Cyclophilin A-Deficient Mice Are Resistant to

Immunosuppression by Cyclosporine
This information is current as
of January 1, 2015.
John Colgan, Mohammed Asmal, Bin Yu and Jeremy Luban
J Immunol 2005; 174:6030-6038; ;
doi: 10.4049/jimmunol.174.10.6030
http://www.jimmunol.org/content/174/10/6030

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 The Journal of Immunology

Cyclophilin A-Deficient Mice Are Resistant to

Immunosuppression by Cyclosporine1

John Colgan,2* Mohammed Asmal,* Bin Yu,* and Jeremy Luban3*†

Cyclosporine is an immunosuppressive drug that is widely used to prevent organ transplant rejection. Known intracellular ligands
for cyclosporine include the cyclophilins, a large family of phylogenetically conserved proteins that potentially regulate protein
folding in cells. Immunosuppression by cyclosporine is thought to result from the formation of a drug-cyclophilin complex that
binds to and inhibits calcineurin, a serine/threonine phosphatase that is activated by TCR engagement. Amino acids within the
cyclophilins that are critical for binding to cyclosporine have been identified. Most of these residues are highly conserved within
the 15 mammalian cyclophilins, suggesting that many are potential targets for the drug. We examined the effects of cyclosporine
on immune cells and mice lacking Ppia, the gene encoding the prototypical cyclophilin protein cyclophilin A. TCR-induced
proliferation and signal transduction by Ppiaⴚ/ⴚ CD4ⴙ T cells were resistant to cyclosporine, an effect that was attributable to
diminished calcineurin inhibition. Immunosuppressive doses of cyclosporine failed to block the responses of Ppiaⴚ/ⴚ mice to

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allogeneic challenge. Rag2ⴚ/ⴚ mice reconstituted with Ppiaⴚ/ⴚ splenocytes were also cyclosporine resistant, indicating that this
property is intrinsic to Ppiaⴚ/ⴚ immune cells. Thus, among multiple potential ligands, CypA is the primary mediator of immu-
nosuppression by cyclosporine. The Journal of Immunology, 2005, 174: 6030 – 6038.

C yclosporine is a cyclic decapeptide produced by the soil
fungi Tolypocladium inflatum. Originally identified in a
screen for novel antibiotics, cyclosporine was shown by
Borel et al. (1, 2) to be a potent and specific inhibitor of T cell
responses to alloantigens. This discovery led to widespread clinical
use of cyclosporine as an immunosuppressant, revolutionizing or-
gan transplantation in humans.
Efforts to identify the cellular receptor for cyclosporine (3) led
to discovery of the cyclophilins, one of three protein families
known collectively as peptidyl-prolyl isomerases (PPIases)4 (4).
Other PPIase families are the FK506-binding proteins (FKBPs)
and the parvulins (4). PPIases catalyze the cis-trans interconver-
sion of peptide bonds N-terminal to proline, an activity that has
been extensively characterized in vitro using model peptides or
denatured proteins as substrates (5). Consistent with a global role
in the folding of nascent proteins in vivo, the distribution of PPI-
ases overlaps with the heat shock protein 70 protein family, being
found in all eubacteria, a few archaebacteria, and all eukaryotes,
(6). PPIases also may regulate the function of mature proteins by
catalyzing isomerization between alternative structures, contribut-
Departments of *Microbiology and †Medicine, Columbia University College of Phy-
sicians and Surgeons, New York, NY 10032
Received for publication December 15, 2004. Accepted for publication March
4, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the Sandler Foundation for Asthma Re-
search, National Institutes of Health Grant RO1 AI 36199, and a Pilot and Feasibility
Grant from the Columbia University Diabetes and Endocrinology Research Center.
2
Current address: Roy J. and Lucille A. Carver College of Medicine, Department of
Internal Medicine, University of Iowa, 375 Newton Road, Iowa City, IA 52246.
3
Address correspondence and reprint requests to Dr. Jeremy Luban, Department of
Microbiology, Columbia University, 701 West 168th Street, New York, NY 10032.
E-mail address: jl45@columbia.edu
4
Abbreviations used in this paper: PPIase, peptidyl-prolyl isomerase; BMDDC, bone
marrow-derived dendritic cell; CypA/B, cyclophilin A/B; FKBP, FK506-binding
protein.
ing to conformational stability, or sterically blocking interactions
between factors (7–10).
Cyclophilin A (CypA), the prototypical member of the cyclo-
philin family, is a highly conserved protein that is maintained at
high levels in mammalian cells (11). Studies of budding yeast have
shown that CypA localizes to both the cytoplasm and nucleus (12,
13). Consisting solely of the conserved PPIase domain, CypA
forms a globular, eight-stranded ␤-barrel with a solvent-exposed
hydrophobic pocket that is the binding site for proline-containing
peptides as well as the enzymatic active site (14). Cyclosporine
binds with subnanomolar affinity to CypA via contacts within the
hydrophobic pocket (15) and inhibits PPIase activity. However,
this effect is thought to be irrelevant for the immunosuppression.
Rather, the complex between cyclosporine and CypA creates a
composite surface that binds to and inhibits calcineurin (16, 17), a
serine-threonine phosphatase that is activated by calcium. Sub-
strates for calcineurin include members of the NF-AT family of
transcription factors (18). Found in the cytoplasm of resting T
cells, the NF-ATs are dephosphorylated upon TCR ligation and
relocate to the nucleus in a functionally active form. In the pres-
ence of cyclosporine, dephosphorylation of the NF-ATs is
blocked, and expression of genes encoding cytokines and other
proteins required for an immune response is inhibited.
Structural and genetic studies have identified amino acids within
CypA that are critical for the formation of a complex with cyclo-
sporine that binds to calcineurin (15, 19 –24). In addition to CypA,
the mouse and human genomes each encode 14 other cyclophilins
(25), most of which are distinguishable from CypA by the presence
of terminal extensions bearing motifs for subcellular localization
or binding to nucleic acids or other proteins. Despite these differ-
ences, the residues in CypA that mediate cyclosporine binding are
highly conserved in other family members (Table I), indicating
that multiple cyclophilins are potential targets for the drug.
The genome of the budding yeast Saccharomyces cerevisiae en-
codes eight different cyclophilins (26), none of which are essential
(27). Several studies have demonstrated that CypA is the primary
mediator of calcineurin inhibition by cyclosporine in this organism
(28, 29). Hence, it might be predicted that mammalian CypA

