A-Deficient Mice Are Resistant to Immunosuppression by Cyclosporine John Colgan, Mohammed Asmal, Bin Yu and Jeremy Luban This information is current as J Immunol 2005; 174:6030-6038; ; of October 2, 2021. doi: 10.4049/jimmunol.174.10.6030 http://www.jimmunol.org/content/174/10/6030

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 that is widely used to prevent organ . Known intracellular ligands for cyclosporine include the , a large family of phylogenetically conserved that potentially regulate folding in cells. Immunosuppression by cyclosporine is thought to result from the formation of a drug-cyclophilin complex that binds to and inhibits , a serine/threonine 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 encoding the prototypical cyclophilin protein cyclophilin A. TCR-induced proliferation and signal transduction by Ppia؊/؊ CD4؉ 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 Ppia؊/؊ mice to Downloaded from allogeneic challenge. Rag2؊/؊ mice reconstituted with Ppia؊/؊ splenocytes were also cyclosporine resistant, indicating that this -property is intrinsic to Ppia؊/؊ 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.

yclosporine is a cyclic decapeptide produced by the ing to conformational stability, or sterically blocking interactions

fungi inflatum. Originally identified in a between factors (7–10). http://www.jimmunol.org/ C screen for novel antibiotics, cyclosporine was shown by Cyclophilin A (CypA), the prototypical member of the cyclo- Borel et al. (1, 2) to be a potent and specific inhibitor of philin family, is a highly conserved protein that is maintained at responses to alloantigens. This discovery led to widespread clinical high levels in mammalian cells (11). Studies of budding yeast have use of cyclosporine as an immunosuppressant, revolutionizing or- shown that CypA localizes to both the and nucleus (12, gan transplantation in humans. 13). Consisting solely of the conserved PPIase domain, CypA Efforts to identify the cellular receptor for cyclosporine (3) led forms a globular, eight-stranded ␤-barrel with a solvent-exposed to discovery of the cyclophilins, one of three protein families hydrophobic pocket that is the for -containing known collectively as peptidyl-prolyl (PPIases)4 (4). peptides as well as the enzymatic (14). Cyclosporine by guest on October 2, 2021 Other PPIase families are the FK506-binding proteins () binds with subnanomolar affinity to CypA via contacts within the and the parvulins (4). PPIases catalyze the cis-trans interconver- hydrophobic pocket (15) and inhibits PPIase activity. However, sion of peptide bonds N-terminal to proline, an activity that has this effect is thought to be irrelevant for the immunosuppression. been extensively characterized in vitro using model peptides or Rather, the complex between cyclosporine and CypA creates a denatured proteins as substrates (5). Consistent with a global role composite surface that binds to and inhibits calcineurin (16, 17), a in the folding of nascent proteins in vivo, the distribution of PPI- serine-threonine phosphatase that is activated by . Sub- ases overlaps with the heat shock protein 70 , being strates for calcineurin include members of the NF-AT family of found in all eubacteria, a few archaebacteria, and all eukaryotes, transcription factors (18). Found in the cytoplasm of resting T (6). PPIases also may regulate the function of mature proteins by cells, the NF-ATs are dephosphorylated upon TCR ligation and catalyzing isomerization between alternative structures, contribut- 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 encoding cytokines and other proteins required for an immune response is inhibited. Departments of *Microbiology and †Medicine, Columbia University College of Phy- sicians