Methods in Cellular Immunity

Paul Zhou
Institut Pasteur of Shanghai, CAS
November, 2005
Cells and molecules in cellular immune
responses
T cells and denderitic cells (DCs)
T cell receptors and TCR signaling
CD28 and B7 family members

Major Histocompatibility Complex (MHC) and peptide complex

Uptake of antigens and pathogens - C-type lectin receptors (CLRs)
Toll-like receptors (TLRs)
Processing of antigens into MHC class I loading compartment
Processing of antigens into MHC class II loading compartment

Experimental procedures
Ex vivo generation of DCs and their characterization
Ex vivo manipulation of DCs for vaccine
Measurements of T cell responses in vitro
Measurements of T cell responses in vivo
Adaptive T cell-based immune therapies
T cells and denderitic cells
 T cells
• One of two major classes of lymphocytes
• Derived from hematopoietic stem cells, undergoing
differentiation in thymus, and then seeded to the
peripheral lymphoid tissues and to circulating
throughout the body
• Two major subsets: α/β TCR and γ/δ TCR
• Two sublineages in α/β TCR T cells: CD4 and CD8
T cells (differ in antigen recognition and regulatory
and effector functions)
• Th1 and Th2 subtypes in CD4 T cells (differ in
cytokine secretion and helper functions)
 Dendritic Cells
• Initial discovery of Langerhans cells in skin by Paul
Langerhans in 1868
• Dendritic cells (DCs) described by Steinman and Cohn in
mouse spleen in 1973
• Special properties of DCs in initiating immunity (i.e. antigen
and pathogen recognition, uptake, process and presentation
as well as pathogen dissemination) were discovered after
depletion of monocytes, macrophages and B cells
• Reside in most peripheral tissues, especially at sites
interface with environment (skins and mucosa); but DCs
migrate to draining lymphoid nodes to activate T cells
• Paradigm of immature (steady) and mature DCs in antigen
capture and presentation, lymphocyte activation and tissue
distribution
• Ex vivo generation of DCs
• Heterogeneity of DCs
 LC DDC-IDC moDC pDC
_________________________________________________

CD1a + + + -
CD1d - + + nr
CD11b - + + -
CD11c + + + -
CD52 - - + +/-
CD83 + + + +
e-cadherin + - - -
CD207, Langerin + - - -
CD208, CD-LAMP + + + -
CD123 + + + ++
BDCA-2,4 nr nr -/+ +
_________________________________________________
LC, DDC-IDC and moDC are also termed myeloid DCs or conventional DCs. pDCs
(plasmacytoid DCs) are also termed lymphoid DCs.
 Immature DC Mature DC
(steady state)
___________________________________

CD80 Low High

CD86 Low High
HLA-DR Low High
Ag capture Efficient not
Ag processing Efficient not
Ag presentation not Efficient
Allo-stimulation Weak Strong
The Dendritic Cells on cytospin
T cell receptor MHC and peptide
complex
TCR, MHC and peptide tri-molecular complex
 T Cell Receptors
• TCR consists of two subsets: α/β and γ/δ.
• Through somatic gene arrangement, the TCR
displays extreme sequence diversity.
• TCR expresses on the surface of T cells in a clonal
fashion, in which to a given T cells only one pair of
α/β or γ/δ TCR is expressed.
• α/β or γ/δ TCR itself does not mediate signaling.
Instead it is intimately associated with CD3
complex (γδ2ε2ζ). It is the latter that mediates TCR
signaling.
• The signal mediated through TCR/CD3 complex is
called the signal one.
Human TCR gene organization
TCR/CD3 complex
Human and mouse CD3 protein structure
ITAM motifs
Structure of PTKs
TCR signaling through PTKs
Downstream of TCR signaling
CD28 and CD80/CD86 family
molecules
T cell activation and proliferation

Two step T cell activation model:

Activating T cells - IL-2 expression and secretion -
binding of IL-2 to its receptor on T cells - T cell
proliferation

However, in some cases engaging TCR with a

cognate peptide/MHC complex leads to no IL-2
expression and release and T cell anergy, which is
refractory further activation, suggesting an additional
signal (signal 2) is needed for T cell activation.
Co-stimulatory molecules between T cells and
APCs
 Effect of CD28 signaling on T cells

• Increase transcription and mRNA stability of IL-2

• Increase expression of anti-apoptotic protein Bcl-XL
• Decrease the threshold of T cell activation by promoting the
formation of an immunological synapse
• Naïve T cells depend more on CD28 signaling than
activated and memory T cells
• Regulate T cells towards Th2 differentiation (IL4, IL5, and
IL-10 producing cells)
• Regulate T cell migration by Increasing CXCR4 expression
and MIP-1α and MIP-1β production; while decreasing CCR5
expression
• Maintain homeostasis of immuno-regulatory CD4+/CD25+ T
cells
 Effect of CTLA-4 (CD152) signaling on T cells
• Unlike CD28, CTLA-4 is expressed on surface
activated T cells. It binds higher to CD80 and CD86
than CD28
• Terminate early events of TCR-mediated signaling
through modulating TCRzeta phosphorylation and
ERK activation
• Inhibit IL-2 production
• Inhibit cell cycle progression
• Affect helper T cells to differentiate into Th2 cells
• Potential mechanisms of CTLA-4-induced immune
tolerance:
1. Directly modulate TCR signaling
2. Competitively block CD28/B7 interaction
3. CD4+CD25+and CTLA-4+ regulatory T cells
IL-2 and its receptors
 Cellular responses to TCR signaling
• Early changes
1. Changes in pH
2. Changes in membrane potential (MTT assay)
3. Fluxes in cyclic nucleotides and calcium (Ca influx assay)

• Late changes
1. Cyto-skeletal changes (morphologic change)
2. Activation of the cytolytic mechanism (CTL and tetramer staining)
3. Gene regulation: CD25, CD69, IL-2, IL-3, IFNγ, GM-CSF, CTLA-4,
MHC class II, VLA-2, 4F2, transferrin receptors, and insulin
receptors, etc. (Elisa, Elispots, intracellular cytokine staining,
activation markers)
4. T cell proliferation (3H-thymidine and CFSE assays)
Major Histocompatibility
Complex (MHC)
Genes encoding MHC class I and class II molecules are
located in a MHC region. In each species this region
covers several mega-bases of DNA (chromosome 6 in
human, chromosome 17 in mouse).