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00

The Journal of Immunology 6031

Table I. Amino acid alignment of mouse cyclophilinsa

Amino Acid Position in PPIA (CypA)

Cyclophilin 54 55 60 61 63 72 101 102 103 111 113 121 122 126

PPIA (CypA) His Arg Phe Met Gln Gly Ala Asn Ala Gln Phe Trp Leu His
PPIB (CypB) ––– ––– ––– ––– ––– ––– ––– ––– ––– ––– ––– ––– ––– –––
PPIC (CypC) ––– ––– ––– ––– ––– ––– ––– ––– ––– ––– ––– ––– ––– –––
PPID (CypD) ––– ––– ––– ––– ––– ––– ––– ––– ––– ––– ––– His ––– –––
PPIE (CypE) ––– ––– ––– ––– ––– ––– ––– ––– Ser ––– ––– ––– ––– –––
PPIF (CypF) ––– ––– ––– ––– ––– ––– ––– ––– ––– ––– ––– ––– ––– –––
PPIG (CypG) ––– ––– ––– ––– ––– ––– ––– ––– Arg ––– ––– His ––– –––
PPIL1 ––– ––– ––– Val ––– ––– ––– ––– ––– ––– ––– ––– ––– –––
PPIL2 ––– ––– ––– ––– ––– ––– ––– ––– Ser ––– ––– Tyr ––– –––
PPIL3 ––– ––– ––– ––– ––– ––– ––– ––– Asn ––– ––– His ––– Tyr
PPIL4 ––– Asn ––– Ile ––– ––– Val ––– Asn ––– Leu Tyr ––– –––
NKTR ––– ––– ––– ––– ––– ––– ––– ––– Arg ––– ––– His ––– –––
RANBP2 ––– ––– Ser Ala ––– ––– Gln ––– ––– Pro Val Gly ––– Gln
J08 Riken ––– ––– ––– ––– ––– ––– ––– ––– Ser ––– ––– ––– ––– –––
F20 Riken Asp Pro Leu Gln Phe Lys Ile Asp Val Ala Thr Leu Lys –––
a
The residues listed mediate contacts between CypA and cyclosporine based on X–ray crystallographic and nuclear magnetic resonance analysis (see Refs. 15 and 20). Dashes
(–––) denote identity to the amino acid at the indicated position in CypA.

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would mediate the immunosuppressive effects of cyclosporine in T
cells. In contrast, CypA and CypB can mediate calcineurin inhi-
bition when overexpressed in transformed T cells (30), suggesting
that both of these proteins have a role in immunosuppression by
cyclosporine in vivo.
Mice lacking Ppia, the gene encoding CypA, have augmented
Th2-type immune responses attributable to increases in the activity
of the Itk, a tyrosine kinase that is inhibited by CypA via recog-
nition of a proline in its Src homology 2 domain (10). In this study,
we present analysis of in vitro T cell function and in vivo immune
responses that shows Ppia⫺/⫺ mice are resistant to immunosup-
pression by cyclosporine.
Materials and Methods
Mice
Type Culture Collection and were maintained, as recommended by the
supplier. Bone marrow-derived dendritic cells (BMDDCs) were generated
as described (32).
Proliferation assays
Cells (2 ⫻ 105/well) were cultured in 96-well round-bottom plates. To
stimulate with plate-bound Ab, wells were incubated for 3 h at 37°C with
Ab in PBS and washed with PBS before cells were added. CD4⫹ T cell
proliferation in response to syngeneic or allogeneic cells was assessed by
mixing irradiated (2500 rad) BMDDCs (1 ⫻ 105/well) with purified CD4⫹
T cells (0.1–3.0 ⫻ 105/well) in 96-well round-bottom plates. Cyclosporine,
methyl-Ile4-cyclosporine, and FK506 in oil emulsion were diluted to 1
mg/ml in DMSO, further diluted in RPMI 1640, and added to wells before
cells were plated. Cultures were pulsed with [3H]thymidine (1 ␮Ci/well;
PerkinElmer Life Science Products) 48 or 72 h (when stimulated with
dendritic cells) after plating, and [3H]thymidine incorporation was mea-
sured 12 h later using a Top Count (Packard Instrument).

