bioreactors (https://www.cescobio.com.tw); (2) CELLine 1000 flasks (https://wheaton.com);
(3) WAVE Cellbag bioreactors (https://www.cytivalifesciences.com); (4) roller bottles; and
(5) shaker flasks. We have tested some of these in both serum-containing and serum-free
media. All of them work and can be used based upon the preferences of individual labs. In
Basic Protocol 2, we describe the system we’re currently using, culture in shaker flasks in
media containing 10% fetal calf serum. Shaker flasks work well for most of the cell lines we
have tested regardless of expression level, and they only require a shaker platform as
opposed to more specialized equipment.
CONSTRUCT DESIGN
LENTIVIRAL EXPRESSION VECTORS—The overall design of our lentiviral class II
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MHC monomer expression vectors is shown in Figure 1. Our expression vector is designed
for use with a second-generation lentiviral packaging system (Barde et al., 2010). The basic
lentiviral elements of the vector are shown in Figure 1A and briefly described in the legend.
More detail about the system can be found in the Barde, Salmon and Trono Current
Protocols chapter referenced above. The grey area in Figure 1A indicates the MHC sequence
to be expressed with associated tags, which are described briefly below and in more detail
later in this section. The class II MHC α and β chains are expressed from separate vectors
which are otherwise identical except for surrogate marker genes (described in more detail
below). Complete vector sequences are available from the authors and vectors for several
common class II MHC alleles will be made available at Addgene. Soluble regions encoding
other α and β chain alleles can be swapped out as necessary using standard molecular
cloning techniques.
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Figure 1B shows the β chain design. A peptide epitope that binds the desired MHC allele
(human CLIP in this example) is encoded 5’ of the β chain, tethered by a flexible linker that
contains an HRV-3C protease recognition site. The tethered epitope can be replaced if
needed using standard cloning techniques. Epitopes are prescreened for high expression
levels of the class II MHC allele and the ability to be cleaved and exchanged with the
desired antigenic epitope. The transmembrane region of the β chain is replaced with a basic
leucine zipper that pairs with the corresponding acidic zipper on the α chain, expressed from
a different vector.
The α chain design is shown in Figure 1C. A BirA recognition site (BSP85) for enzymatic
biotinylation is encoded 3’ of the acidic leucine zipper. There is a C-terminal hexahistidine
tag (6xHis) for purification and ELISA detection (some constructs use a tandem 6xHis tag).
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LENTIVIRAL PACKAGING VECTORS—Two packaging plasmids are required for

lentiviral production. psPAX2 encodes the HIV gag, pol, rev, and tat genes that are necessary
for transcription of the expression vector and virion production. In place of env, a second
vector (pMD2.G) provides the VSV-G gene. Both plasmids are available from Addgene
(https://www.addgene.org).

2A OR NOT 2A—When we first began to use lentiviral vector transduction of 293T cells
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for expression of class II MHC proteins, we adopted the so-called 2A ribosomal stop-start
system for expression of two protein chains from a single open reading frame (Donnelly et
al., 2001). While we relied on the 2A system for a number of years, we subsequently
discovered that in all cases where we directly compared expression levels of the same MHC
molecule produced by cells transduced with a single 2A-containing vector versus cells
transduced with two lentiviral vectors, each coding for separate subunits, we obtained
significantly greater expression from the dual vector strategy. Consequently, while we still
use many first-generation cell lines transduced with 2A-containing vectors for class II MHC
expression, all of our new expression cell lines are prepared by transduction with two
vectors, one for each MHC subunit. This unit describes only the dual vector approach.
SEQUENCES OF CLASS II PROTEINS—The IMGT website has a well-curated

repository of the sequence of MHC alleles from humans (http://www.ebi.ac.uk/ipd/
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imgt/hla/) and from many other species (http://www.ebi.ac.uk/ipd/mhc/), but unfortunately

not the mouse. The haplotypes of the most commonly used laboratory mouse strains are
published in this volume (Kruisbeek, 2001), but the definitive sequences are only available
from generalized databases such as GenBank (http://www.ncbi.nlm.nih.gov/protein) and
UniProt (http://www.uniprot.org). Furthermore, many of these sequences were deposited
before standardized nomenclatures were in place, making even searching for the desired
sequence difficult. Searches of UniProt produce outputs that display allele-level information
but this, for the most part, is not the case with GenBank. The following query of UniProt
generates most of the desired protein sequences: “h 2 class 2” AND organism:”Mus
musculus (Mouse) [10090]”.
With the advent of inexpensive gene synthesis, we no longer isolate molecular clones from
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cDNA, and when a new allele is required we order synthetic genes using codons optimized
for expression in mammalian cells.
EPITOPES - TETHERED VS EXCHANGED—We express all class II MHC proteins

with tethered peptide ligands. With rare exceptions, we use constructs in which the tether
can be proteolytically cleaved by thrombin (1st generation constructs) or HRV-3C (second
generation constructs) and the formerly tethered peptide is replaced with a peptide epitope of
interest in an exchange reaction. Following the cell biology of class II MHC antigen
processing and presentation, the only published examples of constructs designed for
cleavage and exchange use the CLIP peptide (Day et al., 2003; Huang et al., 2003), but the
uncatalyzed rate of CLIP dissociation varies from allele to allele and can be too slow to be of
practical use. For years, this forced us and others (Moon et al., 2011) to prepare new
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expression constructs for every epitope for alleles such as I-Ab.
More recently, we have found that barriers to exchange can be surmounted in either of two
ways. First, exchange competent constructs can use tethered peptides other than CLIP
provided they have a more rapid dissociation rate than CLIP. From a large library of tethered
I-Ab constructs, we used a dissociation screen to find a peptide from mouse myelin basic
protein (MBP63–74, TTHYGSLPQKSQ) that proved useful as an exchange substrate
(unpublished). Shortly thereafter, we discovered that soluble HLA‑DM was an extremely

effective exchange catalyst for I-Ab and many other class II alleles in mouse and humans (as
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expected), and was effective at promoting replacement of CLIP, the MBP peptide, and many
others from most alleles, including I-Ab. With the addition of HLA‑DM to our exchange
protocols, there are very few class II alleles where complexes are not generally accessible by
exchange methodologies.
Finally, while exchange protocols are obviously a more rapid route to production of new
class II MHCp complexes for tetramers, there are notable cases where tethered constructs
may be preferred or required. First, it is conceivable that a genuine peptide epitope may have
only a weak affinity for the restricting class II MHC allele, and a tether might impart
stability to a reagent that would otherwise fall apart or be difficult to isolate. Second, the
open ends of class II MHC peptide binding sites permit some peptides to bind in multiple
registers, and tethers, sometimes accompanied by additional engineered disulfide crosslinks,
provide a means to constrain complexes to a single register of interest (Moon et al., 2011;
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Bankovich et al., 2004; Landais et al., 2009; Stadinski et al., 2010; Chu et al., 2010).
Development of appropriately engineered register-constrained constructs requires cycles of
empirical design and testing and is beyond the scope of this chapter.
SIGNAL SEQUENCES—For nearly all MHC-II α chains, we use their own wild type
signal sequences (with optimized codons). In contrast, all our β chain expression vectors use
the DRB1*01:01 signal sequence followed by the sequence GDTAG. This strategy was
chosen for two reasons, both related to the linked peptide strategy described above: (1) GDT
are the first three amino acids of the DRB1*01:01 mature protein, and we expect that this
would promote appropriate signal sequence cleavage for all linked peptide constructs; and
(2) the AG dipeptide sequence completes a useful Fse I restriction site.
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LEUCINE ZIPPERS AND HIS6 TAGS—We have adopted the synthetic aZip/bZip pair
originally developed by Erin O’Shea and Peter Kim (O’Shea et al., 1993) and later adopted
by Ellis Reinherz for generalized facilitation of heterodimeric subunit (Chang et al., 1994).
Most importantly, Reinherz developed a very useful monoclonal antibody specific for the
aZip/bZip heterodimer (the hybridoma is called 2H11, and we have developed a high-
producing subclone called 2H11.33, available from the authors), and we use the purified
mAb for both affinity purifications of all class II proteins from tissue culture supernatants—
independent of both the species and allele of the fused MHC subunits—and for capture
ELISAs.
Most of our class II proteins also have C-terminal His6 tags for IMAC affinity purification,
but in our hands, proteins purified by IMAC from serum-containing supernatants often
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require a second step of purification, while this is rarely the case for proteins purified on
2H11 affinity columns. More recently, we have found that the 2xHis6 tag adopted by
Luescher and colleagues (Schmidt et al., 2011) provides purity comparable to that obtained
by 2H11 affinity chromatography, and we use the 2xHis6 tag on many newer constructs.
BIRA SUBSTRATE PEPTIDE TAG—An essential core element of MHC tetramer

technology is enzymatic biotinylation of proteins on a BirA substrate peptide (BSP) fused to
the carboxy terminus of one of the chains of the MHC protein (Altman et al., 1996). A large

library of BSPs was originally described by Schatz (Schatz, 1993); for all of our class II
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proteins, we have used peptide 85 from that paper, but other investigators have successfully
used different BSPs. The BirA enzyme is commercially available (Avidity; http://
www.avidity.com, though, as described above, our expression cell line has been engineered
to co-express E. coli BirA, and this provides essentially 100% biotinylated product from all
culture supernatants. Almost all of our constructs have BSP tags on the α chain of the MHC;
this was an arbitrary decision, and other labs have attached BSP tags to the MHC β chain
(Crawford et al., 1998).
SURROGATE MARKERS FOR EXPRESSION—An internal ribosome entry site

(IRES) is placed 3’ to the MHC allele insert to allow co-expression of a surrogate marker,
allowing rapid screening of transduced cell lines and FACS sorting if needed. EGFP was
used as the surrogate marker for BirA expression in the TB-15 and TPM cell lines. For most
of our expression vectors, Thy1.1 is used for the β chain and DsRed Express2 (Strack et al.,
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2008) is used for the α chain. TagBFP is sometimes used (Subach et al., 2008) for the α
chain.
SUPPORT REAGENTS AND EQUIPMENT

SYNTHETIC PEPTIDES—Synthetic peptides are used to load MHC molecules to make
the reagents of interest. In general, peptides should be at least 85% pure; for most suppliers,
this means the peptides will be HPLC purified. Blocking of the peptide termini by
acetylation or amidation is usually tolerated for MHC-II epitopes (but rarely for MHC-I
epitopes), and many class II epitopes can tolerate N- and C-terminal extensions (Margulies
et al., 2013). Most of the peptides that we have loaded are in the range of 12–20 mers, and
we discourage use of peptides that are longer than necessary because they increase the
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likelihood of multiple binding registers.
HLA-DM—HLA-DM is required to catalyze peptide exchange for many of the soluble class
II proteins expressed using the methods described in this chapter, including many mouse and
non-human primate alleles. We express and purify it from the 293T-derived TPM cells using
the same methods as described in this unit. We remove it from exchange reactions by a batch
binding method using beads conjugated to the anti-HLA-DM mAb MaP.DM1 (Hammond et
al., 1998); this protocol is described in Basic Protocol 4.
3C—Early class II MHC constructs with tethered peptide ligands made in our laboratory
contained linkers with a thrombin protease recognition site, but most currently in use
constructs use an HRV 3C protease cleavage site in the linker. HRC 3C protease is available
from many commercial suppliers, but we use a recombinant 3C-GST fusion protein
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produced expressed in our laboratory from a plasmid provided by Kai Wucherpfennig (to be
deposited in Addgene). We generally pre-cleave the class II MHC proteins in large batches
and remove the protease on a glutathione affinity column.
CHROMATOGRAPHY EQUIPMENT—The only truly essential purification protocols in

this article can be performed without any specialty equipment. However, because of the
range of products we produce, the scale of our preparations, and our need for a variety of

polishing procedures, we have a collection of dedicated chromatography workstations in our

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laboratory. Such systems provide numerous advantages, from carefully controlled flow rates,
UV monitoring of eluates, and collection of fractions. We use two different categories of
instruments in our lab: (1) low pressure systems such as the recently discontinued
ÄKTAprime plus system from Cytiva (https://www.cytivalifesciences.com) are used for
most affinity columns (including both antibody and IMAC columns) and preparative gel
filtration columns; and (2) higher pressure systems such as the ÄKTA pure or BioRad NGC
instruments that we use for ion exchange columns, but also for gel filtration. Equivalent
instruments are available from other manufacturers.
QUALITY CONTROL
We use a battery of assays to assess quality of class II MHC proteins, both immediately
following purification from tissue culture supernatants and after peptide exchange. We use
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SDS-PAGE to assess overall purity, high-performance size-exclusion chromatography (HP-

SEC) to assess aggregation, and either mass spectrometry or native isoelectric focusing to
measure peptide exchange. A few of these are described in more detail below.
PEPTIDE EXCHANGE – MASS SPEC AND NATIVE IEF—We now routinely use
HLA-DM-mediated peptide exchange to load class II MHC proteins with synthetic peptides.
Rarely are these peptides labeled, so we had to develop label-free methods to detect loading.
For a number of years, we used native isoelectric focusing gels (Alternative Protocol 2) to
detect loading of MHC proteins with peptides that had a net charge differing from zero (the
method is not particularly useful for uncharged peptide). More recently, we have adopted
assays involving elution of peptides from the MHCp complex followed by mass
spectrometry using either MALDI-TOF or LC-MS (Basic Protocol 5). Each method has
advantages and disadvantages. Native IEF analysis often provided definitive evidence for
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complete exchange, but it is both technically demanding and has blindspots as noted above.
In contrast, the mass spectrometry methods are extremely simple, but in the absence of
prohibitively expensive isotope labeled standards, they do not provide quantitative measures
of peptide binding and so do not distinguish between empty and loaded MHC. This could be
obviated by positive selection of peptide complexes when affinity handles are included on
the exchange peptides (Day et al., 2003; Schmidt et al., 2013), but we have not found it
necessary to do this routinely.
HP-SEC—A number of the methods described in this chapter—notably elution from

antibody affinity columns and peptide exchange procedures—can conceivably lead to
unwanted protein aggregation that could adversely impact multimer staining performance
but that cannot be detected by denaturing methods such as SDS-PAGE. Therefore, as a
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secondary quality control measure, we often use high-performance size-exclusion

chromatography (HP-SEC) to detect aggregate formation, as in Figure 2. HP-SEC methods
are beyond the scope of this unit but are described in detail in Unit 7.10 of Current Protocols
in Protein Science (Begg et al., 1999). When a mixture of aggregated and unaggregated class
II MHC protein is detected by analytical methods, we often choose to perform preparative
scale gel filtration chromatography to remove the aggregates.

BIOTINYLATION ASSAYS—In our Current Protocols in Immunology unit on class I

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MHC tetramer production (Altman and Davis, 2016), we described a streptavidin-gel shift
assay for determining biotinylation levels. For many of the class II MHC proteins produced
using the methods described here, the ɑ and β subunits are not well resolved by SDS-PAGE
—this is likely a consequence of multiple design features such as leucine zippers, BSP and
His6 tags, and peptide ligands tethered to the amino terminus of the class II β chain—and the
gel shift assay is simply not suitable in these cases. Instead, we have developed a
biotinylation assay that begins with streptavidin-bead depletion of biotinylated proteins
followed by SDS-PAGE analysis of protein remaining in the supernatant. This method is
described in Support Protocol 5.
STAINING CONSIDERATIONS WITH CLASS II MHC TETRAMERS

FLUROPHORES FOR CLASS II TETRAMERS—Our unit on class I MHC tetramers
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contained an extensive discussion of fluorophores used for these reagents (Altman and
Davis, 2016). One issue raised there is worth recapitulating here, and that is the beneficial
creation of higher order multimers when protein fluorophores such as PE and APC are
conjugated to streptavidin but that does not occur upon labeling of streptavidin with most
other fluorophores. Conjugation of PE or APC to streptavidin uses heterobifunctional cross-
linking reagents that always create the possibility of multipoint attachment and therefore
effective creation of higher order multimers. The extent of higher order multimers in
commercial preparations of streptavidin-PE or -APC conjugates varies from manufacturer to
manufacturer and depends upon both the details of the conjugation reactions and the post-
conjugation fractionation of the products by size-exclusion chromatography. Using HP-SEC
analysis in our own laboratory, we have found that SA-PE and SA-APC from some
manufacturers tends to contain mostly 1:1 conjugates, while material from other
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manufacturers tends to contain higher order conjugates. In general, we have found that the
higher order conjugates perform significantly better in staining applications, and therefore
we strongly recommend the use of PE and APC fluorophores for class II MHCp tetramers.
We periodically screen SA-PE and SA-APC products from a number of suppliers, and we
currently use the materials from Prozyme/Agilent; this is further discussed in the section on
Critical Parameters below.
STAINING REAGENTS USED IN CONJUNCTION WITH CLASS II MHC

MULTIMERS—Class II MHC multimers are almost always used with a multicolor flow
cytometry panel of reagents that (1) define the major cell lineage of interest (i.e. CD3+ CD4+
T cells), and (2) provide additional phenotypic information about both the multimer positive
and negative populations. Negative discriminators such as CD8, B cell markers such as
CD19 or B220 (in the mouse), or markers for other non-T cell lineages are often included to
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better define the CD3+ CD4+ lineage and to reduce inclusion of cells binding the multimer
non-specifically. Phenotypic markers typically include some that reliably distinguish
between naïve and non-naïve T cells (e.g. CD11a and CD44 in the mouse). Beyond those
basic markers, additional panel members are determined by the research interests of the
investigator and are limited by the available instrumentation. Construction of polychromatic
flow cytometry panels is beyond the scope of this unit.