and Surgeons, New York, NY 10032 Structural and genetic studies have identified amino acids within Received for publication December 15, 2004. Accepted for publication March CypA that are critical for the formation of a complex with cyclo- 4, 2005. sporine that binds to calcineurin (15, 19–24). In addition to CypA, The costs of publication of this article were defrayed in part by the payment of page the mouse and human genomes each encode 14 other cyclophilins charges. This article must therefore be hereby marked advertisement in accordance (25), most of which are distinguishable from CypA by the presence with 18 U.S.C. Section 1734 solely to indicate this fact. of terminal extensions bearing motifs for subcellular localization 1 This work was supported by grants from the Sandler Foundation for Asthma Re- or binding to nucleic acids or other proteins. Despite these differ- search, National Institutes of Health Grant RO1 AI 36199, and a Pilot and Feasibility Grant from the Columbia University Diabetes and Endocrinology Research Center. ences, the residues in CypA that mediate cyclosporine binding are 2 Current address: Roy J. and Lucille A. Carver College of Medicine, Department of highly conserved in other family members (Table I), indicating Internal Medicine, University of Iowa, 375 Newton Road, Iowa City, IA 52246. that multiple cyclophilins are potential targets for the drug. 3 Address correspondence and reprint requests to Dr. Jeremy Luban, Department of The genome of the budding yeast Saccharomyces cerevisiae en- Microbiology, Columbia University, 701 West 168th Street, New York, NY 10032. codes eight different cyclophilins (26), none of which are essential E-mail address: [email protected] (27). Several studies have demonstrated that CypA is the primary 4 Abbreviations used in this paper: PPIase, peptidyl-prolyl ; BMDDC, bone marrow-derived dendritic cell; CypA/B, cyclophilin A/B; FKBP, FK506-binding mediator of calcineurin inhibition by cyclosporine in this organism protein. (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. 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. Downloaded from would mediate the immunosuppressive effects of cyclosporine in T Type Culture Collection and were maintained, as recommended by the cells. In contrast, CypA and CypB can mediate calcineurin inhi- supplier. Bone marrow-derived dendritic cells (BMDDCs) were generated bition when overexpressed in transformed T cells (30), suggesting as described (32). that both of these proteins have a role in immunosuppression by Proliferation assays

cyclosporine in vivo. http://www.jimmunol.org/ Mice lacking Ppia, the gene encoding CypA, have augmented Cells (2 ϫ 105/well) were cultured in 96-well round-bottom plates. To Th2-type immune responses attributable to increases in the activity stimulate with plate-bound Ab, wells were incubated for3hat37°C with Ab in PBS and washed with PBS before cells were added. CD4ϩ T cell of the Itk, a tyrosine kinase that is inhibited by CypA via recog- proliferation in response to syngeneic or allogeneic cells was assessed by nition of a proline in its Src homology 2 domain (10). In this study, mixing irradiated (2500 rad) BMDDCs (1 ϫ 105/well) with purified CD4ϩ we present analysis of in vitro T cell function and in vivo immune T cells (0.1–3.0 ϫ 105/well) in 96-well round-bottom plates. Cyclosporine, responses that shows PpiaϪ/Ϫ mice are resistant to immunosup- methyl-Ile4-cyclosporine, and FK506 in oil emulsion were diluted to 1 pression by cyclosporine. 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 Materials and Methods dendritic cells) after plating, and [3H]thymidine incorporation was mea- by guest on October 2, 2021 Mice sured 12 h later using a Top Count (Packard Instrument).