The 129S6/SvEv Ppia⫺/⫺ mice have been described (10) and deposited
with The Jackson Laboratory Induced Mutant Resource as Stock 5320
(www.jax.org). The 129S6/SvEv Rag2⫺/⫺ mice were from Taconic Farms.
Offspring of Ppia⫹/⫺ mice obtained by backcrossing seven generations
into BALB/c (Taconic Farms) were used as sources of bone marrow for
generating allogeneic dendritic cells. Sex-matched, specific pathogen-free
mice were maintained and used as approved by the Columbia University
Institutional Animal Care and Usage Committee.
Abs and reagents
Purified Ab to CD3 (145-2C11), CD28 (37.51) and IL-2 (JES6-1A12),
FITC anti-CD4, H2Kd and goat anti-rabbit Ab, PE anti-CD69, H2Db and
CD62L, allophycocyanin anti-CD8, biotinylated anti-CD25 and IL-2
(JES6-5H4), streptavidin-allophycocyanin, and alkaline phosphatase were
from BD Biosciences. Affinity-purified anti-67.1 Ab specific for NFATc2/
p/1 (31) was provided by A. Rao (Harvard University, Boston, MA). Rab-
bit anti-phospho-p38 MAPK Ab was from Cell Signaling Technology.
HRP-conjugated goat anti-rabbit Ab was from Promega. Cyclosporine was
from Bedford, methyl-Ile4-cyclosporine from Novartis Pharmaceuticals,
and FK506 from Fujisawa Pharmaceutical. PMA and ionomycin were from
Calbiochem. Anti-CD4 beads and Detachabead were from Dynal Biotech.
Anti-CD8 microbeads were from Miltenyi Biotec. SYBR green was from
Molecular Probes. IL-2-specific molecular beacon was from Midland Cer-
tified Reagent.
Cell preparation
Analysis of IL-2 production by ELISA
Spleen cells (2 ⫻ 105/well) were plated in 96-well round-bottom plates
coated with 10 ␮g/ml anti-CD3 and containing the indicated amounts of
cyclosporine. After 48 h, supernatant from triplicate wells was pooled and
assayed for IL-2 by ELISA, according to a protocol from BD Biosciences.
RT-PCR
CD4⫹ T cells (1 ⫻ 106/well) were added to wells in a 24-well plate coated
with 10 ␮g/ml anti-CD3 and containing the indicated amounts of cyclo-
sporine. RNA isolation, cDNA synthesis, and real-time RT-PCR analysis
were performed, as described (10). Hypoxanthine phosphoribosyltrans-
ferase primers were 5⬘-GGACCTCTCGAAGTGTTGGATAC-3⬘ (for-
ward) and 5⬘-GCTCATCTTAGGCTTTGTATTTGGCT-3⬘ (reverse). IL-2
primers were 5⬘-CCTGAGCAGGATGGAGAATTACA-3⬘ (forward), 5⬘-
TCCAGAACATGCCGCAGAG-3⬘ (reverse), and 5⬘-CGCGCAC
CCAAGCAGGCCACAGAATTGAAAGATTGCGCG-3⬘ (beacon).
Flow cytometry
Staining and wash buffer was PBS containing 3% FBS and 0.1% sodium
azide. Cells were mixed with Ab at concentrations recommended by the
manufacturer, incubated for 20 min on ice, and then washed. Analysis was
performed using a FACSCalibur flow cytometer and CellQuest software
(BD Biosciences).

Spleen and lymph node cell suspensions were depleted of RBC using RBC
lysis buffer (Sigma-Aldrich). CD4⫹ T cells were purified from pooled
lymph node and spleen cells using anti-CD4 beads and eluted with De-
tachabead; purified cells were typically ⬎98% CD4⫹. Cells were cultured
in RPMI 1640 supplemented with 10% FCS, 0.1 ␮M 2-ME, 100 IU/ml
penicillin, 100 ␮g/ml streptomycin, and 2 mM glucose. P815 mastocytoma
cells (TIB-64) and EL4 lymphoma cells (TIB-39) were from American
Surface expression of CD25 and CD69
CD4⫹ T cells (1 ⫻ 106/well) were added to wells in 24-well plates coated
with 10 ␮g/ml anti-CD3 and containing the indicated amounts of cyclo-
sporine or FK506. After 20 h, cells were stained with FITC anti-CD4, PE
anti-CD69, biotinylated anti-CD25, and streptavidin-allophycocyanin for
flow cytometric analysis.
6032 CypA MEDIATES CYCLOSPORINE SENSITIVITY

p38 MAPK activation

CD4⫹ T cells (1 ⫻ 106/well) in 24-well plates were incubated for 20 min
at 37°C in medium alone or medium containing cyclosporine. PMA and
ionomycin were added to 10 and 400 ng/ml, respectively, followed by
incubation for 20 min at 37°C. Cells were washed with PBS, fixed using
Cytofix buffer (BD Pharmingen), and permeabilized using Perm/Wash
buffer (BD Pharmingen). Anti-phospho p38 MAPK Ab was added, fol-
lowed by incubation for 20 min on ice. Cells were washed with Perm/Wash
buffer. FITC-conjugated anti-rabbit Ab was added to cells in Perm/Wash
buffer, followed by incubation for 20 min on ice. Cells were washed with
Perm/Wash buffer and analyzed by flow cytometry.

NF-AT dephosphorylation
A total of 2 ⫻ 106 lymph node cells was incubated for 20 min at 37°C in
medium containing the indicated amounts of cyclosporine. Ionomycin was
added to 400 ng/ml, and samples were incubated for 5 min at 37°C. Cells
were spun down and resuspended in lysis buffer (5% SDS, 30 mM sodium
pyrophosphate, 5 mM EDTA, 2 mM PMSF, 250 ␮M leupeptin, 100 ␮g/ml
aprotinin, and 2 mM sodium orthovanadate), boiled for 5 min, and then
passed through a 26-gauge needle several times. After boiling again for 5
min, 10 ␮g of protein was resolved by SDS-PAGE, transferred to nitro-
cellulose, and probed with affinity-purified anti-67.1 Ab. Reactive species
were visualized using HRP-conjugated anti-rabbit Ab and a chemilumi-

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nescence kit (PerkinElmer).

Cyclosporine treatment
Clinical-grade cyclosporine was diluted fresh daily in PBS and injected i.p.
Control animals were injected with PBS alone. Treatment was given daily
starting the day before injection of P815 cells.