Numerous genes in this region, including

1) MHC class I genes (HLA-A, B, C, E, F, and G; H-2D, L
and K)
2) MHC class II genes (HLA-DR, DP, and DQ; H-2A and E)
3) genes involved in class I and II pathways
4) genes involved in other immunological functions
5) genes seems not to be directly involved in immunological
functions
Genetic map of human and mouse MHC
 Genetic Polymorphism of HLA-A
locus
Serology Alleles Serology Alleles

A1 A*0101,0102 A32(19) A*3201

A2 A*0201?217 A33(19) A*3301?303
A3 A*0301,0302 A34(10) A*3401,3402
A11 A*1101?103 A36 A*3601
A23(9) A*2301 A43 A*4301
A24(9) A*2402?410 A66 A*6601,6602
A25(10) A*2501 A68(28) A*68011?803
A26(10) A*2601?608 A69(28) A*6901
A29(19) A*2901,2902 A74(19) A*7401
A30(19) A*3001 ?004 A*8001
A31(19) A*31012
Genomic organization of MHC class I and II
genes
Structure of MHC class I
Top view of MHC class I or II peptide-
binding cleft and bound peptide

HLA-DR I and Peptide HLA-A2 and Peptide

Uptake of antigens and pathogens
by Dendritic Cells
 Antigen and pathogen recognition and uptake

1. Receptor-mediated endocytosis
FcRs: CD64, CD16, and CD32
Complement receptors: CR3 and CR4
Receptors for heat shock proteins: CD91
Scavenger receptors: CD36
C-type lectin receptors: MMR, DEC205,
Langerin, BDCA-2, and DC-SIGN

2. Phagocytosis: pathogens and apoptotic and

necrotic bodies

3. Macropinocytosis – a constitutive, cytoskeleton-

dependent fluid-phase endocytosis which allows
DCs rapidly and non-specifically to sample large
amount of surrounding fluid.
 Signals that induce DCs to mature

• Receptors for direct pathogen signals: TLRs

• Receptors for indirect sensing of infection

through inflammatory cytokines, internal
cellular compounds and on-going immune
responses: cytokine receptors, TNF
receptor family molecules, CD40, FcR, and
sensors for cell death
 TLRs recognize pathogen-associated molecular
patterns

TRL1 (surface, mDC) Soluble bacterial factor (heterodimer with TRL-2)

TRL2 (surface, mDC) bacterial peptidoglycans, lipoproteins
TRL3 (intra, mDC) viral dsRNA, poly(I:C)
TRL4 (surface, mDC) LPS
TRL5 (surface, mDC) flagellin
TRL6 (surface, mDC) diacyl lipopeptides
TRL7 (intra, pDC) viral ssRNA
TRL8 (intra, mDC) viral ssRNA
TRL9 (intra, pDC) unmethylated CpG motifs
TRL10 unknown
TRL11 unknown

Most of TLR-ligand interactions results in intracellular signaling through

MyD88, which in turn up-regulates CD83, co-stimulatory molecules and
CCR7 and leads to the secretion of IFNα by plasmacytoid DCs or IL-6,
IL-10, TNFα and IL-12 by conventional DCs.
Signaling through TRL7/9 in pDCs
 T cell-based adaptive immune therapy

Propagation
PB Large
With or without a) w/cytokines
Tumor mass T cell or numbers
genetic
TILs (1011 cells)
(patients or manipulation b) w/ anti-CD3
or more
identical &anti-CD8 Abs
twins)

In vitro assays to determine

2) w/o RCV
3) w/ immune functions
4) viability infusion
Measurements of T cell responses in vitro
PB, Spleens,
(Immunized or non-immunized
Lymph nodes,
Infected or non-infected)
Others

Culture w/
Mononuclear cells
b) APCs + Ags
c) Cytokines

T cells or T cell subsets d) Mitogens

e) Superantigens
f) Pharmacological agents
Activated T cells
a) Cytokine production c) Cell proliferation
b) Activation markers d) Killing functions w/ target cells
Manipulation of DCs for Antigen processing & presentation

Induction
through

a) TLRs
Immature DCs b) Inflammatory
2) Subunit Ag loading cytokines

3) Gene delivery c) CD40/CD40L

through viral Mature DCs
d) Others
vectors Peptide Ag
4) mRNA delivery loading
 Ex vivo generate DCs
GM-CSF
Bone Marrow Induction
TNFα through
CD34
Cord Blood Others Immature Mature
Progenit
DCs a) TLRs DCs
PB (cytokine or cells
immobilized) b) Inflammatory
-4 F cytokines
IL S
-C
M c) CD40/CD40L
G
d) Others
PB PBMCs CD14+
Monocytes

Utilities:
Characterization: Antigen presentation to
b) Phenotypes T cells in vivo for
vaccine to induce T cell
c) Functions responses to cancer,
infectious diseases