Ϫ/Ϫ The 129S6/SvEv Ppia mice have been described (10) and deposited Analysis of IL-2 production by ELISA with The Jackson Laboratory Induced Mutant Resource as Stock 5320 Ϫ Ϫ (www.jax.org). The 129S6/SvEv Rag2 / mice were from Taconic Farms. Spleen cells (2 ϫ 105/well) were plated in 96-well round-bottom plates ϩ Ϫ Offspring of Ppia / mice obtained by backcrossing seven generations coated with 10 ␮g/ml anti-CD3 and containing the indicated amounts of into BALB/c (Taconic Farms) were used as sources of bone marrow for cyclosporine. After 48 h, supernatant from triplicate wells was pooled and generating allogeneic dendritic cells. Sex-matched, specific pathogen-free assayed for IL-2 by ELISA, according to a protocol from BD Biosciences. mice were maintained and used as approved by the Columbia University Institutional Animal Care and Usage Committee. RT-PCR Abs and reagents CD4ϩ T cells (1 ϫ 106/well) were added to wells in a 24-well plate coated with 10 ␮ Purified Ab to CD3 (145-2C11), CD28 (37.51) and IL-2 (JES6-1A12), g/ml anti-CD3 and containing the indicated amounts of cyclo- sporine. RNA isolation, cDNA synthesis, and real-time RT-PCR analysis FITC anti-CD4, H2Kd and goat anti-rabbit Ab, PE anti-CD69, H2Db and were performed, as described (10). Hypoxanthine phosphoribosyltrans- CD62L, allophycocyanin anti-CD8, biotinylated anti-CD25 and IL-2 Ј Ј (JES6-5H4), streptavidin-allophycocyanin, and alkaline phosphatase were ferase primers were 5 -GGACCTCTCGAAGTGTTGGATAC-3 (for- ward) and 5Ј-GCTCATCTTAGGCTTTGTATTTGGCT-3Ј (reverse). IL-2 from BD Biosciences. Affinity-purified anti-67.1 Ab specific for NFATc2/ Ј Ј Ј p/1 (31) was provided by A. Rao (Harvard University, Boston, MA). Rab- primers were 5 -CCTGAGCAGGATGGAGAATTACA-3 (forward), 5 - TCCAGAACATGCCGCAGAG-3Ј (reverse), and 5Ј-CGCGCAC bit anti-phospho-p38 MAPK Ab was from Cell Signaling Technology. Ј HRP-conjugated goat anti-rabbit Ab was from Promega. Cyclosporine was CCAAGCAGGCCACAGAATTGAAAGATTGCGCG-3 (beacon). from Bedford, methyl-Ile4-cyclosporine from Pharmaceuticals, and FK506 from Fujisawa Pharmaceutical. PMA and ionomycin were from Flow cytometry Calbiochem. Anti-CD4 beads and Detachabead were from Dynal Biotech. Anti-CD8 microbeads were from Miltenyi Biotec. SYBR green was from Staining and wash buffer was PBS containing 3% FBS and 0.1% sodium Molecular Probes. IL-2-specific molecular beacon was from Midland Cer- azide. Cells were mixed with Ab at concentrations recommended by the tified Reagent. manufacturer, incubated for 20 min on ice, and then washed. Analysis was performed using a FACSCalibur flow cytometer and CellQuest software Cell preparation (BD Biosciences). Spleen and lymph node cell suspensions were depleted of RBC using RBC Surface expression of CD25 and CD69 lysis buffer (Sigma-Aldrich). CD4ϩ T cells were purified from pooled lymph node and spleen cells using anti-CD4 beads and eluted with De- CD4ϩ T cells (1 ϫ 106/well) were added to wells in 24-well plates coated tachabead; purified cells were typically Ͼ98% CD4ϩ. Cells were cultured with 10 ␮g/ml anti-CD3 and containing the indicated amounts of cyclo- in RPMI 1640 supplemented with 10% FCS, 0.1 ␮M 2-ME, 100 IU/ml sporine or FK506. After 20 h, cells were stained with FITC anti-CD4, PE penicillin, 100 ␮g/ml streptomycin, and 2 mM glucose. P815 mastocytoma anti-CD69, biotinylated anti-CD25, and streptavidin-allophycocyanin for cells (TIB-64) and EL4 lymphoma cells (TIB-39) were from American flow cytometric analysis. 