Assessment of tumor cell clearance and CD62L down-regulation

Mice were injected i.p. with 1 ⫻ 107 P815 cells and sacrificed 10 days
later. To assess tumor cell clearance, peritoneal exudates were recovered
by lavage, counted, and stained with FITC anti-H2Kd and PE anti-H2Db for
analysis by flow cytometry. The number of tumor cells recovered was
calculated by determining the percentage of cells staining positive for
H2Kd. To assess surface CD62L expression on T cells, splenocytes were
stained with FITC anti-CD4, PE anti-CD62L, and allophycocyanin anti-
CD8, and analyzed by flow cytometry.
Assay for cytotoxic T cell activity
Mice were injected i.p. with 1 ⫻ 107 P815 cells and sacrificed 10 days
later. CD8⫹ cells were isolated from spleen cells using anti-CD8 mi-
crobeads. Target cells were labeled by mixing 2 ⫻ 107 cells in 0.2 ml of
medium with 0.2 ml (200 ␮Ci) of 51Cr (PerkinElmer Life Science Prod-
ucts), followed by incubation at 37°C for 1 h. Unbound 51Cr was removed
by washing three times with medium. CD8⫹ cells were mixed with 1 ⫻ 104
51
Cr-labeled P815 or control EL-4 cells (final culture vol 0.2 ml) in flat-
bottom 96-well plates. Cells were spun down by brief centrifugation, and
plates were incubated for 3 h at 37°C. Supernatant (20 ␮l) was removed
and analyzed for 51Cr using a Top Count (Packard Instrument). Percent
specific lysis was calculated as follows: (experimental release ⫺ sponta-
neous release)/(maximum release ⫺ spontaneous release). Spontaneous re-
lease was measured by incubating target cells alone in medium. Maximum
release was measured by lysing target cells with 2% SDS.
Reconstitution by adoptive transfer
Splenocytes (1 ⫻ 107/mouse) were injected i.v. into Rag2⫺/⫺ mice. The
next day, both cyclosporine treatment and P815 challenge were initiated.
Results
Proliferation by Ppia⫺/⫺ cells is cyclosporine resistant
To test whether Ppia⫺/⫺ cells have altered sensitivity to cyclo-
sporine, the effects of different doses of drug on splenocyte pro-
liferation induced by plate-bound anti-CD3 were quantified (Fig.
1A). Cyclosporine inhibited proliferation of Ppia⫹/⫹ or Ppia⫹/⫺
splenocytes to a similar degree, with an IC50 of ⬃25 nM and
complete inhibition at ⬃100 nM. In striking contrast, the IC50 of
Ppia⫺/⫺ splenocytes was at least 10-fold higher, and complete
inhibition was not observed using drug doses up to 2.5 ␮M. Sim-
ilar cyclosporine resistance was seen when Ppia⫺/⫺ splenocytes
were stimulated with either Con A or PMA and ionomycin (data
FIGURE 1. Proliferation by stimulated Ppia⫺/⫺ cells is cyclosporine
resistant. A, Proliferative responses of Ppia⫹/⫹, Ppia⫹/⫺, and Ppia⫺/⫺
splenocytes. Wells coated with 10 ␮g/ml anti-CD3 were used to stimulate
cells in the presence of the indicated concentrations of cyclosporine (CsA).
B–D, Proliferative responses of purified Ppia⫹/⫹ and Ppia⫺/⫺ CD4⫹ T
cells. Wells coated with 10 ␮g/ml anti-CD3 were used to stimulate cells in
the presence of the indicated concentrations of cyclosporine (CsA) (B), or
methyl-Ile4-cyclosporine (Me-Ile4-CsA) (C), or FK506 (D).
not shown). Analysis of purified CD4⫹ T cells stimulated with
anti-CD3 showed that the IC50 of cyclosporine for Ppia⫺/⫺ cells
was also at least 10-fold greater than that of Ppia⫹/⫹ cells (Fig.
1B), demonstrating that cyclosporine resistance was intrinsic to T
cells. Similar results were obtained when purified CD4⫹ T cells
were stimulated with anti-CD3 in combination with anti-CD28
(data not shown).
As seen with splenocytes, proliferation by Ppia⫺/⫺ CD4⫹ T
cells was not completely inhibited by any dose of cyclosporine
tested, but was diminished at drug concentrations of 250 nM or
greater. One explanation for this effect is that another cyclophilin
can mediate calcineurin inhibition, but only at high doses of drug.
Alternatively, some calcineurin-independent pathway might be af-
fected under these conditions. To distinguish between these pos-
sibilities, CD4⫹ T cell proliferation in the presence of methyl-Ile4-
cyclosporine was analyzed (Fig. 1C). This cyclosporine derivative
has higher affinity for CypA than the parent compound, but com-
pletely lacks immunosuppressive activity (33). Methyl-Ile4-cyclo-
sporine had no effect on the proliferation of Ppia⫹/⫹ or Ppia⫺/⫺
CD4⫹ T cells at doses equivalent to those at which cyclosporine
caused inhibition. This result indicates that Ppia⫺/⫺ cells are par-
tially sensitive to high-dose cyclosporine due to calcineurin inhi-
bition mediated by another cyclophilin family member.
The Journal of Immunology
FK506 is another immunosuppressive compound used to pre-
vent organ transplant rejection. Structurally unrelated to cyclospor-
ine, FK506 also binds to and inhibits calcineurin, but only as part
of a complex with members of the FKBP family of PPIases (34).
Ppia⫹/⫹ and Ppia⫺/⫺ CD4⫹ T cells stimulated with anti-CD3 had
similar responses to different doses of FK506 (Fig. 1D). These
results show that proliferation by Ppia⫺/⫺ cells is dependent on
calcineurin activity, indicating that cyclosporine resistance is due
to abrogation of calcineurin inhibition rather than gross dysregu-
lation of TCR-induced signaling pathways.
Proliferation by Ppia⫹/⫹ and Ppia⫺/⫺ CD4⫹ T cells in response
to syngeneic and allogeneic BMDDCs was also assessed. The re-
sponses of Ppia⫺/⫺ cells to either syngeneic or allogeneic BMD-
DCs were slightly lower than those of Ppia⫹/⫹ cells (Fig. 2A). To
measure cyclosporine responses, CD4⫹ T cells were mixed with
either Ppia⫹/⫹ or Ppia⫺/⫺ allogeneic BMDDCs in the presence of
different concentrations of drug (Fig. 2B). Ppia⫹/⫹ CD4⫹ T cells
were inhibited to the same extent by cyclosporine regardless of the
genotype of the stimulator cell population. Ppia⫺/⫺ CD4⫹ T cells
showed resistance to cyclosporine when challenged with Ppia⫹/⫹
allogeneic BMDDCs, and this was increased when Ppia⫺/⫺ cells
were used as stimulators. These results suggest that cyclosporine,
via interactions with CypA, can inhibit the function of APCs.
FIGURE 2. The responses of Ppia⫺/⫺ CD4⫹ T cells to allogeneic den-
dritic cells are cyclosporine resistant. A, Proliferative responses of Ppia⫹/⫹
and Ppia⫺/⫺ CD4⫹ T cells mixed with irradiated, syngeneic or allogeneic
Ppia⫹/⫹ BMDDCs. B, Proliferative responses of Ppia⫹/⫹ and Ppia⫺/⫺
CD4⫹ T cells mixed with irradiated, allogeneic Ppia⫹/⫹ or Ppia⫺/⫺ BM-
DDCs (DCs) in the presence of the indicated concentrations of cyclospor-
ine (CsA). Relative proliferation corresponds to [3H]thymidine incorpora-
tion observed relative to that by the control (an identical mixture of CD4⫹
T cells and DCs lacking cyclosporine).
6033
Ppia⫺/⫺ cells are resistant to inhibition of gene expression by
cyclosporine
One well-characterized response to calcineurin activation is induc-
tion of IL-2 expression (35). Ppia⫹/⫹ and Ppia⫺/⫺ splenocytes
were stimulated with anti-CD3, and accumulation of IL-2 in cul-
ture supernatant was determined (Fig. 3A). When no cyclosporine
was added, Ppia⫹/⫹ and Ppia⫺/⫺ cells produced similar amounts
of IL-2. At low doses of cyclosporine (25 nM), IL-2 production by
Ppia⫹/⫹ cells was reduced ⬎10-fold, and was undetectable at
higher concentrations of drug. In contrast, IL-2 production by
Downloaded from http://www.jimmunol.org/ by guest on January 1, 2015
FIGURE 3. Cyclosporine-resistant calcineurin and MAPK signaling in
Ppia⫺/⫺ T cells. A, IL-2 production by splenocytes. Wells coated with 10
␮g/ml anti-CD3 were used to stimulate cells in the presence of the indi-
cated concentrations of cyclosporine (CsA). After 48 h, culture supernatant
was collected, and IL-2 levels were determined by ELISA. B, IL-2 mRNA
expression by CD4⫹ T cells. Wells coated with 10 ␮g/ml anti-CD3 were
used to stimulate purified CD4⫹ T cells in the presence of the indicated
concentrations of cyclosporine (CsA). After 8 h, total RNA was isolated
and reverse transcribed. IL-2 cDNA was quantified by real-time RT-PCR
as normalized to hypoxanthine phosphoribosyltransferase cDNA. C, Sur-
face CD25 and CD69 expression on CD4⫹ T cells. Wells coated with 10
␮g/ml anti-CD3 were used to stimulate cells in the presence of the indi-
cated concentrations of drug. After 16 h, cells were harvested and analyzed
by flow cytometry. D, Immunoblot analysis of NFATp/1c/c2. Lysates were
prepared from lymph node cells preincubated with the indicated concen-
trations of cyclosporine (CsA) and then stimulated for 5 min with iono-
mycin. E, p38 MAPK phosphorylation in CD4⫹ T cells. Cells were pre-
incubated with or without 40 nM cyclosporine (CsA), stimulated with
PMA and ionomycin for 20 min, and then analyzed by flow cytometry.
6034 CypA MEDIATES CYCLOSPORINE SENSITIVITY