Previously, we have noted that class I MHC multimers can participate in Förster resonance
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energy transfer (FRET) interactions with fluorophores attached to CD3, the CD8 co-
receptor, or relevant anti-Vα and anti-Vβ antibodies (Altman, 2004). Similar interactions
can be expected for MHC-II multimers (with the CD4 coreceptor instead of CD8), and
panels should be designed to avoid these interactions.
BASIC PROTOCOL 1 – LENTIVIRUS PRODUCTION AND EXPRESSION LINE

CREATION
This Basic Protocol describes production of lentivirus stocks, transduction of an expression
line (usually 293TPM), and titering of the virus stock. Our protocol and vectors are based on
those described by Didier Trono (Barde et al., 2010; Naldini et al., 1996). Transduction of
the expression cell line is done at an extremely high MOI and uses nearly all of the virus
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produced by the initial transfection. Infection of the expression cell line at high MOI causes
considerable cytotoxicity, and it takes several days for cells to grow out. This protocol
typically produces lines that have uniformly high levels of expression of the lentivirus-
encoded marker genes and such lines do not always need to be sorted or subcloned.
Second generation lentiviral stock production requires Biosafety Level 2 procedures and
institutional approval. All liquid handling should take place in a biosafety cabinet using
pipets and tips with aerosol barriers. Material should only be removed from the cabinet in
closed tubes and flasks or covered plates. Solid waste should be removed in red biohazard
bags and liquid waste should be treated with >10% bleach (final concentration) before
disposal in the sink. All containers and other material should be sprayed with 75% ethanol
before removal from the cabinet. SW28 rotor buckets should be doused with 75% ethanol
and allowed to dry. Transduced cell lines should be treated as BSL-2 indefinitely and fixed
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with paraformaldehyde (2%, 10 min at room temperature) before removal from the lab for
flow cytometry and other procedures other than cell sorting.
MATERIALS
• 2X HEBS precipitation reagent (see recipe)
• 2 M CaCl2 (in DI H2O)
• D10 Media (see recipe)
• D-biotin (see recipe)
• Cryopreservation media (see recipe)
• 2% paraformaldehyde (see recipe)

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• Controlled rate freezing container (such as Thermo Scientific™ Mr. Frosty™

Freezing Container, 51000001)
• HEK-293T/17 cells (293T, ATCC CRL-11268)
• 293TPM or 293TB15 cells (optional, to be deposited in ATCC ).
• Lentiviral expression vector, 1 μg/μl (see Strategic Planning; 90 μg needed)

• pMD2G vector, 1 μg/μl (Addgene #12259; 30 μg needed)

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• psPAX2 vector, 1 μg/μl (Addgene #12260; 60 μg needed)
• 0.2μm PES 50 ml tube cap filter (Thermo Cat# 564–0020)
• T25, T75 and T225 tissue culture flasks
• 6-well and 96-well plates
• 5 ml and 25 ml polypropylene centrifuge tubes
• Tabletop centrifuge
• Ultracentrifuge with swinging bucket rotor (such as Beckman SW28)
• 38.5 mL, Open-Top Thinwall Ultra-Clear Ultracentrifuge Tube (Beckman

344058)
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• Flow cytometry staining buffer (see recipe)
• OX-7 monoclonal antibody (anti-Thy 1.1/CD90.1) conjugated to Pacific Blue

(BioLegend 202522) or other fluorochrome
• 37°C CO2 incubator
DAY 1 – SEED CELLS FOR LENTIVIRUS PRODUCTION

1. Seed the appropriate number of 293T cells for each transfection. For standard
transfections in T225 flasks, seed 2×107 cells per flask in 40 ml D10 at the end
of the day. Culture at 37°C in a 5% CO2 incubator.
DAY 2 – TRANSFECTION WITH LENTI AND PACKAGING VECTORS

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1. If a new batch of 2M CaCl2 or 2X HEBS is made, a transfection optimization

must be performed as described in Reagents and Solutions.
2. Prior to each transfection, allow all reagents and DNA samples to equilibrate to
room temperature for 1 hour. Set up transfection reagents as follows in a 50 ml
tube and mix well:
Lentivector 90 μl (45 μl each for ɑ and β chain)
psPAX2 60 μl
pMD2G 30 μl
2M CaCl2 300–600 μl, determined in step 1

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ddH2O q.s. 4.5 ml
Remove T225 flask from incubator to have on hand in biosafety cabinet.
In the past, we used a more conventional approach of preparing the packaged

lentiviral vectors for the α and β chains in separate transductions. However, we
discovered that preparing a mixed stock as described above produced excellent
results and saved material and time.

3. Using a 10 ml plastic tissue culture pipette attached to a mechanical pipettor, add

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4.5 ml 2x HEBS precipitation solution to the DNA/CaCl2 solution. Hold the tip
of the pipette at the bottom of the tube and gently expel air bubbles into the
solution for 10 s. We have found that this creates dispersed DNA/CaCl2 particles
that are optimal for transfection.
4. Let sit at room temperature for time determined in step 1 (30 sec-5 min) and
then add to cells (do not remove media). Mix to cover cells with gentle rocking/
swirling.
5. Place cells in incubator for 6–8 hours.
6. Aspirate transfection mix from flasks. Without washing, add 40 ml fresh D10.
DAY 4 – SEED CELLS FOR TRANSDUCTION AND TITERING

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1. For each lentiviral stock, 3 wells of a 6-well plate should be seeded with 6×105
cells per well in 3 ml for titering, and 20,000 cells in 100ul should be seeded into
one well of a 96-well plate for transduction of the eventual production line.
The cells for transduction can be 293T, 293TPM or 293TB15 depending on the
desired nature of the expressed protein (see Strategic Planning). It is convenient
but not necessary to use the same cells for the titering plate.
DAY 5 – LENTIVIRUS CONCENTRATION, TRANSDUCTION AND TITERING

1. Harvest lentiviral supernatant at ~60 hrs post media change (the morning of the
third day after transfection).
2. Pellet debris at 4000 × g, 30 min, 4 °C in a tabletop centrifuge.

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3. Filter supernatant through 0.2 μm 50 ml tube cap filter.
4. Carefully pipet the top 37 ml lentiviral supernatant into Beckman ultracentrifuge

tubes for SW28 rotor (take care not to disturb debris pellet at bottom of tube).
5. Place tubes into clean buckets and screw down caps. Weigh opposing buckets
and balance opposing buckets to within 0.1 g by adding appropriate volume of
D10.
6. Pellet virus for 3 hr, 18,000 rpm (42,874 average g), 4°C in SW28 rotor using
max acceleration and deceleration.
If pressed for time, pellet for 1.5 hr, 28,000 rpm (103,745 average g).
7. Remove tubes from buckets and decant supernatants into waste container
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containing 10% bleach. Invert tubes onto clean Kimwipes and allow to drain for
5 mins.
8. Re-suspend viral pellet in 60 μl D10 by gentle, repeated pipetting (20–30 times).
9. Aspirate media from 96-well plate, add 50 μl lentiviral stock, return to incubator
overnight.
10. Aspirate media from 6-well plate(s), add 0.5 ml D10.

11. Prepare a 1:400 dilution of remaining lentiviral stock. Add 50, 25 and 12.5 μl to
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each of 3 wells of the 6-well plate to make 1:4000, 8000 and 16,000 dilutions to
titer the lentiviral stock. Return plate to incubator overnight.
DAY 6 – TRANSDUCTION AND TITERING (CONTINUED)

12. Add 2.5 ml D10 media to each well of the 6-well plate(s). Return to incubator for
48 hours.
13. Add 150 μl D10 media to transduced cells in the 96-well plate. Return to
incubator.
The growth of the transduced cells should be observed daily. Significant cell
death and changes in morphology are to be expected after transduction. Once
cells become >50% confluent in the 96-well plate (3–5 days or longer),
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trypsinize and transfer to a 6-well plate (2 ml D10) for expansion. Continue to

grow the cells until they are ready to be scaled up to a T75 flask. Expand for
several more days and harvest cells for the expression test (Support Protocol 1)
and cryopreservation (resuspend 2–106 cells/ml in cryopreservation media,
freeze 1 ml/vial in a controlled-rate container at −80°C, move to liquid N2
freezer after 2–3 days).
DAY 8 – TITERING (CONTINUED)

14. Harvest and count cells from the 6-well plates used for titering the lentiviral
stock.
15. Prepare cells for analysis by flow cytometry for expression of the appropriate
surrogate marker (i.e. Thy 1.1, eGFP, etc. see Strategic Planning).
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If the chosen surrogate markers are all fluorescent proteins, cells can be fixed for
10 min at room temperature with 2% paraformaldehyde, then washed and
resuspended in flow cytometry staining buffer and analyzed on a flow cytometer.
If cell surface proteins such as Thy 1.1 are used as surrogate markers, wash and
resuspend cells in flow cytometry staining buffer, incubate 20 min on ice with
OX-7 or appropriate fluorochrome conjugated monoclonal antibody, then wash
and follow the procedure above for fluorescent protein marked cells.
16. After analysis, choose a virus dilution with 1–10% of the cells positive for
marker gene expression. These cells are presumed to have had an MOI of 1. It is
assumed that the 6 ×105 seeded cells doubled to 1.2 ×106 overnight.
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Calculate the titer of the virus T as below, where G is percentage of GFP+ cells,
N is the number of cells in the transduction sample, D is the dilution factor of the
virus stock used in the transduction reaction, and T is the titer of the virus in
transduction units per ml.
G × N × D
T=
0.5 ml

For example, where G is 3% GFP+ cells, N is 1.2 × 106 cells, and D is 16,000,
the titer T is 1.15 × 109 transduction units per ml.
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To calculate the MOI used to produce the production line, multiply the volume of
the virus stock used (Vp) by the titer (T) and divide by the number of cells used
(Np).
Vp × T
MOI p =
Np
For example, where Vp is 0.05 ml, T is 1.15 × 109 transduction units per ml, and
Np is 40,000 cells (20,000 cells plated, assumed to double overnight to 40,000
cells), then MOIp is 1440.
The MOI should be somewhere between 500 and 5000. If the cells do not
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recover after the transduction (i.e. they all die), then a second attempt can be
made at transduction with lower titers of LV (by dilution), or with shorter
incubation with the LV supernatant.
SUPPORT PROTOCOL 1 – SIX WELL ASSAY FOR ESTIMATION OF

PRODUCTION CELL LINE YIELD
This Support Protocol is designed to provide a rough estimate of the expression levels of
newly established expression cell lines in a standardized way. It is useful for comparing one
line against another, and for estimating the required scale for production cultures.
MATERIALS
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• Transduced cells from Basic Protocol 1
• 6-well plates
• 96-well ELISA plates
• 37 °C CO2 incubator
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SEED CELLS
1. Harvest and count transduced cells from the T75 flask at the end of Basic
Protocol 1.
2. Add 2×106 cells in 2 ml D10 media to a 6-well plate, one well for each
transduced cell line.

3. Use the remaining cells from the T75 flask in (1) to seed a T225 flask for further
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expansion of the cell line for production (or for cryopreservation if this was not
done at the end of Basic Protocol 1).
4. Return to a 37 °C CO2 incubator for 48 hours.
We have recently begun to setup a duplicate plate that is incubated at 32 °C,

permitting measurement of yield at both 37°C and 32*C..
HARVEST SUPERNATANT FOR ELISA

5. Collect supernatant from 6-well plates and pellet debris 4000g, 30 min, 4 °C in a
tabletop centrifuge. Harvest cells at this time as well (see below).
6. Test supernatant for the yield of soluble class II MHC by ELISA (Hornbeck,
2015).
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The nature of the test will depend on the exact protein you express. If you use the
vector design described in Strategic Planning, you should be able to use a
universal ELISA that avoids the need for MHC allele-specific monoclonal
antibodies. Support Protocol 2 takes advantage of a monoclonal antibody (2H11)
that recognizes the C-terminal leucine zipper for coating (Chang et al., 1994).
Detection can be performed using a monoclonal antibody specific for the 6xHis
tag, or, if the protein is biotinylated during expression, streptavidin HRP.
Observed yields span a range from 0 to 50 mg/l and are highly dependent upon
the allele and the sequence of the tethered peptide. This is further discussed in
the section on “Difficult Alleles” in the Commentary section.
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HARVEST CELLS FOR FLOW CYTOMETRY

7. Harvest and count cells from 6-well plate at the time supernatants are collected.
8. Prepare cells for analysis by flow cytometry for expression of the appropriate
surrogate marker (see Basic Protocol 1 and Strategic Planning).
9. Analyze the transduced cell lines for percentage of cells that are positive for the
appropriate surrogate marker(s).
a. If the MOI for a given transduction is 500–5000, the resulting cell line
will often be nearly 100% positive for each marker. If this is the case,
the cell line can be expanded for protein expression (Basic Protocol 2).
b. If the cells <95% positive, the transduced cell line can be sorted for
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expression of the surrogate marker(s). Choose a sort gate that contains

the 1–5% most positive cells for all markers, then expand the sorted line
for protein expression (Basic Protocol 2).
Complete details for flow cytometry analysis are beyond the scope of this unit.

SUPPORT PROTOCOL 2 – UNIVERSAL ELISA FOR QUANTIFYING

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PROTEINS WITH FUSED LEUCINE ZIPPERS AND HIS-TAGS

All MHC-II proteins in this unit have a synthetic C-terminal leucine zipper that is
recognized by the 2H11 mAb (Chang et al., 1994). The 2H11 mAb, used for affinity
purification of the MHC-II proteins in Basic Protocol 3, is also used as a capture antibody
for the sandwich ELISA described in this protocol. All MHC-II proteins made in this unit
also have a 6xHis that is recognized by one or more monoclonal antibodies commercially
available as HRP conjugates which are used for readout in this ELISA assay
MATERIALS
• Transduced cell line supernatant from Support Protocol 1 or Basic Protocol 3
• 96-well ELISA plates (i.e. Costar #3591)

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• Reagent reservoirs
• 2H11 monoclonal antibody (1 mg/ml in PBS, produced in-house from the

hybridoma (Chang et al., 1994))
• HRP-conjugated anti-6xHis monoclonal antibody (Abcam ab1187)
• Alternate detection: streptavidin HRP (BD 554066)
• ELISA coating buffer (see recipe)
• ELISA wash buffer (see recipe)
• ELISA blocking buffer (see recipe)

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• TMB substrate (Moss, Inc, TMB-E1000)
• Protein for standard curve/positive control, 100 μg/ml in blocking buffer, 25 μl

aliquots at −80°C (see note)
• 37°C incubator
• Multichannel pipettor
• Absorbance plate reader
• Plate washer (optional)
Note: The standard protein can be a class II MHC protein tagged with leucine
zippers and a His6 tag set aside from a successful purification and quantitated by
absorbance at 280 nm. A class II monomer can also be requested from the
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authors for this purpose.
Do not include sodium azide in any of the ELISA buffers as it will inhibit HRP.

PROTOCOL
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COAT PLATE(S)
1. Add 100 μl of 2H11 antibody to 10 ml of coating buffer per plate (10 μg/ml final
concentration. Add 100 μl/well. Incubate at 37°C for 1 hr.
BLOCK PLATE(S)
2. Discard coating solution, then bang and dry plate on paper towel stack. Add 150
μl blocking buffer per well and incubate 1 hour at 37°C.
Plates can also be stored at this step at 4 °C for up to 3 days.
ADD SAMPLES AND STANDARD TO PLATE(S)

3. Prepare standard: Thaw an aliquot of standard protein on ice. Add 20 μl of the
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stock in 980 μl of blocking buffer for 2 μg/ml.
4. Prepare samples: Dilute supernatants 10-fold in blocking buffer.
For samples you expect to have a high concentration of expressed protein (> 1
mg/L) you might need to start with a higher dilution.
5. Wash blocked ELISA plate(s) three times with wash buffer.
A plate washer is highly recommended but a multichannel pipettor can be used if

necessary.
6. Add 150 μl of standard to two duplicate wells in side-by-side rows in the 1st
column of a 96-well ELISA plate. Add 100 μl blocking buffer to the remaining
wells in the same rows.
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7. Add 150 μl of diluted samples to the remaining wells in the first column. Add
100 μl blocking buffer to the remaining wells in the same rows.
8. For three-fold serial dilutions, pipet 50 μl from the first column with a
multichannel pipettor, add to the next column and mix by pipetting up and down
several times. Repeat until diluted sample is added and mixed in the 12th column.
Dispose of remaining 50 μl sample.
For the standard rows, Leave the last (12th) column as a “blank” (no sample).
9. Incubate 1 hour, 37°C.
10. Wash plate(s) three times with wash buffer.

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ADD DETECTION ANTIBODY

11. Add 100 μl anti-6xHis HRP (1:20,000 in blocking buffer) to all wells.
Streptavidin HRP (1:20,000) can be used for detection instead if the protein is
biotinylated.
12. Incubate 1 hour, 37°C.
13. Wash three times with wash buffer.