6032 CypA MEDIATES CYCLOSPORINE SENSITIVITY p38 MAPK activation CD4ϩ T cells (1 ϫ 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 ϫ 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 were visualized using HRP-conjugated anti-rabbit Ab and a chemilumi- Downloaded from 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. http://www.jimmunol.org/ Assessment of tumor cell clearance and CD62L down-regulation Mice were injected i.p. with 1 ϫ 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 Ϫ/Ϫ stained with FITC anti-CD4, PE anti-CD62L, and allophycocyanin anti- FIGURE 1. Proliferation by stimulated Ppia cells is cyclosporine ϩ ϩ ϩ Ϫ Ϫ Ϫ CD8, and analyzed by flow cytometry. resistant. A, Proliferative responses of Ppia / , Ppia / , and Ppia / splenocytes. Wells coated with 10 ␮g/ml anti-CD3 were used to stimulate by guest on October 2, 2021 Assay for cytotoxic T cell activity cells in the presence of the indicated concentrations of cyclosporine (CsA). ϩ/ϩ Ϫ/Ϫ ϩ Mice were injected i.p. with 1 ϫ 107 P815 cells and sacrificed 10 days B–D, Proliferative responses of purified Ppia and Ppia CD4 T later. CD8ϩ cells were isolated from spleen cells using anti-CD8 mi- cells. Wells coated with 10 ␮g/ml anti-CD3 were used to stimulate cells in crobeads. Target cells were labeled by mixing 2 ϫ 107 cells in 0.2 ml of the presence of the indicated concentrations of cyclosporine (CsA) (B), or medium with 0.2 ml (200 ␮Ci) of 51Cr (PerkinElmer Life Science Prod- methyl-Ile4-cyclosporine (Me-Ile4-CsA) (C), or FK506 (D). ucts), followed by incubation at 37°C for 1 h. Unbound 51Cr was removed by washing three times with medium. CD8ϩ cells were mixed with 1 ϫ 104 51 Cr-labeled P815 or control EL-4 cells (final culture vol 0.2 ml) in flat- ϩ bottom 96-well plates. Cells were spun down by brief centrifugation, and not shown). Analysis of purified CD4 T cells stimulated with Ϫ/Ϫ plates were incubated for3hat37°C. Supernatant (20 ␮l) was removed anti-CD3 showed that the IC50 of cyclosporine for Ppia cells and analyzed for 51Cr using a Top Count (Packard Instrument). Percent was also at least 10-fold greater than that of Ppiaϩ/ϩ cells (Fig. specific lysis was calculated as follows: (experimental release Ϫ sponta- 1B), demonstrating that cyclosporine resistance was intrinsic to T neous release)/(maximum release Ϫ spontaneous release). Spontaneous re- ϩ lease was measured by incubating target cells alone in medium. Maximum cells. Similar results were obtained when purified CD4 T cells release was measured by lysing target cells with 2% SDS. were stimulated with anti-CD3 in combination with anti-CD28 (data not shown). Reconstitution by adoptive transfer As seen with splenocytes, proliferation by PpiaϪ/Ϫ CD4ϩ T Splenocytes (1 ϫ 107/mouse) were injected i.v. into Rag2Ϫ/Ϫ mice. The cells was not completely inhibited by any dose of cyclosporine next day, both cyclosporine treatment and P815 challenge were initiated. tested, but was diminished at drug concentrations of 250 nM or greater. One explanation for this effect is that another cyclophilin Results Ϫ/Ϫ can mediate calcineurin inhibition, but only at high doses of drug. Proliferation by Ppia cells is cyclosporine resistant Alternatively, some calcineurin-independent pathway might be af- To test whether PpiaϪ/Ϫ cells have altered sensitivity to cyclo- fected under these conditions. To distinguish between these pos- sporine, the effects of different doses of drug on splenocyte pro- sibilities, CD4ϩ T cell proliferation in the presence of methyl-Ile4- liferation induced by plate-bound anti-CD3 were quantified (Fig. cyclosporine was analyzed (Fig. 1C). This cyclosporine derivative 1A). Cyclosporine inhibited proliferation of Ppiaϩ/ϩ or Ppiaϩ/Ϫ has higher affinity for CypA than the parent compound, but com- ϳ 4 splenocytes to a similar degree, with an IC50 of 25 nM and pletely lacks immunosuppressive activity (33). Methyl-Ile -cyclo- ϳ ϩ/ϩ Ϫ/Ϫ complete inhibition at 100 nM. In striking contrast, the