Ppia⫺/⫺ cells was modestly decreased by the presence of 25 nM
cyclosporine, and remained detectable at drug concentrations up to
750 nM. These results were confirmed by RT-PCR analysis of IL-2
mRNA (Fig. 3B). In the absence of drug, steady-state levels of
IL-2 mRNA were similar in Ppia⫹/⫹ and Ppia⫺/⫺ cells. Addition
of 25 nM cyclosporine greatly reduced IL-2 mRNA in Ppia⫹/⫹
cells, but had only a slight effect on IL-2 mRNA levels in Ppia⫺/⫺
cells. Higher concentrations of cyclosporine caused gradual reduc-
tions in the amounts of IL-2 mRNA detected in Ppia⫺/⫺ cells, but
the levels seen were still well above those in Ppia⫹/⫹ cells at drug
concentrations up to 250 nM.
Two other genes that are regulated by calcineurin encode the
IL-2R ␣-chain (CD25) and the very early activation Ag CD69
(18). Cell surface expression of these proteins on CD4⫹ T cells
stimulated with anti-CD3 was therefore assessed (Fig. 3C). When
no cyclosporine was present, Ppia⫹/⫹ and Ppia⫺/⫺ cells expressed
similar levels of CD25 and CD69. Addition of 25 nM cyclosporine
was sufficient to completely block expression of both proteins on
Ppia⫹/⫹ cells, but caused only a slight decrease in the percentage
of Ppia⫺/⫺ CD4⫹ T cells expressing high level CD25 and CD69.
exudate cells (Fig. 4A). In the absence of cyclosporine treatment,
Ppia⫹/⫹ and Ppia⫺/⫺ mice eradicated the tumor cells with similar
kinetics (Fig. 4B). Ppia⫹/⫹ mice treated with 30 mg/kg/day cy-
closporine were unable to clear the tumor cells, which expanded
and completely overtook the endogenous cell population by day 10
after injection (Fig. 4, C and D). In striking contrast, Ppia⫺/⫺ mice
receiving the same dose of cyclosporine cleared the tumor cells as
efficiently as animals given PBS instead of drug.
Cyclosporine resistance in Ppia⫺/⫺ mice correlates with
preservation of T cell responses
Eradication of P815 tumor cells as an allogeneic challenge requires
Ag-specific CD8⫹ T cell responses (37). Robust, specific killing
by CD8⫹ T cells from Ppia⫹/⫹ and Ppia⫺/⫺ mice primed with
P815 cells was observed (Fig. 5A). The cytotoxic activity of cells
from Ppia⫺/⫺ mice was consistently lower activity than those from
Ppia⫹/⫹ mice, suggesting that CD8⫹ T cell responses in Ppia⫺/⫺
mice are slightly impaired. CD8⫹ T cells from Ppia⫹/⫹ mice
treated with cyclosporine completely lacked killing activity, but