Wash six times if streptavidin HRP is used.

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DEVELOP ELISA WITH HRP SUBSTRATE

14. Take out the stock bottle of TMB substrate from the cold room 30 minutes before
the end of the HRP step. Take 10 ml/plate into a 50 ml tube covered with foil,
leave at RT. Keep the stock bottle back in Refrigerator. Pour the substrate from
the tube into a trough and immediately add to all the wells. Incubate room
temperature for 30 minutes or less.
Watch color develop in the wells, it is important that the plate is not over
developed because the standard curve will not be accurate.
15. Add 100 μl of 0.5 M HCl. Read at 450 nm within 10 minutes on a plate reader.
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BASIC PROTOCOL 2 – CULTURES FOR PRODUCTION OF CLASS II MHC

PROTEINS
We have tested a large variety of production culture formats over the last 20 years. The
method described below—growth of 293T-derived cells in shake flasks in media containing
10% serum—is a bit unusual and results in cell growth in large clusters, but we have found
that the method produces excellent yields, is extremely simple, and is surprisingly
permissive of extensive serial passage. All other methods we have tested also produce good-
to-excellent yields, and you should not hesitate to adopt variations that are more common in
your laboratory.
We find that per-volume yields from many of our cell lines improves significantly when cells
are cultured at 30–32°C, most likely due to increased stability of the expressed protein. This
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requires an initial two passages at 37°C to bulk up the culture followed by moving the flask
to a lower temperature (ideally refrigerated) CO2 incubator for the subsequent passages. We
do find that culture in shake flasks is less permissive of serial passaging, so we monitor
yields of every harvest by performing ELISA assays (described in Support Protocol 2) on
clarified supernatants; when yields per harvest drop significantly, we terminate the
production run.
MATERIALS
• Transduced 293T cell line (from Basic Protocol 1)

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• Pluronic F-68 surfactant 100x (Thermo Fisher 24040032)
• FreeStyle 293 Expression Media (optional, Thermo Scientific 12338018)
• T225 tissue culture flasks
• Sterile, polypropylene shaker flasks (such as Fisher Scientific PBV2000 2L

flask, or Corning 431684 5L flask)


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• 250 ml-1L centrifuge bottles
• 1L 0.2 μm vacuum filter units
• Floor model centrifuge
• 37°C CO2 incubator
• Optional: 30–32°C CO2 incubator (30°C will likely require a refrigerated

incubator)
EXPAND CELLS AND SEED IN SHAKER FLASK

1. Expand transduced cell line in two T225 flasks and grow until near confluency
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(~2 × 108 cells).
2. Seed cells in a 2L shaker flask in 1L D10 with 500 μl of the biotin stock solution
and Pluronic F-68 (an anti-foaming agent) and place on shaker (125 rpm) in
37°C CO2 incubator for 8 – 10 days.
Many proteins will be easier to purify using more expensive serum-free

(FreeStyle 293) media, so you might want to compare these media for shaker
flask production. We have not observed significant differences in expression
levels.
HARVESTING AND REFEEDING

3. Warm D10 with biotin and Pluronic F-68 (or serum-free) media to room
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temperature. Stop Shaker for 20 – 30 min to allow cells to settle to the bottom of
the flask.
Cells in either media will form large clusters over time. These clusters contain
viable cells that produce high levels of recombinant protein in our system. They
also aid in harvesting, as they settle quickly to the bottom of the shaker flask and
do not require centrifugation for feeding and serial passage.
4. Slowly decant supernatant into clean centrifuge bottles. Avoid disturbing the
settled cell clusters as much as possible, although some cells will be transferred
with the supernatant.
5. Add 1L of prewarmed media from step 3 and return shaker to incubator for 7
days.
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Cultures can be harvested and refed two or more times. The maximum number of
harvests varies with the cell line (ranging from 3–12) and should be monitored
by ELISA for a decrease in expression levels.
6. Harvested supernatant should be cleared by centrifugation at the highest rcf

possible for your available centrifuge bottles and rotor, followed by 0.2 μm
filtration.

7. Proceed to purification of the supernatant as soon as possible (Basic Protocol 3

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or Alternate Protocol 1).
We have found that supernatants can be stored at 4 °C for 1–2 months prior to
purification without degradation of the MHC-II or its tags. We do not necessarily
recommend this, but it does permit strategic pooling of harvests from a
production run.
It is advisable to test harvested supernatants by ELISA (Support Protocol 2)

before purification.
8. Optional: After the second supernatant harvest, cells can be refed as above and
moved to a 30–32 °C incubator. Individual cell lines should be tested by ELISA
following each harvest to determine whether this is beneficial. The first two
passages need to be performed at 37°C to bulk up the culture; at 30–32 °C, the
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cells are less permissive to serial passage.
If expression levels measured by ELISA increase by three-fold when the

temperature is reduced, we make culture at the reduced temperature the standard
for that specific cell line.
BASIC PROTOCOL 3 – PURIFICATION OF CLASS II MHC PROTEINS BY

ANTI-LEUCINE ZIPPER AFFINITY CHROMATOGRAPHY
Using the methods of this unit, class II MHC proteins are secreted into the supernatant of
cultures of engineered derivatives of HEK-293 cells and must be purified for further
processing. All class II MHC proteins produced by the NTCF have attached leucine zippers
(Chang et al., 1994) and most have either a single or a tandem His6 tag (Schmidt et al.,
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2011), providing a choice of affinity handles for purification. In general, the antibody
affinity column described here provides better purity than the IMAC method described in
Alternative Protocol 1, especially when purifying proteins from supernatants containing
10% serum. Obviously, the antibody column method also requires production and
purification of substantial quantities of the 2H11 antibody, and this effort should be taken
into account when choosing between the two primary affinity purification methods.
In the NTCF, production runs from an expression line usually generate multiple supernatant
harvests generated by serial passage. Each harvest is stored at 4 °C until ready for
purification (performed at 4 °C). Multiple harvests from a single production culture run are
often pooled before purification. The estimated binding capacity of the 2H11 antibody
column is 1–1.5 mg of class II MHC per ml of packed column resin, and it is often helpful to
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perform ELISAs (Support Protocol 2) to estimate the concentration of class II MHC in each
harvest before deciding how many harvests to pool for purification.
MATERIALS
• 1X PBS with 0.05% Azide.
• Wash Buffer – 1 M NaCl/PBS wash buffer (see Reagents and Solutions)

• 0.1M Glycine pH 2 .8 (see Reagents and Solutions)

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• Elution Buffer- 30 mM Sodium phosphate pH 11.3 (see Reagents and Solutions)
• Neutralization buffer - 1 M Sodium Acetate pH 6 (see Reagents and Solutions)
• 10 ml 2H11 antibody conjugated Sepharose. (see Reagents and Solutions)
• Econo-Column® Chromatography Columns, 1.5 × 10 cm for packing 2H11

Sepharose (BioRad Catalog #7371512) with flow adaptors (BioRad catalog
#7380016) compatible for AKTAprime system.
• Amicon Ultra-15 10K Centrifugal ultrafiltration devices (Catalog # UFC901024)
• Bottle top sterile filter units 0.2 μm (Thermo Scientific Nalgene Catalog # 595–
4520)
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• Microcentrifuge tubes (USA Scientific # 1615–5500)
• 14 ml Collection tubes (VWR Catalog # 60818–618)
• ÄKTAprime plus chromatography system (Cytiva; discontinued)
• NanoDrop-2000 spectrophotometer
PROTOCOL
PREPARING SAMPLE AND CHROMATOGRAPHY SYSTEM FOR A
PURIFICATION RUN
1. Pool culture supernatants from Basic Protocol 2, step 6) so that the estimated
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total amount of MHC-II matches the estimated binding capacity of the 2H11
column.
For example, if there are 6 × 1 liters of culture supernatant, each with an ELISA-
estimated concentration of MHC-II of 3 mg/liter, 3 liter pools should be created
for running on a 10 ml 2H11 column. A 10 ml column has a binding capacity of
15 mg MHC protein.
2. Centrifuge harvests at 3000 × g for 30 min at 4 °C and filter through 0.2 μm

bottle top filters.
3. Prepare the chromatography system for a purification run.
As noted elsewhere, we use the ÄKTAprime plus system from Cytiva and all the
affinity purifications done at 4 °C.
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a. Pump 50 ml diH2O through each of four buffer lines.
b. Prime each of the buffer lines into the respective buffers as follows:
i. Line 1: 1XPBS with 0.05% Azide.
ii. Line 2: 1M NaCl/PBS.
iii. Line 3 or 4: 0.1M glycine, pH 2.8.

iv. Line 4: Elution buffer.

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c. Connect 10 ml 2H11 Sepharose column and equilibrate with 5 column

volumes of PBS.
2H11 Sepharose columns are stored in PBS with 0.05% sodium aziide
when not in use.
4. Set up the chromatography system to monitor the OD280 of the column flow
through.
5. Place sample line in the supernatant to be purified. The column flow through line
should be directed to a clean bottle or other suitable collection vessel.
Take care not to introduce air bubbles into the lines.
We often perform ELISA assays on column flow throughs to check for

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overloading. In most cases, this does not happen.
LOADING AND WASHING THE COLUMN

6. Load the sample at a flow rate of 5 ml/min. Carefully monitor the level in the
sample reservoir throughout so as not to introduce bubbles, especially near the
completion of loading.
Volumes of the supernatants can vary from 30 ml to 3000 ml based on the

expression level of the specific MHC allele to not exceed binding capacity of the
column.
7. Just before the sample is completely loaded onto the column, add 15 ml of PBS
to the sample reservoir to ensure that all of the sample is loaded onto the column.
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8. Repeat step 6 with an additional 80 ml of PBS.
9. Wash the column with 10 column volumes (100 ml) of PBS/NaCl buffer.
10. Wash the column with 10 column volumes of PBS/Azide.
11. Set up the fraction collector and collection tubes.
a. Dispense 2 ml neutralization buffer in each clean collection tube where

the eluted proteins will be collected.
a. Place these tubes into the appropriate positions of fraction collector.
b. Set the fraction collector to collect 10 ml fractions, based upon an

elution flow rate of 5 ml/min.
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ELUTION OF THE COLUMN

12. Elution of proteins.
a. Switch chromatography system to the Elution Buffer line.
b. Elute the MHC-IIp at a flow rate of 5 ml/min, collecting fractions as

above, all the while monitoring the A280 of the eluate.

13. Regenerate the affinity columns.

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a. Strip any remaining protein by running 100 ml of 0.1M Glycine buffer.
b. Wash the column with 20 column volumes of PBS.
c. Store the column with PBS/0.05% sodium azide.
14. Pool the protein containing fractions.
The chromatogram provides an excellent guide to the protein containing

fractions. From a 10 ml column, the protein typically elutes in a total volume of
20–40 ml.
PROCESSING PURIFIED PROTEIN

15. Concentrate the protein using centrifugal ultrafiltration filters to a volume of 1 ml
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in each filter. The filters are spun as per the manufacturer’s instructions. For the
Amicon Ultra-15 10K Centrifugal ultrafiltration devices, this is 3000 × g for 15–
20 minutes.
The filtration devices must be set up in balanced pairs. Add up to 15 ml of

pooled fractions to each of 2 centrifugal ultrafiltration devices. When the volume
of the eluted protein pool is more than 30 ml, repeated cycles of concentration
are performed.
16. Exchange the buffer of the protein for Monomer Storage Buffer (MSB) by two
cycles of centrifugal ultrafiltration.
When all of the protein has been concentrated to a volume of approximately 1 ml

in each of the filters, add MSB to fill the reservoir (typically, this requires ~14
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ml). Make sure the filter cap is secure and gently invert to mix before
centrifuging. Repeat one time for two cycles of buffer exchange.
17. Pool the retentate from each of the filters in a clean polypropylene tube.
18. Measure the absorbance of the protein by UV spectrophotometry.
19. See Support Protocol 3 for more details.
20. Calculate the concentration of the protein using the estimated extinction
coefficient and adjust to 2 mg/ml as described in Support Protocol 3.
21. Save an aliquot of 20 μl for subsequent analysis of biotinylation by Support

Protocol 5. Store this aliquot at −80 °C.
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22. Prepare aliquots of the protein for cryostorage.
Aliquots are stored at −80 °C for future peptide exchange. In the NTCF, a single-
use aliquot contains 125 μl of protein at a concentration of 2 mg/ml. We often
store larger aliquots in multiples of 125 μl that can be used for multiple peptide
exchanges (for example, a 1 ml aliquot can be used for 8 exchange reactions).
We often perform cleavage of the tether with the 3C protease and store the
material as “pre-cleaved”.

23. Snap freeze the aliquots in liquid nitrogen and store in a −80 °C freezer.
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We have used aliquots that are over 2 years old with no observable change in
effect.
ALTERNATE PROTOCOL 1 – IMAC PURIFICATION OF HIS-TAGGED CLASS

II MHC
The Immobilized Metal Affinity Chromatography (IMAC) method described here results in
protein purities ranging from 60–90% depending on the production yield of the expressed
allele and the choice of serum versus serum-free media for production. In general, the anti-
zipper affinity column method described in Basic Protocol 3 provides better purity, but it
does require substantial effort to produce the columns. When we do use purification by
IMAC, a second step of purification is usually performed either by gel filtration or ion-
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exchange. Protocols for gel filtration and ion-exchange chromatography can be found
elsewhere in the Current Protocols series (Hagel, 1998; Williams and Frasca, 1999).
MATERIALS
• Culture supernatants from Basic Protocol 2, step 6
• Binding buffer - 20mM HEPES pH 7.4, 150mM NaCl, 2mM Imidazole (see
Reagents and Solutions)
• Elution Buffer - 20 mM HEPES pH 7.4, 150mM NaCl, 500mM Imidazole (see

• HisTrapFF Crude columns (Cytiva Catalog #17528601)

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• 20X MES SDS running buffer (Thermo Fisher Catalog # NP0002–02)
• NUPAGE LDS sample buffer 4X (Thermo Fisher Catalog # 1187053)
• NUPAGE 12% Bis-Tris gels (Thermo Fisher Catalog # NP0342)
• GelCode Blue Stain Reagent (Thermo Fisher Catalog #24592)
• Collection tubes
• AKTA Primeplus system (Cytivia # 11001313) or equivalent. This system was

discontinued in 2019.
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• SureLock electrophoresis cell (Thermo Fisher Catalog # E10001)
• NanoDrop™ 2000 spectrophotometer or equivalent (Thermo Fisher Catalog #

ND-2000).

PROTOCOL
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DAY 1: AKTA PURIFICATION RUN

1. Warm culture supernatants from Basic Protocol 2, step 6 to be purified to room
temperature. While they are warming up, prepare binding buffer and elution
buffer.
In the NTCF, culture supernatants are stored at 4 °C, often for weeks without
apparent degradation of protein or loss of tags. We perform all IMAC
purifications at room temperature to avoid precipitation issues with Imidazole.
Warming the supernatants to room temperature minimizes on-column degassing.
2. Wash all buffer and sample lines with 50 ml diH2O.
The chromatography system lines and pumps are stored in 20% ethanol when not
in use.
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3. Prime the buffer and sample lines with 50 ml of appropriate buffers. The sample
line and buffer line 1 are primed with binding buffer, buffer line 2 is primed with
elution buffer, and buffer line 3 is primed with diH2O.
4. Connect the 5 ml HisTrapFF column to the system, wash it with 5 column

volumes of diH2O followed by 5 column volumes of binding buffer.
All wash steps can be done at a flow rate of 5 ml/min.
5. Setup the chromatography system to continually monitor UV absorbance at 280

nm.
6. Set up the column flow through line into a clean collection reservoir of
appropriate size.
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7. Place sample line in supernatant to be purified.
completion of loading to about 90% of the sample volume.
WASH, ELUTION, COLUMN REGENERATION, AND STORAGE

9. Transfer the sample line to a fresh bottle of binding buffer and continue washing
the column with at least 15 column volumes of binding buffer. Verify that the
signal on the in-line UV detector has returned to baseline.
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10. Setup a fraction collector to collect 10 ml fractions. Switch the column flow
through valve to the fraction collector.
11. Run a 250 ml elution gradient of 0–30% elution buffer at a flow rate of 1 ml/min.
12. Elution of MHC monomer is monitored by absorbance at 280 nm.
13. Following the gradient, column is washed with a second gradient of 30–90% of
elution buffer in 5 column volumes of 25 ml.

14. Washing of the column is continued with 25 ml of 90% of Elution buffer.

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15. After the elution, wash the column with 10 column volumes of binding buffer.
The column is now ready for the next purification run.
16. For long term storage, switch the system to pump 10 column volumes of water
through the column followed by 10 column volumes of 20% ethanol. Store the
column at 4 °C.
A 1 liter run should take about 7 hours.
DAY 2: ANALYSIS OF FRACTIONS

17. From the 280 absorbance on the chromatogram, select fractions likely to contain
the eluted MHC protein for analysis by SDS-PAGE. From each fraction, take 10
μl for analysis on SDS-PAGE.
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18. Analyze the fractions by SDS-PAGE (Gallagher, 2012).
19. Based on the SDS-PAGE analysis, combine the fractions that contain the protein
of interest (usually 3–10 fractions of 10 ml each).
20. Concentrate the sample and perform a buffer exchange by centrifugal

diafiltration.
a. Add up to 15ml of pooled sample to Amicon-15 ultra filtration devices

and spin as per manufacturer’s instructions (3800 RPM for 15–20
mins).
b. Concentrate to about 1 ml and fill Amicon filters with appropriate

buffer for polishing step (gel filtration or anion exchange) and spin
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down again.
c. Repeat step b once time. The final volume of the concentrated, buffer-
exchanged sample should be about 1ml.
d. Remove the buffer exchanged protein from the spin filters and measure
both the volume and the protein concentration.
e. Adjust the protein concentration to 2 mg/ml with the appropriate buffer.
f. Proceed to the next purification step if warranted.
A sample chromatogram and associated SDS-PAGE analysis of selected fractions is shown

in Figure 3.
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SUPPORT PROTOCOL 3 - PROTEIN CONCENTRATION MEASUREMENTS

AND ADJUSTMENTS
This brief Support Protocol describes how to measure protein concentrations by UV
spectrometry and how to perform concentration adjustments.

We calculate concentrations of purified MHC-II from absorbance at 280 nm values using the
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method of Gill and von Hippel (Gill and Hippel, 1989) with updated extinction coefficients
as described by Edelhoch (Edelhoch, 1967) and described by the ProtParam tool at https://
web.expasy.org/protparam/.
Protein molecular weights are calculated from the amino acid sequence as described in the
ProtParam tool. We ignore the contribution of carbohydrates. Most of the MHC-II proteins
produced in this unit have mass extinction coefficients in the range of 1.3–1.6.
The NTCF has long adopted storage of purified MHC at a concentration of 2 mg/ml as a
standard. Single use aliquots typically contain 100 μl of protein and are ready for
multimerization. Prior to storage at −80 °C, samples are snap frozen in liquid nitrogen.
MATERIALS
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ND-2000).
• Buffers as applicable.
PROTOCOL
1. Measure the absorbance of the protein by UV spectrophotometry.
Good technique is critical for obtaining accurate absorbance data. Be sure the
stage is cleaned with ddH2O. Be sure the NanoDrop is blanked with undiluted
buffer matched to the sample buffer. To be accurate, absorbance at 280nm must
be between 1 and 10.0. Higher or lower values will require further dilution or
concentration of the sample, respectively. The absorbance at 260 and 280 is taken
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simultaneously. The OD260 should be ½ to ¾ that of the OD280. Values outside

this range may indicate a problem with the sample, so these data should be
carefully noted.
2. Calculate the concentration of the protein using the estimated extinction

coefficient (Edelhoch, 1967; https://web.expasy.org/protparam).
3. Adjust the concentration of the protein to 2 mg/ml.
a. If the concentration of the protein is below 2 mg/ml, this will require

further concentration of the protein, either in the same filtration device
or one of the smaller volume units available from Amicon or other
suppliers.
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b. If the concentration is above 2 mg/ml, calculate the volume of storage

buffer to be added using the equation below, where Va is the volume of
buffer to be added, Vm is the measured volume of the concentrated
protein before adjustment, and Cm is the measured concentration of the
protein before adjustment.

Cm
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Va = Vm ( − 1)
2
SUPPORT PROTOCOL 4 – POLISHING PURIFICATION BY ANION

EXCHANGE CHROMATOGRAPHY
This Ion Exchange Chromatography (IEC) Support Protocol is typically used in cases where
the MHC-II protein is first purified by IMAC and if the initial purity is < 90%. In some
cases, we have used preparative gel filtration chromatography (Hagel, 1998) as an alternative
to this IEC protocol.
MATERIALS AND REAGENTS

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• MHC-II samples from Alternate Protocol 1, step 20
• Binding buffer - 20mM Tris pH 8 (see Reagents and Solutions)
• Elution Buffer - 20 mM Tris pH 8, 1M NaCl (see Reagents and Solutions)
• Monomer Storage buffer (see Reagents and Solutions)
• Mono Q 5/50GL columns (Cytiva Catalog #17516601)
• GelCode Blue Stain Reagent (Thermo Fisher Catalog #24592)

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• 5 ml Collection tubes (VWR Catalog # 60818–281)
• ÄKTApurifier UPC10 system (Cytiva #29257810) or equivalent. This system

was discontinued in 2019.

ND-2000).
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PROTOCOL
In the protocol below, we limit loading to ≤ 12 mg on a 1 ml Mono Q column.
1. Prepare MHC-II samples from Alternate Protocol 1, step 20 for anion exchange
chromatography by buffer exchange using centrifugal diafiltration.

a. Fill the 15 ml centrifugal ultrafilter containing the concentrated MHC-II

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sample with 20mM Tris pH 8. Mix by gentle inversion. Concentrate

sample to 1 ml at 3500g at 4 °C.
b. Repeat step a twice.
c. Make up the sample to 10 mL with 20 mM Tris pH 8 in a 15ml

polypropylene conical tube.
This step is necessary to remove salt from the previous purification step or from
the storage buffer.
2. Prepare the chromatography system for the purification run.
We use the ÄKTApurifier system from Cytiva (discontinued in 2019). Suitable

equivalents can be used.
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a. Prime each of the Buffer lines with respective buffers as follows –
i. Line 1: Binding/wash buffer (20mM Tris pH 8)
ii. Line 2: Elution Buffer (20mM Tris pH 8, 1M NaCl)
b. Connect 1 ml MonoQ column and equilibrate with 5 column volumes

of binding buffer.
c. Flush the Sample line with 10ml of binding buffer.
d. Set up collection tubes in the Fraction collector.
e. Set up the chromatography system to monitor absorbance at 280 nm.
3. Prepare samples for loading: Place 15ml conical tube with sample in sample tube
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stand, and position sample line in the tube so that it reaches the bottom. Ensure
that the tube is secured so that it remains on the bottom of the tube as sample is
loaded and does not draw air into the sample pump.
4. Load sample at 1ml/min.
5. Wash the column with 10 column volumes of binding buffer.
6. Set the fraction collector to collect 1 ml fractions, based upon an elution flow
rate of 1 ml/min.
ELUTION OF PROTEINS AND ANALYSIS BY SDS-PAGE

7. Run a 20 ml elution gradient from 0–25% of elution buffer at a flow rate of 1ml/
min.
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This corresponds to a gradient of 0–0.25 M NaCl in 20 mM Tris, pH 8.0 buffer.
8. Elution of MHC monomer is monitored by absorbance at 280 nm.
9. Following the gradient, column is washed with 25% of elution buffer in 5

column volumes of 5 ml.

10. Washing of the column is continued with a second gradient of 25–100% elution
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buffer in 5 ml.
11. Continue washing the column with 5 column volumes of 100% elution buffer.
12. After the elution, equilibrate the column with 5 column volumes of binding
buffer.
13. From the 280 absorbance on the chromatogram, select fractions likely to contain
the eluted MHC protein for analysis by SDS-PAGE. From each fraction, take 10
μl for analysis on SDS-PAGE.
Basic protocols for SDS-PAGE are widely available (Gallagher, 2012). Follow
the manufacturers recommendation for your equipment.
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PREPARE PURIFIED PROTEIN FOR STORAGE

14. Based on the SDS-PAGE analysis, combine the fractions that contain the protein
of interest (usually 3–6 fractions of 1 ml each).
15. Concentrate the sample and perform a buffer exchange by centrifugal

diafiltration.
a. Add up to 4ml of pooled sample to Amicon-4 ultra-filtration devices

and spin as per manufacturer’s instructions (3800 RPM for 15–20
mins).
b. Concentrate to about 1 ml and fill Amicon filters with monomer storage

buffer and spin down again.
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c. Repeat step b once again. The final volume of the concentrated, buffer-
exchanged sample should be about 0.5–1ml.
d. Remove the buffer exchanged protein from the spin filters and measure
both the volume and the protein concentration.
e. Adjust the protein concentration to 2 mg/ml with monomer storage

buffer as described in Support Protocol 3.
f. Snap freeze the aliquots in liquid nitrogen and store in a −80 °C freezer.
SUPPORT PROTOCOL 5 - ESTIMATING BIOTINYLATION PERCENTAGE BY

STREPTAVIDIN PRECIPITATION
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We measure the extent of biotinylation of class II MHC monomers by a streptavidin

precipitation (SAP) protocol, followed by SDS-PAGE (SAP-Gel). Biotinylated class II MHC
proteins are incubated at room temperature with either streptavidin-coated (SA) or control
beads. Samples are then spun down in a microcentrifuge and the supernatants are analyzed
by SDS-PAGE. Good biotinylation is seen as the complete (or nearly complete) absence of
protein in the samples incubated with SA beads. To assure that equal amounts of material
were added to each lane, a loading control protein (RNase A) is added to each sample in the

assay. For biotinylated class II MHC proteins, this method is more suitable than the
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streptavidin gel shift assay we described in the class I MHC production protocol (Altman
and Davis, 2016)) because for many of our engineered class II proteins, the two subunits
often run together on SDS-PAGE. Also, since all samples are visualized by SDS-PAGE after
biotinylation, an assessment of the integrity of the protein can also be made.
MATERIALS AND REAGENTS

• Purified MHC-II protein sample from Basic Protocol 3, step 22 or Alternate
Protocol 1, step 20 or Support Protocol 4, step 15.
• 10X PBS (Cellgro Catalog #46013CM)
• Sepharose 4B beads (Sigma-Aldrich Catalog # 4B200–100ml)
• Streptavidin-Agarose beads (Sigma Catalog #S1638–5ml)

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• RNAse A (Sigma-Aldrich Catalog #R6513). 2 mg/ml stock of RNAase A is

made in 1X PBS and is stored at −80 °C in aliquots of 50 μl.
• 2mg/ml purified MHC
• Tabletop centrifuge (Eppendorf Centrifuge 5415C)
PROTOCOL
WASH BEADS—Sepharose 4B beads are supplied in 20% ethanol and streptavidin beads
are supplied in PBS containing azide. Before use, both beads have to be washed with 1X
PBS.
1. Pipette 0.5 ml suspension into 1.5 ml microcentrifuge tubes.

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2. Centrifuge at 2000 × g for 5 minutes to pellet beads.
3. Aspirate the supernatant and resuspend the beads in an equal volume of 1 × PBS.
4. Repeat steps 2 and 3 for a total of 3 times.
STREPTAVIDIN PRECIPITATION
5. Label two 0.6 ml microcentrifuge tubes per MHC-II sample that is being tested,
〝−〞 and “+”. Pipette 2.5 μl of 2mg/ml RNAse A into each of the labeled tubes.
6. Pipette 5 μl each of the MHC-II protein (stock concentration is 2 mg/ml) into the
labeled tubes. Mix gently by tapping the tube.
7. With a scissors or razor blade, cut the tip of a pipette tip that will be used to
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pipette the beads. Wash the tip with 1X PBS two times.
This is necessary because the unmodified orifice on most 200 μl tips is too
narrow to pipette beads.
8. Add 50 μl of the Sepharose 4B beads into the tube labeled - and 50 μl of the
streptavidin beads into the tube labeled +. Mix by gently tapping the tube. Leave

at room temperature for 1 hour. Reaction mix is pipetted up and down every 10
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minutes making sure that beads are at the bottom of the tube.
9. At the end of the incubation, centrifuge the tubes for 5 minutes at 2000 × g.
10. Carefully without touching the beads, aspirate 7 μl of the respective supernatant
and dispense into fresh tubes, again labeled – and +.
ANALYZE SUPERNATANTS FROM PRECIPITATION BY SDS-PAGE

11. Add 2.5 μl of the 4X gel loading buffer and 0.5 μl of 1M DTT. Boil samples for 5
minutes and load on an SDS-PAGE gel (Gallagher, 2006).
An example of this assay is shown in Figure 4.
BASIC PROTOCOL 4 - PEPTIDE EXCHANGE

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The MHC-II proteins that are expressed using the protocols in this unit are designed to
permit loading with appropriate synthetic peptide ligands. There are two discrete steps
involved: (1) cleavage of the linker between the N-terminus of the MHC-II β chain and the
expression-construct coded peptide ligand; and (2) loading of the synthetic peptide ligand,
usually mediated by a combination of below neutral pH and catalysis by HLA-DM (Denzin
and Cresswell, 1995). These two steps are dissociable, permitting batch cleavage of the
linker containing an HRV-3C protease recognition site in the tethered constructs to yield
stable but exchange-competent complexes with the formerly tethered peptide (FTP) ligand
that can be loaded with exchange peptides at a later time. Batch cleavage of the linker also
facilitates removal of the 3C protease, provided as a recombinant fusion to GST (available
commercially from many sources) and removed on a GSTrap FF affinity column.
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MATERIALS
• 10X PBS (CellGro Catalog # 46–013-CM)
• GSTrap FF column, 5 ml (Cytiva 17513101)
• GST Elution Buffer – 50 mM Tris pH 7.5 with 10 mM reduced glutathione (see

• PreScission Protease (Cytiva Catalog # 27084301). This product is a GST fusion

to the HRV 3C protease. We often used a similar in-house expressed and purified
product.
• Monomer Storage buffer (see Reagents and Solutions)

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• 10X 3C Cleavage buffer (see Reagents and Solutions)
• GelCode Blue stain Reagent (Thermo Fisher Catalog #24592)

• Amicon® Stirred Cell 400mL (Catalog #: UFSC40001, https://

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www.emdmillipore.com)
• Ultrafiltration membranes (Millipore Ultracel 10kDa # PLGC07610)
• Amicon Ultra-15 or Ultra-4 10K Centrifugal ultrafiltration devices (Catalog #

UFC901024, UFC801096)
• Screw-capped microcentrifuge tubes (Fisher Scientific Catalog #02681338)
• Cryolabels.
• AKTA Primeplus system

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• Nano drop spectrophotometer
• 0.5 M Citrate buffer (see Reagents and Solutions section)
• 10% Octyl glucoside (see Reagents and Solutions section)
• 5 M NaCl (see Reagents and Solutions section)
• Leupeptin mg/ml (see Reagents and Solutions section)
• Pepstatin mg/ml (see Reagents and Solutions section)
• 0.5 M EDTA (see Reagents and Solutions section)
• 0.1 M PMSF (see Reagents and Solutions section)

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• Anti-DM Sepharose beads (see Reagents and Solutions section)
• 2 mg/ml MHC monomer stocks from Basic Protocol 3, step 22
• 2 mg/ml HLA-DM stock (produced in-house, using the methods in this chapter)
• Peptide stocks (≥ 85% pure)
CLEAVAGE PROTOCOL
CLEAVE THE MHC-IITP WITH 3C-GST PROTEASE
1. Create a master MHC-II protein stock consisting of 50 mg of MHC-IItp at a
concentration of 2 mg/ml. Set aside 5 μl of this for use as an uncleaved control
for SDS-PAGE.
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In the NTCF, we store nearly all purified MHC proteins at −80 °C at a standard
concentration of 2 mg/ml. For this protocol, we sometimes pool proteins derived
from different purification batches, always produced from a single expression
cell line. For this protocol, we describe cleavage of 50 mg of MHC-IItp, but the
quantities in the table below can be scaled up or down.
2. Set up overnight cleavage reactions according to the quantities in the table below.

The HRV-3C enzyme is a cysteine protease and many commercial suppliers

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recommend inclusion of dithiothreitol (DTT) as a reducing agent in reaction

buffers to achieve optimal and sustained reactivity. For this application, we have
found addition of DTT is not necessary and have omitted it because of unverified
concerns over unwanted reduction of disulfides in the MHC-II proteins.
Component Volume
10X 3C buffer 25 ml
2mg/ml MHC Monomer 25 ml
2mg/ml 3C protease 1.25 ml
Water 198.75 ml
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3. Incubate the cleavage reaction overnight (10–15 hrs) at 25 °C.
REMOVAL OF 3C-GST PROTEASE

4. Before loading the sample onto the GSTrap FF column, set aside 10 μl
corresponding to 4 μg of MHC-II for SDS-PAGE analysis.
5. Run all chromatography steps below at 4 °C.
Our chromatography system is in a cold room, so we run the GSTrap column at 4

°C. This step could also be run at room temperature. If you choose to run it at 4
°C, chill the cleavage reaction in an ice water bath before loading the column.
6. Setup the chromatography system to continually monitor UV absorbance at 280

nm.
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7. Connect a 5 ml GSTrap FF Column to the chromatography system.
8. Prime buffer line 1 with PBS and buffer line 2 with GST Elution Buffer.
9. Wash the column with 5 column volumes (25 ml) of PBS at a flow rate of 5 ml/
minute.
10. Set up the column flow through line into a clean collection reservoir of
appropriate size.
This purification depletes the 3C-GST protease from the sample. The desired
product does not bind to the column. We collect the flow through into a 250 ml
glass bottle. Alternatively, flow through can be collected in 15 ml fractions in
polypropylene tubes.
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11. Place buffer line 1 in the cleavage reaction sample.
completion of loading.