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Increasing doses of drug further reduced the proportion of Ppia⫺/⫺
cells expressing both markers, but a population staining positive
for CD25 and CD69 remained detectable at cyclosporine concen-
trations up to 250 nM. As expected, doses of FK506 sufficient to
inhibit proliferation of CD4⫹ T cells (Fig. 1D) completely blocked
expression of CD25 and CD69 on both Ppia⫹/⫹ and Ppia⫺/⫺ cells.
These results show that the expression of calcineurin-regulated
target genes by Ppia⫺/⫺ cells is cyclosporine resistant.

Calcineurin and MAPK pathways in Ppia⫺/⫺ cells are resistant

to cyclosporine
In response to increased intracellular calcium levels, calcineurin
dephosphorylates the cytoplasmic forms of the NF-ATs, a modi-
fication that is blocked by cyclosporine (18). Immunoblot analysis
of lysates prepared from lymph node cells stimulated with iono-
mycin in the absence of cyclosporine showed that the extent of
NFATc2/p/1 dephosphorylation was similar in Ppia⫹/⫹ and
Ppia⫺/⫺ cells (Fig. 3D). Pretreatment with 25 nM cyclosporine
completely abolished NFATc2/p/1 dephosphorylation in Ppia⫹/⫹
cells treated with ionomycin. In contrast, dephosphorylation of
NFATc2/p/1 could be detected in ionomycin-stimulated Ppia⫺/⫺
cells pretreated with concentrations of cyclosporine as high as 750
nM. Thus, based on direct analysis of a physiologic substrate, cal-
cineurin activity in Ppia⫺/⫺ cells is cyclosporine resistant.
Cyclosporine can suppress activation of p38 MAPK in T cells
via inhibition of calcineurin-independent pathways (36). Whether
CypA is required for this effect was therefore assessed (Fig. 3E).
In the absence of cyclosporine, PMA and ionomycin induced sim-
ilar levels of p38 phosphorylation in Ppia⫹/⫹ and Ppia⫺/⫺ CD4⫹
T cells. Addition of 40 nM cyclosporine completely inhibited
phosphorylation of p38 in Ppia⫹/⫹ cells, but had no effect on the FIGURE 4. Allogeneic tumor cell clearance in Ppia⫺/⫺ mice is not sup-
level of p38 phosphorylation in Ppia⫺/⫺ cells. Higher amounts of pressed by cyclosporine. A, Assessment of P815 allotumor cell clearance
cyclosporine (200 nM) suppressed p38 phosphorylation in by flow cytometry. Surface expression of host (H2b) and P815 tumor cell
Ppia⫺/⫺ cells (data not shown), suggesting that another cyclophi- (H2d) MHC I in peritoneal exudate cells recovered on the indicated days
lin can substitute for CypA, but only at high doses of drug. These after i.p. injection of 1 ⫻ 107 tumor cells into wild-type mice. B, Normal
results show that CypA mediates inhibition of p38 MAPK activa- allotumor cell clearance in Ppia⫺/⫺ mice. The percentage of peritoneal
exudate cells expressing P815 tumor cell MHC I was quantified by flow
tion by cyclosporine.
cytometric analysis on the indicated days after tumor cell injection, as
Ppia⫺/⫺ mice are resistant to immunosuppression by shown in A. C and D, Allotumor cell growth in cyclosporine-treated mice.
cyclosporine C, Surface expression of host and tumor cell MHC I in peritoneal exudate
cells from mice injected with 1 ⫻ 107 tumor cells on day 0 and then given
To test whether CypA is required for the effects of cyclosporine in either PBS or 30 mg/kg/day cyclosporine (CsA) for 10 days. D, The ab-
vivo, H2b recipient mice were challenged with allogeneic H2d solute number of tumor cells recovered was derived from flow cytometric
P815 mastocytoma cells delivered by i.p. injection. Tumor cell analysis quantifying the percentage of peritoneal exudate cells expressing
clearance was monitored by flow cytometric analysis of peritoneal tumor cell MHC I, as shown in D.
The Journal of Immunology
FIGURE 5. Allotumor cell-specific T cell responses
in Ppia⫺/⫺ mice are resistant to cyclosporine. A, Cyto-
toxic activity from P815 tumor cell-primed mice. Kill-
ing by CD8⫹ T cells purified from mice primed with
P815 tumor cells 10 days earlier was assayed using
51
Cr-labeled P815 (Allo) or EL4 (Syn) cells as targets.
B, Cytotoxic activity elicited from mice treated with cy-
closporine. Mice were primed with P815 tumor cells on
day 0 and given either PBS or 30 mg/kg/day cyclospor-
ine (CsA) for 10 days. Killing by CD8⫹ T cells purified
from treated mice was assayed using 51Cr-labeled P815
cells as target. C, CD4⫹ and CD8⫹ T cell activation in
allotumor cell-primed mice. Surface expression of
CD62L on the indicated cell populations from unprimed
mice (Naive) or tumor cell-primed mice given either
PBS or 30 mg/kg/day cyclosporine (CsA).
cells from Ppia⫺/⫺ mice given the same dose of drug were po-
tently and specifically cytotoxic (Fig. 5B). Relative to Ppia⫺/⫺
mice given PBS, the specific killing activity generated in cyclo-
sporine-treated Ppia⫺/⫺ mice was greater, indicating that cyclo-
sporine can paradoxically augment CD8⫹ T cell responses in these
animals.
The cell surface marker CD62L is down-regulated when naive T
cells are stimulated (38). Low level expression of CD62L is main-
tained on effector and memory T cell populations and correlates
with cytolytic activity by CD8⫹ T cells (39). Following injection
of tumor cells, CD62L expression was decreased on CD4⫹ cells
and CD8⫹ cells from the spleens of Ppia⫹/⫹ and Ppia⫺/⫺ mice to
a similar extent (Fig. 5C). Down-regulation of CD62L on CD4⫹ T
cells most likely reflects a specific response to the injected cells,
because Th cell can accelerate the clearance of P815 cells (37, 40).
When 30 mg/kg/day cyclosporine was administered during the tu-
mor cell challenge, CD62L expression remained high on either
CD4⫹ or CD8⫹ cells from Ppia⫹/⫹ mice, but was low on both cell