13. Just before the sample is completely loaded onto the column, add 15 ml of PBS
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to the sample reservoir to ensure that all of the sample is loaded onto the column.
14. Repeat step 13 with an additional 20 ml of PBS.
15. Transfer the flow through line to a waste container.
3C-GST ELUTION, COLUMN REGENERATION, AND STORAGE

16. Transfer the sample line to a fresh bottle of PBS and continue washing the
column with at least 10 column volumes of PBS. Verify that the signal on the in-
line UV detector has returned to baseline.
17. Switch the chromatography system to pump GST Elution buffer through the
column. Setup the column outlet to collect the first 2 column volumes of eluate
to enable verification of 3C-GST removal.
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18. Elute the 3C-GST protease with 10 column volumes of GST Elution Buffer.
19. Elution of 3C protease is monitored by 280 absorbance.
20. After the elution, switch the system to pump 10 column volumes of PBS through
the column.
21. For long term storage, switch the system to pump 10 column volumes of water
through the column followed by 10 column volumes of 20% ethanol. Store the
column at 4 °C.
CONCENTRATION, BUFFER EXCHANGE, AND STORAGE—The cleaved protein

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must be concentrated, and the reaction buffer must be exchanged for Monomer Storage
Buffer (MSB). We use a combination of Amicon stirred cells and centrifugal ultrafiltration
devices to accomplish this.
22. Install the ultrafiltration membrane in the Amicon stirred cell.
23. Rinse the membrane by adding 20 ml of MSB, pressurizing the stirred cell and
stir for 10–15 minutes. Release the pressure, discard the residual buffer, and
repeat this procedure once.
24. Concentrate the protein as described in the stirred cell manual to an estimated
concentration of 4 mg/ml.
As the volume of retentate in the stirred cell approaches 15–20 ml, turn the stir
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rate down to prevent foaming. In addition, do not concentrate below 10 ml to

avoid the risk of drying out the stirred cell or over-concentrating the protein.
25. When the volume of the protein solution in the stirred cells is between 10–15 ml,
stop the stirred cell and add 10 volumes of MSB. Reconcentrate as in step 24.
26. Repeat 25 above to complete the buffer exchange process.

27. Remove the buffer exchanged protein from the stirred cell and measure both the
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volume and the protein concentration. If the protein concentration is < 2 mg/ml,
concentrate it further in a centrifugal ultrafiltration device.
28. Adjust the protein concentration to 2 mg/ml by the addition of MSB.
29. Set aside 5 μl for analysis by SDS-PAGE.
30. Prepare 1 ml aliquots of the cleaved protein in screw-capped microcentrifuge

tubes and apply appropriate cryolabels.
The peptide exchange reactions described below each use 100 μl of MHC-II (2
mg/ml), so it is sometimes useful to store aliquots in size multiples of 100 μl.
31. Snap freeze the tubes in liquid nitrogen and store at −80 °C.
32. Analyze the pre- and post-cleaved material by SDS-PAGE.

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The molecular weight of the typical formerly tethered peptide (i.e. hCLIP) is
about 3,400 Daltons. In general, loss of this peptide from the amino terminus of
the β chain is easily seen by SDS-PAGE. A sample of this analysis is shown in 5.
PEPTIDE EXCHANGE PROTOCOL

33. Day 1: Set up exchange reaction
a. Take out peptide stocks from storage at −80 °C to thaw at room

temperature for least an hour before starting the exchange reaction.
Peptides are normally stored in DMSO which freezes at 19 °C.
b. Remove cleaved MHC-II stocks from the −80 °C freezer and thaw.
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Keep on ice until the exchange reaction is set up.
c. Make a master mix as indicated in the table below.
Human (μl) Mouse (μl)

5M NaCl 20 20
0.5M Citrate buffer pH 5.2 100 100
0.5M Citric Acid - 25
10% Octyl Glucoside 100 -
1mg/ml Leupeptin 2 2
1mg/ml Pepstatin 1 1
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0.5M EDTA 4 4
0.1M PMSF 5 5
We perform exchange reactions with human class II alleles at pH 5.5

with octyl glucoside and with mouse alleles at pH 5.0 without octyl
glucoside. For class II MHC from other species, it is necessary to
optimize the exchange conditions on a per-allele basis.

d. Add master mix into 1.5 ml Eppendorff tube..

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e. Add 20–40μl of peptide stocks to the master mix. Vortex the sample to
mix.
We use 75 fold molar excess of peptide for loading mouse class II

alleles and 25 fold molar excess of peptide for human alleles.
f. Add 100 μl of cleaved MHC-II. Mix by pipetting gently.
g. Add 25 μl of HLA-DM (stock concentration of 2 mg/ml). Mix by

pipetting. Make up total reaction volume to 1 ml with water.
We use HLA-DM for class II MHC peptide exchanges for all species.
We have verified that it works on many mouse class II alleles.
34. Incubate for 22–24 hrs at an allele specific temperature as shown in the Table
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below.
Alleles Incubation Temperature

All human alleles 30 ºC
Mouse I-Ab 33º C
Mouse I-A(d) 25 ºC
Mouse I-A(g7) 25 ºC
Mouse I-A(s) 25 ºC
35. Day 2: Removal of DM

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a. After incubation, neutralize the pH by adding 200μl of 1M phosphate

buffer pH7.5 and centrifuge exchange reaction sample at 4 °C for 15
minutes at 13000 × g.
This step removes material that may have aggregated during the
exchange reaction.
b. While sample is being centrifuged, transfer 100 μl anti-DM sepharose

beads to a clean 1.5 ml Eppendorf tube labeled for the intended sample.
c. Without disturbing any pelleted material, pipette the supernatants of

each sample carefully into Eppendorff tubes with beads. Mix gently by
pipetting up and down.
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d. Place tubes in a rotator and incubate for 30 minutes at 4 °C.
e. Decant the sample in to a clean 5 ml spin column. Elute by gravity into

an ultrafiltration concentrator or collect the eluate by centrifuging the
column for 2min at 1000 × g into a 15 ml conical tube and transfer the
eluate into an ultrafiltration concentrator (this fraction contains MHC
protein free from DM that is bound to the resin). Wash the columns
with 0.5 ml of 1× PBS, pool the eluate with sample from step d.

f. Start concentrating this fraction by standard centrifugal ultrafiltration,

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followed by buffer exchange with MSB twice.
g. Measure absorbance at 280 nm and calculate the protein concentration.

Adjust the concentration of the MHC monomer to 2 mg/ml.
h. These MHC monomers are now ready for multimerization.
36. For long-term storage, these MHC monomers are snap frozen in 0.1 ml aliquots
and stored at – 80 °C until multimerization.
BASIC PROTOCOL 5. ANALYSIS OF PEPTIDE EXCHANGE BY MATRIX-

ASSISTED LASER DESORPTION/IONIZATION (MALDI) MASS
SPECTROMETRY
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The methods used to produce class II MHCp complexes in this unit—expression of MHC-II
with a tethered peptide ligand followed by protease cleavage of the tether and exchange with
a single synthetic peptide—ensure that the peptide-binding site of the final product can only
exist in one of three states: (1) it can be empty, (2) it can be occupied by the formerly
tethered peptide (FTP), or (3) it can be occupied by a synthetic exchange peptide (this
ignores the possibility of peptide binding in discrete registers (Bankovich et al., 2004;
Landais et al., 2009; Stadinski et al., 2010)). The FTP can easily be distinguished from the
synthetic exchange peptide by elution followed by simple mass spectrometry methods using
a MALDI-TOF instrument (Sandoval, 2014)) as described in the method below (LC-MS
methods can also be used as an alternative). In favorable circumstances, peptide-loaded
MHC-II can be distinguished from empty MHC-II by nIEFGE described in Alternate
Protocol 2, but completely generalizable methods for detecting empty MHC-II remain to be
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developed.
MATERIALS
• 2mg/ml MHC-IIp stocks (from Basic Protocol 4, step 36).
• 1% trifluoroacetic acid (see Reagents and Solutions)
• Methanol (Sigma Aldrich Catalog # 9000688–1L)
• 96-well Natural Polypropylene, V bottom plates (Falcon Catalog #353263)
• Pierce C18 tips, 10 μl bed (Thermo Fisher Catalog # 87782)
• 0.2 ml 8 tube PCR strips (BioRad # TLS0801)

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• 0.2 ml Flat PCR tube 8-Cap strips (BioRad catalog # TCS0803)
• 10mg α-Cyano-4-hydroxycinnamic acid (Sigma-Aldrich Catalog # C8982)
• Bruker Daltonics MTP 384 Target plate Grand STL (Catalog # 8280784)
• Bruker Daltonics Ultraflex-II MALDI-TOF/TOF system

All solvents are mass spectrometry grade. In addition, it is highly recommended to use
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polypropylene tubes and plates and to avoid polystyrene (which can contribute to polymer
noise in the mass spectra).
PROTOCOL
1. Elute peptide from MHC-IIp (from Basic Protocol 4, step 36):
a. Add 6 μL of the post exchanged MHC-IIp (stored at a standard

concentration of 2 mg/ml) to 2 μL of 1% trifluoroacetic acid and 12 μL
of deionized water in a polypropylene PCR tube.
b. Incubate for 2 hours at 70 °C in GeneAmp PCR system or equivalent.
2. Desalt eluted peptide sample:
According to the manufacturer’s specification, the tips we use have 10 μl of C18

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resin. In the conditioning and equilibration steps below, a sufficient volume of

liquid is drawn into the tip to the top level of the C18 resin. To accomplish this,
the pipettor is set to 20 μl, but when withdrawing liquid, it is not fully released.
a. Condition C18 tips. Add 125 μl of methanol into required number of

wells of a polypropylene 96 well plate. Insert C18 tips on a pipettor set
at 20 μL and aspirate 100 % methanol slowly until the solution reaches
the top portion of the tips. Discard the solution and repeat this
procedure for a total five number of times.
a. Equilibrate C18 tips in TFA solution: Add 125 μl of 0.1% TFA into
wells of the same 96 well plate and aspirate 0.1 % TFA in ddH2O
slowly until the solution reaches the top portion of the tips. Discard the
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solution and repeat this procedure for a total number of five times. The
tips are now conditioned and ready for samples.
b. Bind sample to C18 tips. Set the pipettor at 20 μL and aspirate the
sample solution from the PCR tubes slowly until the solution reaches
the top portion of the tips. Dispense the solution back to the solution
and repeat this procedure for a total number of 10 times.
c. Wash the C18 tips with 0.1 % TFA in ddH2O. Aspirate and dispense
0.1 % TFA in ddH2O water through each C18 tip for a total five
number of times.
3. Elute bound peptides from C18 tips. To elute bound peptides, add 5 μl of
methanol to a fresh Eppendorff tube and elute the peptides by aspirating and
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dispensing slowly into the tube.
4. Spot the matrix and sample on the MALDI plate.
a. Spot 0.5 μL of α-Cyano-4-hydroxycinnamic acid matrix (10mg/ml in

50% Acetonitrile with 0.1% Trifluoroacetic acid) onto each of the
requires spots on the target plate. Let it air dry for few minutes.

b. Spot 0.5 μL of the eluted sample on the matrix. Let it air dry for few
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minutes to crystallize.
5. Data acquisition. After crystallization, the plate was loaded into a Bruker
Daltonics Ultraflex-II MALDI-TOF/TOF system. Reflectron positive ion mode is
used for data acquisition with a scan range of 500 to 5000 Daltons and
FlexAnalysis software is used to analyze the data. Sample data is shown in
Figure 6.
The details on use of the MALDI-TOF instrument and analysis of the data are
beyond the scope of this chapter. Generally, these can be obtained from the
instrument manufacturer or a core facility director.
As noted above, mass spectrometry analysis of the peptides can be performed on

a variety of alternate systems. When using an LC/MS system, it is rarely
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necessary to optimize the chromatographic separation of discrete peptides

because the possible sample components are limited to so few species.
ALTERNATE PROTOCOL 2 - NATIVE ISOELECTRIC FOCUSING TO

VALIDATE MHC-II PEPTIDE LOADING
When loading MHC-II with synthetic peptides that have a non-zero net charge, loading can
often be verified by native isoelectric focusing gel electrophoresis. For this protocol to be
useful, it is essential that the MHC-II proteins have been expressed in a cell line that lacks
the enzyme ɑ−1,3-mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase (GNT-I;
gene name MGAT1) that is responsible for the conversion of high-mannose N-linked
glycans to hybrid and complex forms; expression in such a cell line reduces charge
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heterogeneity of the MHC-II protein (Reeves et al., 2002). Even when a GNT-I- cell line is
used for expression, highly purified MHC-II proteins run as 3–4 prominent bands on native
IEF gels (the source of this remaining heterogeneity remains unknown).
There are a number of advantages and disadvantages when comparing the native IEF to mass
spectrometry-based methods (MS) for determination of peptide loading. The primary
advantage of the IEF method is that in favorable cases it can distinguish between loaded and
empty MHC-II and provides a semi-quantitative measure of peptide occupancy. In contrast,
in the absence of prohibitively expensive stable isotope labeled standards, the MS methods
can only distinguish between the presence or absence of a peptide and cannot detect
mixtures of peptide-loaded and empty MHC-II. There are a number of disadvantages to the
IEF method: (1) it cannot be used to detect binding of peptides with a net zero charge (often
extended to peptides with a charge of ±1), and (2) it requires considerable skill and practice
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to produce high quality gel runs. While this method is still in occasional use in our
laboratory, it has largely been supplanted by MS methods.
MATERIALS
• XCell SureLock Mini-Cell (Thermo Fisher Scientific, Catalog # EI0001)
• Novex™ pH 3–7 IEF gels (Thermo Fisher Scientific, Catalog # EC6645)

• Novex™ IEF Anode Buffer 50X (Thermo Fisher Scientific, Catalog # LC 5300)
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• Novex™ IEF Cathode buffer 10X (Thermo Fisher Scientific, Catalog # LC5370)
• Novex™ IEF pH 3–7 sample buffer 2X (Thermo Fisher Scientific, Catalog #

LC5371)
• IEF Marker (SERVA Electrophoresis, Catalog # 3921201)
• Trichloroacetic acid 100%(w/v) Aqueous solution (Spectrum, Catalog #T-184)
• Gel Code Blue staining Reagent (Pierce Catalog # 24592)
• XCell SureLock mini-cell electrophoresis unit (Thermo Fisher Scientific,

Catalog #EI0001)
• Power supply (Thermo Fisher Scientific, Catalog #FB1000 or equivalent

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programmable power supply)
• Photo documentation system
PROTOCOL
Assembly, loading, and running of native IEF gels is best done in a cold room at 4 °C.
1. Assemble the gel apparatus according to the manufacturer’s instructions with

either one or two IEF gels.
A buffer dam is used when running only one gel.
2. Fill the upper buffer chamber with chilled 200 ml 1X Cathode buffer and the
lower buffer chamber with chilled 500 ml 1X Anode buffer.
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3. Mix 3 μl of peptide exchanged MHC-IIp at a concentration of 2mg/ml with 2 μl

of 1X PBS and 5 μl of 2X IEF sample buffer. Load the samples. Load 5 μl of the
IEF marker standards.
4. Attach the leads to the power supply and run the gel. The gel running conditions
are: 100V constant for 1 hour, 200V constant for 1 hour, and 500V constant for
30 minutes.
5. Using the gel knife supplied with the electrophoresis unit, pry open the plates
and carefully transfer the gel with the gel plate into a wide plastic box containing
sufficient 12% TCA to cover the gel. Once the gel is safely in solution, slowly
detach the gel from plastic gel plate.
IEF gels contain 5% acrylamide and can easily tear, so extra care is needed while
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handling them.
Gloves should be worn throughout this procedure.
6. Fix the gel in 12% TCA for 30 minutes with gentle shaking.
7. Discard the TCA solution and wash the gel with ddH2O once.
8. Add Gel code blue staining reagent just enough to cover the gel. Stain for about
30 minutes or until the bands are clearly visible.

9. Add water to destain for about 30 minutes with gentle shaking.

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10. For photo documentation, carefully transfer the gel onto a gel plate (ridged side
facing down). Lift the plate and transfer the gel on to a transilluminator gel box
by slowly sliding it. Take a picture of the gel and save it in a notebook and/or on
a file server.
Figure 7 gives an example of expected results.
BASIC PROTOCOL 6 – MULTIMERIZATION

The protocol we use for multimerization of biotinylated MHCp was described in our unit on
class I MHCp multimer production (Altman and Davis, 2016) and has not changed since our
introduction of the MHC multimer method (Altman et al., 1996). Due to the importance of
multimerization to the entire approach (and for the convenience of readers of this unit), the
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multimerization section of the previous unit is reproduced here with only minor
modifications and these introductory notes. We highly recommend restricting fluorophore
usage for MHC-II tetramers to PE and APC, for reasons discussed below in the section
“Critical Parameters / Streptavidin Conjugates”.
MATERIALS
• Biotinylated peptide-loaded MHC monomer (see Basic Protocols)
• Fluorochrome-labeled streptavidin (typically SA-PE (PJRS25–1) and SA-APC

(PJ27S-1) from Agilent, formerly Prozyme)
• Amber polypropylene screw-cap 1.5–2 ml tubes
• Box with cover

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• Additional reagents and equipment for calculating molar quantities of

biotinylated MHC (see Basic Protocol 3)
MULTIMERIZATION PROTOCOL
1. Calculate the total moles biotinylated MHC in the purified stock (see Basic
Protocol 3).
As noted in Basic Protocol 3, it is recommended that biotinylated MHC/peptide

complex be stored in 100 μl aliquots at a concentration of 2 mg/ml. The
calculation of the number of moles of the MHC/peptide complex depends upon
the molecular weight of the components (ɑ chain, β chain, and peptide), and is
usually in the range of 58 to 60 kDa. As an example, if the molecular weight is
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60 kDa, then the following calculation applies:
2 mg 1g 1 mol
0.1 ml × × × × %Biotinylation = 3.33 × 10−9 mol
ml 1000 mg 60,000 g
2. Calculate the concentration of biotin binding sites in the streptavidin stock.

To perform this calculation, it is necessary to know (1) the concentration of the

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streptavidin conjugate, (2) whether the concentration is expressed in terms of

streptavidin content alone or in terms of the total mass of streptavidin conjugate,
and (3) the ratio of label to streptavidin. As an example, Agilent/Prozyme
provides streptavidin-PE conjugates at a concentration of 1 mg/ml. The
concentration refers to the mass of the total conjugate, not just the streptavidin
moiety. We assume that the material from Agilent/Prozyme has a 1:1 ratio of
phycoerythrin:streptavidin (this is very hard to measure directly). The average
molecular weight of a 1:1 streptavidin-PE conjugate is 300,000 g/mol; therefore,
the concentration of biotin binding sites is:
1 mg 1g 1000 ml 1 mol 4 BBS

× × × × = 1.33 × 10−5 mol/liter
1 ml 1000 mg liter 300,000 g mol
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This translates into 1.33 × 10–11 mol/μl or BBS/μl.
Table 1 lists the average number of biotin binding sites for additional streptavidin
conjugates, assuming that the concentration in terms of milligrams/milliliter
refers to the total conjugate.
3. Calculate the volume of streptavidin conjugate that must be added to give a 1:1
ratio of biotinylated MHC to biotin binding sites.
For example, when adding streptavidin-PE to 200 μg biotinylated MHC, the

proper total volume of streptavidin-PE is:
3.33 × 10−9 mol bioMHC

= 250 μl SAP E
molBBS
1.33 × 10−11 SAP E
μl
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4. Add the streptavidin-PE in 1/10 vol aliquots (25.0 μl in the example above)
stepwise, incubating 10 to 15 min between each addition in the dark (e.g., in a
drawer) at room temperature.
If the volume of streptavidin required has been overestimated, this procedure will
produce the maximum concentration of multimers because all biotin-binding
sites will be saturated after the early additions.
5. Store the tetramer in an amber polypropylene tube at 4°C in a covered box.
The stability of each tetramer at 4°C varies with the MHC allele and especially
with the peptide ligand. Most class II MHCp tetramers last for at least 3 to 6
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months when stored at 4°C. When working with an inherently unstable tetramer,
prepare smaller aliquots of the frozen biotinylated monomers and prepare fresh
tetramers for each experiment.
Alternatively, most tetramers can be stored at −80°C if a cryo-protectant is

added, such as glycerol at concentrations from 5–16% and 0.5% BSA (Hadrup et
al., 2015).

BASIC PROTOCOL 7 – STAINING CELLS WITH CLASS II MHC TETRAMERS

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Optimal conditions for staining with class II MHC tetramers are substantially more
idiosyncratic than conditions for either standard monoclonal antibody or class I MHC
tetramer staining. The key parameters that must be optimized for every class II tetramer/
sample pair are: (1) tetramer concentration, (2) temperature, and (3) time. For example,
while most class I tetramers work when used at 4–25 °C for 15–30 minute incubations, it is
not uncommon for class II tetramers to work only at 37 °C for incubation times between 1–2
hours. A few consequences of this are that it may be more difficult to use whole blood
staining protocols and that live/dead cell discrimination methods are highly recommended.
Advice is given below for an initial optimization experiment that must be performed for
specific sample types and tetramers. More details as well as advice on choice of
fluorochromes and other surface stains are provided in the sections on Strategic Planning
and Critical Parameters.
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MATERIALS
• Cell sample such as human peripheral blood mononuclear cells (PBMCs) or
mouse splenocytes potentially containing T cells specific for the MHC-IIp
tetramer of interest
• Flow cytometry staining buffer (see recipe)
• 4% Paraformaldehyde fixation buffer (i.e. BioLegend Fixation Buffer, 420801)
• Class II MHC tetramer(s) (see Basic and Support Protocols)
• Other surface and intracellular staining reagents as appropriate for your sample
(see Strategic Planning and Note below)
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• Dead cell discrimination marker (such as Invitrogen LIVE/DEAD™ Fixable

Aqua Dead Cell Stain Kit, L34957, optional)
• 96-well round bottom plates (optional)
• 5 ml 12×75 mm round bottom tubes (check for compatibility with available flow
cytometer)
• Eppendorf tubes
• Microfuge
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• 37°C incubator
PROTOCOL
1. Sample preparation will depend on the source of cells but should result in a
single cell suspension in staining buffer with at least 2 × 106 cells or more per
sample.

If there are many samples, it might be more convenient to adapt this protocol to
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staining in round bottom 96-well plates rather than the sample tubes for the
cytometer.
In addition, fewer cells can be used if there is an expectation that the antigen-
specific cells might be present at frequencies above 0.2% of CD4+ T cells.
2. Prepare a diluted working stock of tetramer. Make a 1:10 dilution of tetramer in

staining buffer, spin at 12,000g in a microfuge for 10 min, and carefully remove
the supernatant to a fresh tube.
Tetramer master stocks stored at 4 °C, especially those labeled with PE or APC,
often accumulate aggregates over time. These aggregates will contribute to
background staining and are largely removed by centrifugation. It is best to
perform this on a 1:10 dilution of the master stock and not the master stock itself.
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3. Add tetramer to samples in tubes or plates and incubate at a predetermined final

dilution and incubation time and temperature. A typical final staining volume at
this step is 100 μl. All incubations at this and subsequent stages should be
shielded from ambient light.
For a new tetramer, an initial optimization experiment must be performed.

Typical final working dilutions for class II MHC tetramers prepared according to
the protocols in this chapter range from 1:100 to 1:500, usually closer to 1:100.
Incubation should be tested on ice, at room temperature and 37°C for 1–2 hours.
Higher temperatures and longer incubations might increase specific staining but
often increase background staining, so a judgment must be made about the best
conditions.
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4. After tetramer incubation, prepare a cocktail of remaining surface staining

reagents (q.s. 100 μl/sample in staining buffer) and add to samples. Incubate on
ice for 20 minutes.
Washing at this step is not necessary, and the staining cocktail can be added
directly to the sample without centrifugation. The final sample volume at this
step is doubled to 200 μl to facilitate mixing.
5. Pellet cells in a tabletop centrifuge, 5 min at 500 g.
6. Wash cells 1–2 times with staining buffer, pellet each time, 5 min at 500 g.
7. Resuspend cells in 2% paraformaldehyde, incubate 10 min at room temperature.
Omit this step when live cell sorting will be performed.

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8. Pellet cells for 10 min at 500 g.
Fixed cells do not pellet as well as unfixed, hence the longer centrifugation time.
9. Wash cells with staining buffer, pellet for 10 min at 500 g, and resuspend in 400
μl staining buffer for analysis on a flow cytometer.
Plan to collect data on a larger than usual number of cells assuming that positive
cells will be rare. We prefer to collect at least 1–2 million events.

REAGENTS AND SOLUTIONS

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2X HEBS precipitation reagent (500ml)

HEPES 6.24g
NaCl 8.18g
Na2HPO4 0.11g
Dissolve in ddH2O. q.s. to 450ml with ddH2O and pH to exactly 7.05.
q.s. to 500ml with ddH20 and filter sterilize.
Perform transfection optimization test (described after next solution).

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2M CaCl2 (500ml)
CaCl2•2H2O 147g
Dissolve in 450 ml ddH2O.
q.s. to 500ml with ddH2O and filter sterilize.
Perform transfection optimization test.
Calcium phosphate transfection of 293T cells works very well when properly optimized.
Following this protocol routinely yields a final titer of 1.0×109–5.0×109 LV/ml. Set aside a
large bottle of filter-sterilized MilliQ water and 2M CaCl2 at room temperature for use in
transfections only. Make a 500ml batch of 2X HEBS and store at 4°C, after successful
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transfection test (see below). Each batch of HEBS is good for a maximum of 9 months, at
which time it should be discarded, and a new batch made.
For each batch of 2X HEBS, a short optimization must be performed to determine the proper
concentration of CaCl2 to use. This is done in 2 steps. First, set up a transfection as
described in the table below (T75 size), and determine how long it takes for the CaPO4
precipitate to form. Start with 150ul of 2M CaCl2 per transfection. Mix reagents, bubble for
exactly 10 seconds, and then take 20ul onto a standard slide or hemocytometer and view
under a microscope at 200X. A good precipitate will appear as a very fine grainy mist
throughout the solution and will be difficult to see initially. Take and examine a 20ul sample
on hemocytometer every 30s (don’t look at the first 20ul over time as the kinetics of
precipitate formation are different on the slide vs. in the tube). The time point after bubbling
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at which visible precipitate appears should be noted. This roughly corresponds to the
optimal time for transfection. At later time points, the precipitate becomes visibly large and
clumpy and results in poor transfection.
Component T75 (15ml tube)

lentivector (1 μg/ml) 30 μl

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Component T75 (15ml tube)

psPAX2 (1 μg/ml) 20 μl
pMD2G (1 μg/ml) 10 μl
2M CaCl2 150 μl*
ddH20 q.s to 1500 μl
Mix well before addition of 2X HEBS

2X HEBS 1500 μl
*
As discussed in the text, this can be decreased when necessary depending upon the quality of the observed precipitate
If the precipitate immediately becomes large and clumpy after the addition of 2X HEBS,
then the transfection should be set up again with less CaCl2 (e.g. use 120ul CaCl2 per
transfection). If no visible precipitate forms after 10 mins, then the transfections should be
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set up again with more CaCl2. Continue to adjust CaCl2 until a very fine precipitate forms
somewhere between 30s and 5 mins after bubbling. Note this time. This will be the amount
of time required for precipitate formation for each transfection for the batch of 2X HEBS
being tested and should not change over time.
Having identified a working CaCl2 volume, set up another transfection to precisely identify
the optimum incubation time after mixing. Set up a transfection (T75 size) and take 300ul
aliquots onto cells (in a 6-well plate, seeded the day before) at several time points before and
after the putative optimum. Follow the rest of the transfection protocol and analyze the
transfected cells 24 hours later by flow cytometry or other appropriate method. This will
give you the exact time point for optimal transfection after mixing.
D10 Media
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1L DMEM (Corning 10–017-CM)
110ml FCS
11ml 100X Antibiotic Antimycotic solution (Corning 30–004-CI)
11ml 100X L-Glutamine solution (25–005-CI)
Cryopreservation media
FCS 9 ml
DMSO 1 ml
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D-biotin (100 mM)

D-biotin 1 g (Fisher Scientific BP2321)
Tris Base (Sigma T-6066)
Dissolve 1 g D-biotin (entire vial) in 40 ml 200 mM Tris Base (do not adjust pH). Filter
sterilize and store aliquots at −20°C.

Flow cytometry staining buffer

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PBS 500 ml
BSA 10 g
Sodium azide 0.5 g
ELISA coating buffer

Sodium bicarbonate 2.92 g
Sodium carbonate 1.7 g
q.s. 1 L with deionized water. Adjust pH to 9.6. Store at 4°C.

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ELISA wash buffer (PBS, 0.05%Tween20)

10xPBS 300 ml
Tween-20 1.5ml
q.s. 3 L with deionized water.
ELISA blocking buffer (PBS, 0.05%Tween20, 1%BSA)

Make 50 ml of 10% BSA with PBS.
q.s. 500 ml with ELISA wash buffer. Can be stored at 4°C for one week or frozen at −20°C
in 50 ml aliquots.
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Do not add sodium azide to ELISA buffers as it will inhibit HRP.
1M sodium phosphate stock solution pH 11.3 (500ml)

78.01g monobasic sodium phosphate dihydrate (Sigma 71505).
350 ml ddH2O.
pH to 11.3 by adding 5M NaOH.
Make up to 500ml with ddH2O.
2H11 Elution buffer (30mM sodium phosphate pH 11.3) (500ml)

30ml 0.5M sodium phosphate stock solution pH 11.3.
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470ml ddH2O.
1.2 mL of 1M Phosphoric Acid.
Filter sterilize.

1M Glycine, pH 2.8 stock solution

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700 mL ddH2O
75.06g glycine (Acros)
pH to 2.8 with HCl
Make up to 1L with ddH2O
0.1M Glycine, pH 2.8

100mL of 1M glycine, pH 2.8
900mL of ddH2O
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Filter Sterilize
5M NaCl stock solution

292.2g NaCl (Sigma S3014).
Make up to 1L with ddH2O.
1M NaCl/PBS wash buffer

200ml 5M NaCl.
800ml 1X PBS.
Filter sterilize.
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0.5M EDTA pH 8.0 (500ml)

93.06g of EDTA (Sigma 03677).
4g NaOH pellets.
400ml ddH2O.
Stir vigorously on magnetic stir plate. The EDTA will not go into solution until the solution
becomes slightly alkaline. After the EDTA and NaOH pellets dissolve the pH will be ~7.5,
make final adjustments to pH with concentrated NaOH.
Make up to 500ml with ddH2O.

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Leupeptin (1mg/ml lab stock should already be made and aliquoted).

Dissolve 50mg leupeptin (1 new vial) in 50ml ddH2O; 0.2 micron filter and store in 1ml
aliquots at –20°C.

Pepstatin (1mg/ml lab stock should already be made and aliquoted).

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Dissolve 50mg pepstatin (1 new vial) in 50ml 100% methanol and store in 1ml aliquots at –
20°C.
Monomer storage buffer (MSB) (500ml)

0.5ml Pepstatin.
0.5ml Leupeptin.
2.0ml 0.5M EDTA pH8.0.
50 ml 10X PBS
447ml ddH2O.
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Filter sterilize.
2H11 Neutralization buffer (1M Sodium Acetate pH6.0) (500ml)

41.02g sodium acetate, anhydrous (Sigma 71183).
400ml ddH2O.
pH to 6.0.
Make up to 500ml and filter sterilize.
5% Sodium azide stock solution (1L).

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Toxic, work in fume hood when preparing master stock.
Empty entire contents of 50g bottle of sodium azide (Sigma 71289) into 1L of ddH2O. This
solution is now 100X concentrated for most applications (1/100 gives 0.05% final).
PBS Azide (0.05%) (1 L)

Add 10mls of 5% sodium azide stock solution to 1L 1X PBS.
Filter sterilize and store in @ 4°C
IEF 1X Anode buffer (0.5 L)

10ml 50X IEF Anode buffer.
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490ml ddH2O. Chill at 4 °C
IEF 1X Cathode buffer (0.2 L)

20ml 10X IEF Cathode buffer.
180ml ddH2O. Chill at 4 °C

12% TCA (0.5 ml)

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60 ml 100% TCA solution
440 ml ddH2O.
10X 3C Cleavage buffer (50 ml)

25 ml 1M Tris HCl pH 7.5
15 ml 5M NaCl
10 ml ddH2O.
1% Trifluoroacetic acid (Thermo Scientific LC-MS grade Catalog # 85183)

Add 1 ml to 99 ml of deionized water to a final volume of 100 ml.
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0.5M Citric acid

10.5g Citric acid (Sigma Aldrich C1909).
Dissolve in100 ml ddH2O.
0.5M Sodium Citrate

14.7g Citric acid (Sigma S4641).
Dissolve in100 ml ddH2O.
0.5M Citrate buffer pH5.2

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30.5 ml 0.5M Citric acid.
69.5 ml 0.5M Sodium Citrate.
Anti-DM Sepharose beads

About 115 mg of in-house purified anti-DM antibodies are conjugated to 15 ml of CNBr-
activated Sepharose Fast flow (Sigma-Aldrich C5338) following the standard protocol by the
manufacturer. Antibody coupled Sepharose resin is stored as 50% suspension in PBS azide.
Prior to using the beads for DM depletion, they are washed in PBS twice.
COMMENTARY
BACKGROUND INFORMATION
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A BRIEF HISTORY OF MHC TETRAMERS—The study of antigen-specific T cell

responses was transformed in the mid-1990s by the introduction of three single-cell
methodologies which have largely replaced long-established bulk methods such as the
lymphoproliferation and chromium release assays. Two of the assays measure T cell
functions—the cytokine ELISpot (Klinman, 2008) and cytokine flow cytometry (CFC, also
known as intracellular cytokine staining, or ICS) (Yin et al., 2015; Foster et al., 2007)—
while the third, major histocompatibility complex (MHC) multimer staining, is a physical

method for detecting T cells bearing a repertoire of antigen receptors specific for a single
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MHC peptide complex (MHCp) (Altman et al., 1996). The production and use of class I
MHCp tetramers has been discussed in multiple articles in this volume (Altman and Davis,
2016; Toebes et al., 2009; Hansen et al., 2009; Skinner and Haase, 2004), while the use of
class II tetramers for enrichment of antigen-specific CD4+ T cells has been discussed in a
unit from Kotov and Jenkins (Kotov and Jenkins, 2019).
HIGHLIGHTS FROM PREVIOUS UNITS OF SPECIAL RELEVANCE TO THIS

UNIT—The production and use of MHC multimers has been discussed in many Current
Protocols in Immunology units. Highlights from those chapters that are particularly relevant
to this chapter include:
• A discussion of a variety of multimer formats (Altman and Davis, 2016).
• A discussion of fluorophores used for MHC multimers, including variation

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across suppliers of streptavidin conjugated to protein fluorophores such as

phycoerythrin (PE) and allophycocyanin (APC) (Altman and Davis, 2016).
• A section on “Tricks to improve tetramer stains” (Altman and Davis, 2016).
• A section on cryopreservation of multimers (Altman and Davis, 2016).
• In situ staining (Skinner and Haase, 2004).
COMPARISON OF EXPRESSION OF CLASS II MHC TO CLASS I MHC—While

production of class I MHC peptide complexes for multimers is dominated by a single
method and associated variants — in vitro folding of subunits produced as inclusion bodies
in E. coli and solubilized in denaturants as described in Chapter 17.3 of this volume (Altman
and Davis, 2016; Toebes et al., 2009) — a wide variety of expression systems have been
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described for production of class II MHC peptide complexes.
There are two reasons for the predominance of in vitro folding for production of MHC-Ip.
First, with the exception of the single-chain trimer approach (Hansen et al., 2009), it is the
only established practical method for ensuring the peptide binding site is occupied with a
single peptide, a property that is essential for the tetramer avidity effect; in contrast, class I
molecules secreted from eukaryotic cells (especially mammalian cells) are likely to be filled
with a diverse spectrum of endogenous peptides, many of which will be resistant to
exchange. Second, for class I MHC molecules, in vitro folding in the presence of a suitable
peptide ligand generally works, and it does so across a wide spectrum of alleles, all in the
absence of added chaperones.
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In contrast, with a few reported exceptions covering a few alleles (Altman et al., 1993;
Frayser et al., 1999; Justesen et al., 2009; Braendstrup et al., 2013), in vitro folding of class
II MHC proteins generally does not work, at least not well. While the reasons for this remain
unknown, it has forced investigators to explore a wide variety of eukaryotic cell substrates
for the production of class II MHC proteins as secreted, native proteins, using organisms
from the yeast Pichia pastoris to human cell lines. A few examples are reviewed in Table 2
below.