types from Ppia⫺/⫺ mice. These results indicate that T cell re-
sponses to allograft challenge in Ppia⫺/⫺ mice are resistant to
cyclosporine.
Assessment of the magnitude of cyclosporine resistance in
Ppia⫺/⫺ mice
To roughly determine the difference in cyclosporine sensitivity be-
tween Ppia⫹/⫹ and Ppia⫺/⫺ mice, tumor cell clearance in animals
treated with different doses of cyclosporine was assessed (Fig. 6A).
A dose of 30 mg/kg/day was sufficient to block tumor cell clear-
ance in Ppia⫹/⫹ mice, while administration of 70 mg/kg/day com-
bined with the tumor cell challenge led to premature sacrifice, with
evidence that the injected tumor cells had infiltrated the liver (data
6035
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not shown). In contrast, cyclosporine doses of at least 70 mg/kg/
day were required to inhibit tumor cell clearance in Ppia⫺/⫺ mice,
which were able to tolerate drug doses up to 90 mg/kg/day and
showed no overt signs of tumor growth in the liver.
The killing activity of CD8⫹ T cells from Ppia⫺/⫺ mice treated
with different doses of cyclosporine was also analyzed (Fig. 6B).
Cells from mice given 50 mg/kg/day cyclosporine were potently
cytotoxic, while those from animals treated with higher doses of
drug had reduced activity. Still, the killing activity by CD8⫹ T
cells from Ppia⫺/⫺ mice receiving the 90 mg/kg/day drug was
greater than that by cells from Ppia⫹/⫹ mice given 3 times less
drug, which had essentially no activity.
Cyclosporine resistance is transferred with Ppia⫺/⫺ splenocytes
Two mechanisms could explain the cyclosporine resistance of
Ppia⫺/⫺ mice. Cyclosporine might be unable to inhibit immune
responses because the necessary receptor is not expressed in target
cells. Alternatively, cyclosporine resistance might be secondary to
abnormal metabolism of the drug due to the absence of CypA in
other nonimmune tissues. To distinguish these possibilities, the
effects of cyclosporine on Rag2⫺/⫺ mice reconstituted with
Ppia⫹/⫹ or Ppia⫺/⫺ splenocytes were evaluated. Ppia⫹/⫹ and
Ppia⫺/⫺ splenocytes reconstituted the immune system of Rag2⫺/⫺
recipients with similar efficiencies (Fig. 7A). In the absence of
cyclosporine treatment, mice reconstituted with either Ppia⫹/⫹ or
Ppia⫺/⫺ cells eradicated the tumor cells with similar efficiency
(Fig. 7B). When mice were treated with cyclosporine, the tumor
cells expanded in mice reconstituted with Ppia⫹/⫹ cells, but were
cleared in those that received Ppia⫺/⫺ cells. These results dem-
onstrate that the lack of CypA expression in immune cells results
in cyclosporine resistance.
6036
FIGURE 6. Assessment of the magnitude of cyclosporine resistance in
Ppia⫺/⫺ mice. A, Allotumor cell growth in Ppia⫹/⫹ and Ppia⫺/⫺ mice
treated with different doses of cyclosporine (CsA). The absolute number of
tumor cells recovered 10 days after i.p. injection of 1 ⫻ 107 cells was
derived from flow cytometric analysis by quantifying the percentage of
peritoneal exudate cells expressing allogeneic MHC I (see Fig. 4, C and D).
†, Signifies that tumor cell number was not determined because the mice
were sacrificed prematurely. B, Cytotoxic activity elicited from Ppia⫺/⫺
mice treated with different doses of cyclosporine. Mice were primed with
tumor cells on day 0 and given the indicated amounts of cyclosporine
(CsA) for 10 days. Killing by CD8⫹ cells from treated mice was assayed
using 51Cr-labeled P815 cells as target.
Discussion
Our results demonstrate that, despite the fact that mammalian cells
express multiple cyclophilins with demonstrated or predicted af-
finity for cyclosporine, CypA is the predominant, if not the sole,
mediator of the drug’s immunosuppressive effects. Although cells
and mice lacking Ppia remain partially sensitive to high doses of
cyclosporine, the concentrations of drug required for these effects
are well above that required to bring about immunosuppression in
the wild-type counterparts. Our findings also confirm that the mo-
lecular basis for immunosuppression by cyclosporine is the for-
mation of a drug-CypA complex that inhibits calcineurin.
Determination of the crystal and solution structure of CypA
bound to cyclosporine has identified 15 aa within CypA that me-
diate contacts with the drug (15, 20). Alignment of the 15 cyclo-
philins encoded in the mouse genome (Table I) reveals that CypB,
C, and F share identity with CypA at all of these residues, while 4
other cyclophilins, CypD, E, Ppil1, and Riken clone J08, contain
single substitutions. CypA, B, and C all bind with high affinity to
cyclosporine in vitro (30), whereas secreted CypB augments cel-
lular uptake of cyclosporine via interaction with cell surface bind-
ing sites (41). These observations argue that multiple cyclophilins
have the potential to interact with cyclosporine in cells. From our
CypA MEDIATES CYCLOSPORINE SENSITIVITY
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FIGURE 7. Cyclosporine resistance is transferred with Ppia⫺/⫺ spleno-
cytes. A, Ppia⫹/⫹ and Ppia⫺/⫺ splenocytes reconstitute Rag2⫺/⫺ mice with
similar efficiencies. Flow cytometric analysis of splenocytes from Rag2⫺/⫺
mice injected i.v. with either PBS (None) or 3 ⫻ 107 splenocytes 10 days
earlier. B, Surface expression of host and allogeneic tumor cell MHC I on
peritoneal exudate cells from reconstituted Rag2⫺/⫺ mice. Animals were
primed with tumor cells on day 0 and given daily injections of PBS or 30
mg/kg cyclosporine (CsA) for 10 days.
studies, we cannot rule out the possibility that ligands other than
CypA contribute to the immunosuppressive effects of cyclospor-
ine. Nevertheless, our results clearly demonstrate that CypA is