COMMON MHC-II EXPRESSION DESIGN FEATURES—During early attempts to

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express soluble class II MHC molecules, many investigators encountered problems

associated with the stability of the desired product, manifested as either low expression
yields or a tendency of the proteins to aggregate. These have been addressed in three distinct
ways. First, it has become a nearly universal practice to add heterologous leucine zippers to
each of the class II subunits, promoting heterodimerization and stabilization of the final
product (Scott et al., 1996). Any of a variety of zippers may be used, with the choice largely
depending upon the need for and availability of antibody reagents to specific zippers.
Second, Kappler’s lab developed an approach for tethering class II MHC peptide ligands to
the amino terminus of the β chain and found that class II MHC molecules with filled peptide
binding sites were significantly less prone to aggregation compared to the corresponding
empty proteins (Kozono et al., 1994). Additional variations on this approach have been
demonstrated where the tethered peptide may be proteolytically released, permitting
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exchange for synthetic peptides (Day et al., 2003; Huang et al., 2003). While the peptide
tethering approach is widely used, it is not universal, as demonstrated by the large body of
class II tetramer work from the Nepom and Kwok labs that uses class II molecules expressed
without tethers that are loaded with synthetic peptides. Finally, many labs adopted
expression systems such as baculovirus vectors or the Drosophila S2-cell system using cell
substrates that grow well at temperatures below 30 °C.
CHOOSING A CLASS II MHC EXPRESSION SYSTEM—The key eukaryotic cell

systems use for expression of class II MHC are listed in Table 2; space considerations do not
allow a comprehensive listing of all reports of class II expression systems, and we apologize
to investigators whose efforts we have not cited.
In 2021, it is no longer true that production of class II tetramers itself constitutes a

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significant bottleneck in characterization of class II-restricted T cells by multimer staining.

But with so many choices now available for producing class II MHC peptide complexes, a
number of questions now arise. Does one system provide more efficacious reagents? Does
one system provide better yields? Is one system “easier” than another? We believe the
answer to all these questions is “maybe, but probably not, and there is not even a hint of
evidence that the answer is yes”. As a consequence, this means that when a lab wants to set
up class II MHC tetramer production, they should choose the method they are most familiar
with and have the most expertise in.
When we set out to develop a class II MHC tetramer production system for the NIH
Tetramer Facility, we based our strategy on three key criteria. First, we wanted a system that
provided stable production lines in the absence of drug selection. Second, we wanted a
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system that provided high yield production lines without having to go through the laborious
and time-consuming process of preparation and screening cell clones after a process of gene
amplification (Kingston et al., 2002). Third, we wanted a system that provided constitutive
expression so that we would not have to go through cycles of cell expansion followed by
expression induction. Finally, we were not concerned with either GMP/GLP regulations or
issues associated with animal sera. None of the previously established systems summarized
in Table 2 met all of these criteria, so we elected to develop the lentiviral vector transduced
HEK293T cell expression system described in this chapter.

CRITICAL PARAMETERS
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Critical parameters in this chapter fall into one of three categories: (1) the qualities of the
production cell line, (2) the qualities of the MHC-II protein, (3) the qualities of the
streptavidin-fluorophore conjugates.
PRODUCTION LINES—For the production cell line, three parameters are critical: (1) the
validity of the sequence of the designed expression constructs, (2) the frequency of
transduced cells in the production line, and (3) the absence of cross-contamination of
expression lines. All expression vectors must be validated by DNA sequencing. Transduced
cell lines are analyzed by flow cytometry to assess the frequency of transductants through
analysis of marker genes contained in the lentiviral constructs, with translation directed by
IRES elements. When these analyses indicate either a substantial fraction of non-transduced
cells (for either MHC-II subunit vector) or a substantial spread in expression of the marker
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gene, sublines are prepared by cell sorting, gating on the cells that express the highest levels
of the marker genes. Once established, production lines are remarkably stable and tolerate
extensive serial passaging, indicating there is negligible negative selection against high
expression cells. When we observe a significant decrease in expression levels from an
extensively passaged production line, we discard it and start from a new aliquot of
cryopreserved cells from the initial cell bank.
MHC-II PROTEIN—At the MHC-II protein level, a number of parameters are critical: (1)
purity, (2) percentage biotinylation, (3) exchange competence, and (4) fractional occupancy
by the exchange peptide (related to item 3). In general, less contamination of the product is
observed when the recombinant proteins are purified by the anti-zipper affinity column than
by IMAC, and as for all protein purification, fewer contaminants are seen when production
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levels are higher. In most cases, multimer staining efficacy is not impaired by contaminants,
and so high levels of purity are not absolutely essential, but they may result in more
reproducible reagents. Biotinylation levels of the BSP-tagged proteins expressed from our
engineered HEK293T.TPM line are usually greater than 95%, so poor biotinylation is rarely
a problem. This applies even when production culture supernatants are stored at 4 °C for 1–2
months, a pleasantly surprising result.
When we first developed our MHC-II expression system, more often than not we prepared
expression vectors in which every epitope was expressed as its own tethered construct.
Subsequently, we developed both high efficiency exchange protocols and assays to measure
peptide exchange and these greatly streamlined production of MHC-IIp multimer reagents.
In general, we start with constructs tethered to the CLIP peptide (Day et al., 2003), but not
all alleles bind to the canonical CLIP peptide with high affinity (Sette et al., 1995) and some
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of these constructs express poorly, so alternatives are sometimes necessary. And while the
biology of MHC-IIp antigen presentation obviously points towards the use of CLIP
constructs, the efficacy of DM-assisted release of a bound MHC-II peptide ligand is far from
unique to CLIP, so alternate tethered peptides do not present any theoretical barrier at all to
identification of exchange-competent constructs. There’s no getting around empirical testing
of a number of constructs, but for most alleles, the success rate is high. Some alleles have
been identified as DM-insensitive, and this is frequently but not always a barrier to

development of peptide exchange protocols (Belmares et al., 2002; Fallang et al., 2008;
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Zhou et al., 2016).
CONTROL PEPTIDES—It is essential to have one or more positive control peptides for
exchange assays. When using mass spectrometry to measure exchange, the specific
characteristics of the peptide (beyond high affinity binding) are not so important, but when
using IEF assays it is essential that the positive control peptide have a non-zero net charge,
with multiple positive charges usually being preferable. High affinity binding peptides for a
large number of alleles can be found in the IEDB (http://www.iedb.org).
STREPTAVIDIN CONJUGATES—Setting aside use of explicitly engineered higher order

multimers, we highly recommend that all class II MHCp “tetramers” use either PE or APC
fluorophores (and their tandems, which we rarely use because of their significantly higher
expense). Streptavidin is conjugated to PE and APC using heterobifunctional cross-linking
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reagents such as Sulfo-SMCC. The product of this reaction is a mixture of various species,
with ratios dependent upon numerous reaction conditions, but it is impossible to precisely
control the reactions to exclusively create defined 1:1 conjugates. This heterogeneous
mixture typically includes conjugates with multiple SAs linked to one or more
phycobiliproteins; these are the core of effectively higher order multimers. The reaction
conjugates are often fractionated on size exclusion columns, and different suppliers select
different fractions for their products; some appear to select lower order conjugates, while
others select fractions that contain higher-order conjugates. The effects of this are shown in
Figure 8 below, reproduced from (Altman and Davis, 2016). Clearly, in this case, better
staining is obtained with the product that contains the largest fraction of higher order
conjugates (Manufacturer A). Even in Figure 8, close inspection shows that the enhanced
staining efficiency is accompanied by an increase in background staining of the multimer-
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negative population, but with careful preparation and titration of the reagents (discussed in
Basic Protocol 7), optimal performance can be achieved.
To our knowledge, conjugation of streptavidin to other commonly used fluorophores—from

small molecule dyes such as fluorescein and the Alexa dyes (Ramachandiran et al., 2007) to
the newer polymer dyes in the Brilliant series from BD Biosciences—cannot create higher
order multimers, and such conjugates are likely to consist of almost exclusively true
tetramers. As a result, we generally do not recommend use of these dyes for class II MHCp
tetramers, although in some cases Brilliant dyes such as BV421 can yield reagents with
acceptable performance, most likely due to their exceptional brightness.
TROUBLESHOOTING
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DIFFICULT ALLELES—Expression levels of MHC-II alleles achieved by the methods

described in this chapter span 2 orders of magnitude, from 0.1 mg/l for poorly expressed
constructs to 10–40 mg/l for highly expressed constructs. Even for a single allele, expression
levels can be highly dependent upon the sequence of the tethered peptide ligand, something
we have observed for the mouse I-Ab allele, for which we made hundreds of distinct tethered
peptide constructs before we developed efficient exchange methods.

When we encounter poor expression levels, we attempt two approaches to improve them.
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The easiest variable to test is the temperature of the expression cultures, and we frequently
find improved expression levels when production lines are cultured at 30–32 °C. If this does
not work, we prepare new expression constructs with different tethered peptides, usually
choosing from high affinity binding peptides in the IEDB database (http://www.iedb.org);
we work these up with 4–5 epitopes at a time. This process is laborious and time-consuming,
but we currently do not have a developed alternative such as an expression library screen,
which would likely require a completely new construct engineering approach.
We have attempted to isolate higher expression level subclones from a number of lentiviral
vector transduced cell lines, but these efforts have never yielded significantly improved
levels of expression.
Finally, there are approaches to improving expression levels we are only beginning to
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explore. We could adopt insect cell expression for poorly expressed alleles, hoping that a
further reduction in culture temperature might improve yields. Alternatively, a novel and
promising alternative to the use of leucine zippers to promote heterodimerization has been
described that was inspired by the bi-specific antibody literature that uses fusions of MHC
subunits to complementary Fc mutant chains engineered to contain either a “knob” or a
“hole” (Serra et al., 2019). In addition, there may be opportunities to further engineer the
HEK293T-derived expression line to enhance expression yield, perhaps through increased
expression of chaperones or factors involved in the protein secretion pathway.
CELL LINE TESTING—Once established, production cell lines expressing MHC-II have
proven remarkably stable and retain their characteristic expression levels even after
extensive in vitro passaging. In rare cases when expression levels do decline, our usual
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response is to restart a production run from a new thaw of cryopreserved cells established at
the time of creation of the cell line. When warranted, a variety of laboratory tests can be
performed, including: (1) flow cytometry to examine marker gene expression, (2)
mycoplasma testing, and (3) PCR or RT-PCR and sequencing of amplicons from the
lentiviral constructs to test for potential cell line contamination.
PURIFICATION—Both the anti-zipper affinity chromatography and IMAC protocols are

highly robust and generally give excellent purities. Problems can arise when expression
levels for a particular allele are low. This creates a conundrum, wherein a “polishing”
purification step such as described in Support Protocol 4 can improve purity but at the cost
of decreased overall yield. Our most common response is to create larger supernatant pools
from more production culture passages. If IMAC is the only practical purification option,
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purity can be improved by using constructs that have tandem His-tags (Schmidt et al., 2011)
instead of the conventional His6 tag.
BIOTINYLATION—When poor biotinylation is seen, we attempt in vitro biotinylation

using purified BirA enzyme as described (Altman and Davis, 2016), but often we discard the
batch and start over from new supernatants.

PEPTIDE EXCHANGE—Troubleshooting peptide exchange reactions can be broken down

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into two categories: (1) establishment of a general method for an allele (and associated
formerly tethered peptide from the expression construct), and (2) problems associated with
specific synthetic peptides.
Sette and colleagues have published peptide binding assays for a large collection of human
and mouse class II MHC alleles, with allele specific parameters such as buffers, pH,
included detergents, temperatures, and index peptides (Sidney et al., 2013). The methods
described there are often sufficient, but the methods described here differ from them in four
areas: (1) we are concerned with loading MHC molecules that lack transmembrane domains
and are therefore soluble, while their methods generally use detergent-solubilized proteins;
(2) the Sette assays require index peptides with radio-iodinated tyrosine residues, while we
have no such requirement; (3) the Sette assays do not use HLA-DM as an exchange catalyst,
while we do; and (4) the concentration of the MHC and peptide components in the Sette
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assays are optimized for determination of IC50 values in competitive inhibition assays under
conditions where IC50’s approximate dissociation constants, while we are concerned with
complete replacement of the formerly tethered peptide with exchange peptides.
When developing our own optimized peptide loading conditions, an assay capable of
measuring results across a number of variables is essential. And while our final protocol uses
unlabeled peptides, assay development is greatly aided by use of labeled peptides. We prefer
nonradioactive methods, and due to the presence of biotin on our expressed proteins, we
cannot use biotinylated peptides. Instead, we use index peptides with an N-terminal
fluorescein label—often based upon the Sette index peptides, but sometimes chosen from a
list of high affinity peptides from the IEDB—which we detect using an anti-fluorescein
antibody 4–4-20 (Kranz and Voss, 1981) as the detection antibody in a sandwich ELISA. A
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full description of this assay is beyond the scope of this chapter.
Once robust exchange methods are established for a given allele, problems can arise with
loading of individual peptides for a number of reasons. First, there are many epitope/
restriction-element combinations in the T cell assay section of the IEDB where
corresponding MHC/peptide binding data are not available, and some of these combinations
may be too low affinity to prepare stable MHCp complexes for multimers. Second, many
peptide epitopes are hydrophobic and poorly soluble in aqueous solutions, complicating
loading protocols. In such cases, loading under more dilute conditions may be helpful.
Finally, there may be problems with the quality of synthetic peptides, including post-
synthesis reactions such as oxidation, often a problem with sulfur containing residues on
peptides stored in DMSO. In such cases, the quality of a peptide stock can be re-analyzed by
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reversed phase HPLC and mass spectrometry, or a fresh batch of the peptide can be ordered.
Finally, a small number of epitope/allele combinations have been identified where a single
peptide binds to a specific MHC-II allele in multiple registers. This was discussed above in
the section “Epitopes - tethered vs exchanged” and will not be further discussed here, except
to say that addressing this effect takes a large amount of empirical work and testing.

STAINING—As noted earlier, getting good staining results with class II MHC tetramers is
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usually more challenging than with monoclonal antibodies or even class I MHC tetramers.
When developing staining protocols with a new tetramer, it is crucial to test multiple staining
temperatures (usually room temperature or 37°C for class II), staining times (1–2 hr) and
dilutions. Staining conditions should be initially worked out with a minimal staining panel
including the markers described in Strategic Planning and intracellular stains should be
omitted. After staining is optimized, the panel can be expanded and the effect of
permeabilization/fixation methods on tetramer staining determined.
Background staining is a major obstacle to good results not only due to the typical low
frequency of specific CD4+ T cells but also because of the long incubation at 37 °C that is
usually required. The best negative controls for staining are a sample from an unimmunized/
unexposed subject or an MHC-II reagent with an irrelevant epitope. Gating out cells that
non-specifically bind tetramers (CD8+ T cells and B cells, monocytes/macrophages to a
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lesser extent) is key. Tetramers form aggregates in solution over time (particularly APC and
PE) that will add to background staining and can be largely removed by centrifugation as in
the Basic Protocol. Avoid the temptation to use an overly high concentration of tetramer to
increase positive staining, as this will also increase background. For extremely rare T cells,
tetramer-based enrichment might be required (Kotov and Jenkins, 2019). In addition, some
investigators have used a dual labeling approach in which cells are stained with two
tetramers with the same MHC-IIp but different fluorophores to discriminate true antigen-
specific cells from cells non-specifically binding one of the tetramers (Mpande, 2018).
Note that in some cases an MHC-II reagent that is successfully produced with a bona fide
epitope will not stain. It is possible that these reagents have too low of an avidity for specific
TCR to bind stably, or that the epitope can bind MHC in multiple conformations as
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described above in Strategic Planning. This is difficult to troubleshoot and requires

considerable dedication to the study of a particular epitope.
UNDERSTANDING RESULTS
Because of the large scope and breadth of the MHC-II reagents we produce, we have
developed a custom relational database to track every step of our production process, and
this helps us to spot trends and identify cases where troubleshooting is needed.
CELL LINE PRODUCTION—Once production cell lines are established with uniformly
high levels of marker gene expression, they are tested for MHC-II expression levels using
the Six Well Assay described in Support Protocol 1 and the ELISA in Support Protocol 2. If
expression levels are low (0.1 – 0.4 mg/l), we may (1) test alternate constructs with new
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tethered peptides, (2) simply increase production culture volumes (sometimes at 30–32 °C),
or (3) both.
EXPRESSION AND PURIFICATION—We track expression levels from production

cultures using two methods: (1) ELISAs of supernatants at each passage, and (2) purification
yields. Since our production methods use serial passage techniques, it is helpful to track
yields at each passage, and when yields drop significantly, we terminate a production run
and start a new one from freshly thawed cell bank aliquots. We also track production quality

by SDS-PAGE, and when analyses show deviations from the expected molecular weights
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(which doesn’t happen often), we discard the affected batch.
We do not have hard-and-fast rules for when polishing purification steps are implemented.
As noted above, higher levels of impurities are seen when yields are low, and additional
purification steps further reduce yields. Often, we will proceed with peptide exchange
reactions and if they are of sufficient quality, we will not perform additional purification
steps.
PEPTIDE EXCHANGE—Analysis of post-exchange bound peptides by mass spectrometry

can yield a number of results. When only the formerly tethered peptide (FTP) is present, we
conclude that the exchange reaction did not work. In this case, we re-examine the quality
control data for the exchange peptide, and if it indicates no problem with the quality of the
peptide, we repeat the exchange reaction. If it fails again, we often abandon the product.
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When only the exchange peptide is present, we conclude that the exchange reaction worked
and proceed with multimerization. When a mixture of the FTP and the exchange peptide is
present, we conclude that we have achieved partial exchange. In some cases, we proceed
with multimerization, while in others we will attempt to optimize exchange by increasing
the concentration of the peptide in the exchange reaction or by increasing the length of the
incubation. In some cases, we see only unexpected masses, and we usually repeat these
exchange reactions. Finally, sometimes the mass spectra are blank, which also indicates a
failed exchange reaction, but one in which the FTP has been successfully removed.
One drawback of our current implementation of the mass spectrometry method for
measuring peptide exchange is that it does not provide data on the fraction of MHC-II
proteins in the preparation that have an empty peptide binding site. New methods remain to
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be developed to measure fractional occupancy.
STAINING—The populations of T cells detected by MHC-II multimer staining represent a

lower bound of the total population specific for the allele/epitope combination. Reasons for
potentially only detecting a fraction of the total antigen-specific population include: (1) use
of staining conditions that could be further optimized (Dolton et al., 2015); (2) the multimer
may represent only a single epitope register, while functional assays with antigen-presenting
cells (APCs) pulsed with peptides are likely to detect T cells recognizing all potential
registers; and (3) low affinity T cells may be present that are only detectable by other
methods such as 2-dimensional (2D) affinity measurements (Martinez and Evavold, 2015) or
by higher order multimer staining. Even measuring the upper bound of the total epitope-
specific T cell population is difficult because (1) not all T cells that bind multimers score in
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a particular functional assay such as cytokine flow cytometry, and (2) 2D affinity
measurements require highly specialized equipment present in only a few labs and the assay
is not amenable to low frequency T cell populations. The immunological importance of any
of these discrepancies is difficult to assess.
TIME CONSIDERATIONS
Vector preparation, if necessary, takes 5–10 days. More time (2–3 weeks) is needed if
synthetic genes are required. Production of lentiviral stocks and cell transduction takes about

a week followed by 2–3 weeks to scale up the cell culture and test expression. ELISAs can
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be done in 6 hours. After seeding production cultures, supernatants are harvested every week
and purifications can begin after 2–4 weeks depending on expression levels. Supernatants
can be purified in less than 7 hours (loading of the column overnight is required for larger
volumes) and purified protein can be processed and analyzed for purity and biotinylation by
SDS-PAGE the following day. For peptide exchange, MHC-II monomer can be cleaved in
bulk, protease removed and tested for cleavage in three days. Peptide exchange requires 2–4
hr work over two days with an overnight incubation. Analysis of exchange can be done in 1–
2 days by mass spectrometry or IEF gel. Multimer production using labeled streptavidin and
biotinylated MHC-II monomer takes two hours. Tetramer staining and fixation of samples
can be done in 4 hours not including sample preparation time and analysis by flow
cytometry.
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Acknowledgements
This work was supported by NIH contracts for the NIH Tetramer Facility (75N93020D00005 and 272201300006C)
with additional support from the TBRU-ASTRA (5U19AI111211), the Emory CFAR Immunology Core
(5P30AI050409), and the Yerkes National Primate Research Center (5P51OD011132). We also thank the Emory
Comprehensive Glycomics Core (ECGC, UL1TR000454) for access to mass spectrometry instruments and the
Emory Integrated Genomics Core (UL1TR002378) for expression plasmid production.
We thank past and current members of the Tetramer Facility that are not co-authors for their outstanding dedication
over the course of 20 years. We also thank Kiran Gill and Barbara Cervasi for cell sorting.
We thank Ellis Reinherz, Peter Cresswell, and Kai Wucherpfennig for provision of hybridomas and plasmids.
Finally, we thank a number of experts in class II MHC and T cell immunology for invaluable scientific input
through workshops or informal discussions over the years, including David Margulies, Luc Teyton, Mark Davis,
John Kappler, Fran Crawford, William Kwok, Larry Stern, Betsy Mellins, Rafi Ahmed, Brian Evavold, Peter
Jensen, Marc Jenkins, and Marion Pepper.
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INTERNET RESOURCES WITH ANNOTATIONS

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https://tetramer.yerkes.emory.edu
The NIH Tetramer Core Facility is run by the authors of this unit and provides custom
MHC tetramer to investigators across the world.
http://www.iedb.org
The Immune Epitope Database contains information on MHC-restricted T cell responses

and MHC-peptide binding interactions.
https://www.ebi.ac.uk/ipd/
The Immuno Polymorphism Database contains information on MHC sequences for

humans and many other species.
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http://www.allelefrequencies.net
The Allele Frequency Net Database provides tables of HLA allele frequencies for defined
populations across the world.
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Figure 1. Design of lentiviral vectors for class II peptide/MHC protein expression.

A. The 5’ long terminal repeat (LTR), packaging signal (Ψ) rev-response element (RRE)
and 3’ self-inactivating (SIN) LTR allow one round of lentivirus replication and
transduction. Class II MHC ɑ or β chain expression is driven by the CMV enhancer/
promotor. An internal ribosome entry site (IRES) allows coexpression of a surrogate marker
(eGFP, Thy 1.1, etc.) to facilitate screening and fluorescence-activated cell sorting of
transduced cell lines. The woodchuck Hepatitis virus posttranscriptional regulatory element
(WPRE) enhances transgene expression. B. β chain transgene design. SS; signal sequence.
3C; HRV-3C cleavage site in a 20 aa flexible linker. C. ɑ chain transgene design. BSP85;
BirA recognition site.
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Figure 2. HP-SEC analysis of peptide exchange reactions for two alleles.

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3 μg each of post exchanged DRB1*0401 or DPB1*0201/DPA*0103 is analyzed on a gel

filtration column (TSKgel G3000SWXL) attached to a Shimadzu HPLC at 1ml/min flow
rate with PBS as the mobile phase. Each run is monitored for presence of higher order
aggregates as seen with DPB1*0201.

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Figure 3. Representative chromatogram of an IMAC run and the corresponding SDS-PAGE

analysis of the eluted fractions.
Supernatant from a production run of a cell line expressing Mamu-DRB*w201 was purified
on 5 ml HisTrap FF Crude column and the fractions corresponding to the 280 nm
absorbance peak are analyzed on SDS-PAGE. Fractions 8–13 that contain the eluted protein
are then pooled and buffer exchanged.
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Figure 4. Analysis of biotinylation of Class II MHC protein by streptavidin precipitation (sap)

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followed by SDS-PAGE.
Purified I-Ab protein is assessed for extent of biotinylation by sap-assay followed by SDS-
PAGE analysis. Either Sepharose 4b (−) or streptavidin (+) beads were added to purified I-
Ab protein and incubated for one hour at room temperature with intermittent mixing. After
brief centrifugation, supernatants are boiled in loading buffer and run on SDS_PAGE. RNase
A protein, used as loading control is as indicated. The absence of I-Ab protein with the
added streptavidin beads indicates complete biotinylation.

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Figure 5. Representative SDS-PAGE analysis of 3C cleavage.

Purified DRB1*0401 is cleaved with 3C protease for 14 hours at 25°C and the cleaved MHC
is then processed on GSTrap FF column to remove the 3C protease. Protease cleavage is
then monitored on SDS-PAGE by analyzing the fractions of uncleaved (U), protease cleaved
prior to (Pre) or after (post) processing on GSTrap column.
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Figure 6. MALDI analyses of I-Ab loaded with peptides.

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I-Ab with formerly tethered GDTAGTTHYGSLPQKSQGSGGSGGSGLEVLFQ peptide

was mock exchanged (expected molecular mass of 3081) or exchanged with
VRVSYYGPKTSPVQ (expected molecular mass of 1580), RSRYLTAAAVTAVLQ
(expected molecular mass of 1620) or EYLSFSCYLSVT (expected molecular mass of
1411).

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Figure 7. Native IEF gel of I-Ab loaded with peptides with distinct net charges.
I-Ab with tethered TTHYGSLPQKSQ peptide (plus additional linkers) was uncleaved (U),
HRV-3C-cleaved (C), or cleaved and then incubated in peptide exchange reactions (all other
lanes). Peptides in exchange reactions in labeled lanes are as follows: (M) mock/no-peptide
control, (+4) LPKPPKPVSKMRMATPLLMGALPM, (+3) KPVSKMRMATPLLMQAL,
(+2) DITYKVHLATPINSR, (+1) DIYKGVYQFKSV, (0) PGVMYAFTTPLISFF, (−2)
SQEYSGSVANEANVY. All exchange reactions contained HLA-DM, which was removed
as described in Basic Protocol 4, prior to analysis.
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Figure 8. The source of streptavidin-PE conjugates can have a dramatic effect on staining.
The class II MHCp complex of I-Ab/LCMV.GP66–77. DIYKGVYQFKSV was mixed with
streptavidin PE from three different suppliers and used to stain splenocytes from a C57B6
mouse 8 days after infection with LCMV Armstrong. All stains had the same concentration
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of I-Ab/LCMV.GP66–77. (A) FACS plots of cells gated on CD4+ positive cells inside a
lymphocyte scatter gate. (B) Commercial streptavidin-conjugated PE (SA-PE) from various
manufacturers (a, b, or c) were analyzed by size-exclusion chromatography on a 7.8 mm I.D.
× 30 cm TSKgel G4000swxl (TOSOH Bioscience LLC) column with sodium phosphate
buffer, pH 7.2, as the mobile phase at a flow rate of 1 ml/min. Relative fluorescence profiles
of the conjugates are as shown. Standard proteins for calibration of the column include
thyroglobulin (669 kDa), ferritin (440 kDa), and aldolase (158 kDa); all were obtained from

GE Healthcare (Cytvia). This figure and the associated legend are reproduced from (Altman
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and Davis, 2016).

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Table 1.
Average Number of Biotin Binding Sites for Streptavidin Conjugates

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Label Molecular weight Concentration (mg/ml) BBS (mol/μl)

Phycoerythrin 300,000 1 1.33 × 10−11
Allophycocyanin 164,000 1 2.44 × 10−11
FITC 60,000 1 6.667 × 10−11

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Table 2.
Choosing a eukaryotic expression system for class II MHC proteins

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Cell substrate Vector Control Reference

Pichia pastoris Plasmid, integrated into host cell genome AOX1 promoter, induced by methanol (Kalandadze et al., 1996)
Sf9 cells (moth) Baculovirus Viral infection (Stern and Wiley, 1992)
Metallothionein promoter, induced by

S2 cells (Drosophila) Plasmid CuSO4 (Novak et al., 1999)
Plasmid, using glutamine synthase gene

CHO cells Constitutive (Day et al., 2003)
amplification
HEK293 Lentivirus Constitutive This report

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MHC II Hek 2021

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Production of Class II MHC Proteins in Lentiviral Vector-

Corresponding 404-727-5981, jaltman@emory.edu.

293TB15. Second, using Cas9/CRISPR technology, we prepared a second generation

Curr Protoc. Author manuscript; available in PMC 2022 February 01.

LENTIVIRAL PACKAGING VECTORS—Two packaging plasmids are required for

Curr Protoc. Author manuscript; available in PMC 2022 February 01.

SEQUENCES OF CLASS II PROTEINS—The IMGT website has a well-curated

imgt/hla/) and from many other species (http://www.ebi.ac.uk/ipd/mhc/), but unfortunately

EPITOPES - TETHERED VS EXCHANGED—We express all class II MHC proteins

expression constructs for every epitope for alleles such as I-Ab.

Curr Protoc. Author manuscript; available in PMC 2022 February 01.

BIRA SUBSTRATE PEPTIDE TAG—An essential core element of MHC tetramer

Curr Protoc. Author manuscript; available in PMC 2022 February 01.

SURROGATE MARKERS FOR EXPRESSION—An internal ribosome entry site

SUPPORT REAGENTS AND EQUIPMENT

likelihood of multiple binding registers.

CHROMATOGRAPHY EQUIPMENT—The only truly essential purification protocols in

Curr Protoc. Author manuscript; available in PMC 2022 February 01.

polishing procedures, we have a collection of dedicated chromatography workstations in our

SDS-PAGE to assess overall purity, high-performance size-exclusion chromatography (HP-

HP-SEC—A number of the methods described in this chapter—notably elution from

secondary quality control measure, we often use high-performance size-exclusion

Curr Protoc. Author manuscript; available in PMC 2022 February 01.

BIOTINYLATION ASSAYS—In our Current Protocols in Immunology unit on class I

STAINING CONSIDERATIONS WITH CLASS II MHC TETRAMERS

STAINING REAGENTS USED IN CONJUNCTION WITH CLASS II MHC

Curr Protoc. Author manuscript; available in PMC 2022 February 01.

BASIC PROTOCOL 1 – LENTIVIRUS PRODUCTION AND EXPRESSION LINE

• 2 M CaCl2 (in DI H2O)

• D10 Media (see recipe)

• D-biotin (see recipe)

• Cryopreservation media (see recipe)

• 2% paraformaldehyde (see recipe)

• Controlled rate freezing container (such as Thermo Scientific™ Mr. Frosty™

• HEK-293T/17 cells (293T, ATCC CRL-11268)

• 293TPM or 293TB15 cells (optional, to be deposited in ATCC ).

• Lentiviral expression vector, 1 μg/μl (see Strategic Planning; 90 μg needed)

Curr Protoc. Author manuscript; available in PMC 2022 February 01.

• pMD2G vector, 1 μg/μl (Addgene #12259; 30 μg needed)

• psPAX2 vector, 1 μg/μl (Addgene #12260; 60 μg needed)

• 0.2μm PES 50 ml tube cap filter (Thermo Cat# 564–0020)

• T25, T75 and T225 tissue culture flasks

• 6-well and 96-well plates

• 5 ml and 25 ml polypropylene centrifuge tubes

• Ultracentrifuge with swinging bucket rotor (such as Beckman SW28)

• 38.5 mL, Open-Top Thinwall Ultra-Clear Ultracentrifuge Tube (Beckman

• Flow cytometry staining buffer (see recipe)

• OX-7 monoclonal antibody (anti-Thy 1.1/CD90.1) conjugated to Pacific Blue

• 37°C CO2 incubator

DAY 1 – SEED CELLS FOR LENTIVIRUS PRODUCTION

DAY 2 – TRANSFECTION WITH LENTI AND PACKAGING VECTORS

1. If a new batch of 2M CaCl2 or 2X HEBS is made, a transfection optimization

Lentivector 90 μl (45 μl each for ɑ and β chain)

2M CaCl2 300–600 μl, determined in step 1

ddH2O q.s. 4.5 ml

Remove T225 flask from incubator to have on hand in biosafety cabinet.

In the past, we used a more conventional approach of preparing the packaged

Curr Protoc. Author manuscript; available in PMC 2022 February 01.

3. Using a 10 ml plastic tissue culture pipette attached to a mechanical pipettor, add

5. Place cells in incubator for 6–8 hours.