required for immunosuppression by cyclosporine at physiologi-
cally relevant concentrations of the drug.
It is widely accepted that a complex formed between cyclospor-
ine and cyclophilins causes immunosuppression by binding to and
inhibiting calcineurin (34). Studies in yeast of the effects of cy-
closporine on calcineurin-dependent pathways have provided
strong support for this model (22, 28, 29, 42). Our data show a
strong correlation between the maintenance of calcineurin activity
and cyclosporine resistance in lymphocytes.
CypA, B, and C all form complexes with cyclosporine that in-
hibit calcineurin activity in vitro (30, 43). Moreover, CypB is sig-
nificantly more efficient than CypA at mediating calcineurin inhi-
bition in vitro (21). The unique importance of CypA for
calcineurin inhibition demonstrated in this study must therefore
reflect factors other than affinity for cyclosporine or calcineurin.
CypA is found at high concentrations in cells, and a pool of CypA
must be accessible for interaction with both cyclosporine and cal-
cineurin. CypB is localized to membrane components of the se-
cretory pathway and may be sequestered due to spatial distribution
or competing interactions with other cellular factors. An analogous
situation seems to apply to the FKBP family of PPIases and the
calcineurin inhibitor FK506. Although at least three FKBPs can
form complexes with FK506 that inhibit calcineurin, studies using
mutant mice have shown that FKBP12 is the sole mediator of the
drug’s effect on T cells in vitro (44).
Our data clearly show that CypA is the predominant cyclospor-
ine receptor. Yet, cells lacking CypA are not completely resistant
to the drug, because high levels of cyclosporine can inhibit T cell
The Journal of Immunology
responses. Although likely to be nonphysiologic, these effects sug-
gest that another cyclophilin can mediate calcineurin inhibition
when high concentrations of cyclosporine are present. Previous
studies demonstrated that either CypA or CypB is capable of me-
diating cyclosporine inhibition of calcineurin when overexpressed
in a transformed human T cell line (30). However, because CypB
is sequestered in the secretory pathway, it is difficult to envision
how it might interact with calcineurin. One possible explanation is
that high concentrations of CypA disrupt the transport of CypB
into the secretory system so that more of this protein is accessible
for calcineurin binding. Support for this idea comes from studies
showing that cyclosporine alters the trafficking of CypB (45).
Previous studies have indicated that cyclosporine can inhibit
p38 MAPK activation via what appears to be a calcineurin-inde-
pendent pathway (36). In contrast, a more recent report shows that
FK506, but not cyclosporine, can inhibit p38 activation in mam-
malian cells subjected to osmotic stress (46). The fact that both
cyclosporine and FK506 have been observed to inhibit p38 sup-
ports the hypothesis that this effect involves calcineurin, despite
evidence to the contrary. In contrast, p38 MAPK activation ap-
pears independent of calcium in transformed B cells (53), which is
consistent with the possibility that both FK506 and cyclosporine
affect p38 activity by a mechanism that does not involve cal-
cineurin. Our experiments demonstrate that CypA mediates inhi-
bition of p38 MAPK by cyclosporine in T cells, but do not resolve
the question of how this comes about.
Although the molecular mechanism by which cyclosporine
causes immunosuppression has been studied in detail, the cell
types and factors affected by the drug have not been completely
defined. Our analysis shows that effective T cell responses coin-
cide with cyclosporine resistance, indicating that T cells are major
targets for the drug. Previous studies have established that cyclo-
sporine inhibits expression of multiple cyokines and cell surface
markers by T cells, most or all of which are regulated by the
NF-AT proteins (18). Mice deficient for both IL-2 and IL-4, or
IL-2 and IFN-␥, which are all cyclosporine-sensitive genes, are
still capable of rejecting allografts (47, 48). These findings, and the
demonstration that allograft rejection in these mutant animals can
be delayed by immunosuppressive agents, imply that cyclosporine
must affect additional factors to suppress graft rejection.
One such target might be CD154 (CD40L), the mutation of
which disrupts interactions between T cells and APCs and com-
promises responses to alloantigens (49, 50). In addition to showing
that APC function is critical for allograft rejection, this finding puts
forth the possibility that APC may also serve as targets for cyclo-
sporine. Our data showing that the ability of allogeneic dendritic
cells to drive CD4⫹ T cell proliferation is affected by cyclosporine
in a CypA-dependent manner support this idea, as do reports sug-
gesting that dendritic cell development and mature cell function
are inhibited by the drug (51, 52). The cyclosporine resistance of
Ppia⫺/⫺ mice provides a tool to further test this possibility and,
more generally, gain a better understanding of the molecular path-
ways that must be targeted to suppress allograft rejection. Ppia⫺/⫺
mice also provide a means to determine whether calcineurin inhi-
bition mediated by CypA is the cause of adverse side effects as-
sociated with cyclosporine therapy.
Acknowledgments
We thank Anjana Rao for supplying Ab, and members of the Luban lab for
helpful discussions.
Disclosures
The authors have no financial conflict of interest.
6037
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