IC50 of sporine had no effect on the proliferation of Ppia or Ppia PpiaϪ/Ϫ splenocytes was at least 10-fold higher, and complete CD4ϩ T cells at doses equivalent to those at which cyclosporine inhibition was not observed using drug doses up to 2.5 ␮M. Sim- caused inhibition. This result indicates that PpiaϪ/Ϫ cells are par- ilar cyclosporine resistance was seen when PpiaϪ/Ϫ splenocytes tially sensitive to high-dose cyclosporine due to calcineurin inhi- were stimulated with either Con A or PMA and ionomycin (data bition mediated by another cyclophilin family member. The Journal of Immunology 6033

FK506 is another immunosuppressive compound used to pre- PpiaϪ/Ϫ cells are resistant to inhibition of by vent organ transplant rejection. Structurally unrelated to cyclospor- cyclosporine ine, FK506 also binds to and inhibits calcineurin, but only as part One well-characterized response to calcineurin activation is induc- of a complex with members of the FKBP family of PPIases (34). tion of IL-2 expression (35). Ppiaϩ/ϩ and PpiaϪ/Ϫ splenocytes ϩ/ϩ Ϫ/Ϫ ϩ Ppia and Ppia CD4 T cells stimulated with anti-CD3 had were stimulated with anti-CD3, and accumulation of IL-2 in cul- similar responses to different doses of FK506 (Fig. 1D). These ture supernatant was determined (Fig. 3A). When no cyclosporine Ϫ/Ϫ results show that proliferation by Ppia cells is dependent on was added, Ppiaϩ/ϩ and PpiaϪ/Ϫ cells produced similar amounts calcineurin activity, indicating that cyclosporine resistance is due of IL-2. At low doses of cyclosporine (25 nM), IL-2 production by to abrogation of calcineurin inhibition rather than gross dysregu- Ppiaϩ/ϩ cells was reduced Ͼ10-fold, and was undetectable at lation of TCR-induced signaling pathways. higher concentrations of drug. In contrast, IL-2 production by Proliferation by Ppiaϩ/ϩ and PpiaϪ/Ϫ CD4ϩ T cells in response to syngeneic and allogeneic BMDDCs was also assessed. The re- sponses of PpiaϪ/Ϫ cells to either syngeneic or allogeneic BMD- DCs were slightly lower than those of Ppiaϩ/ϩ cells (Fig. 2A). To measure cyclosporine responses, CD4ϩ T cells were mixed with either Ppiaϩ/ϩ or PpiaϪ/Ϫ allogeneic BMDDCs in the presence of different concentrations of drug (Fig. 2B). Ppiaϩ/ϩ CD4ϩ T cells were inhibited to the same extent by cyclosporine regardless of the Ϫ/Ϫ ϩ genotype of the stimulator cell population. Ppia CD4 T cells Downloaded from showed resistance to cyclosporine when challenged with Ppiaϩ/ϩ allogeneic BMDDCs, and this was increased when PpiaϪ/Ϫ cells were used as stimulators. These results suggest that cyclosporine, via interactions with CypA, can inhibit the function of APCs. http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 3. Cyclosporine-resistant calcineurin and MAPK signaling in PpiaϪ/Ϫ 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 CD4ϩ T cells. Wells coated with 10 ␮g/ml anti-CD3 were used to stimulate purified CD4ϩ 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- FIGURE 2. The responses of PpiaϪ/Ϫ CD4ϩ T cells to allogeneic den- face CD25 and CD69 expression on CD4ϩ T cells. Wells coated with 10 dritic cells are cyclosporine resistant. A, Proliferative responses of Ppiaϩ/ϩ ␮g/ml anti-CD3 were used to stimulate cells in the presence of the indi- and PpiaϪ/Ϫ CD4ϩ T cells mixed with irradiated, syngeneic or allogeneic cated concentrations of drug. After 16 h, cells were harvested and analyzed Ppiaϩ/ϩ BMDDCs. B, Proliferative responses of Ppiaϩ/ϩ and PpiaϪ/Ϫ by flow cytometry. D, Immunoblot analysis of NFATp/1c/c2. Lysates were CD4ϩ T cells mixed with irradiated, allogeneic Ppiaϩ/ϩ or PpiaϪ/Ϫ BM- prepared from lymph node cells preincubated with the indicated concen- DDCs (DCs) in the presence of the indicated concentrations of cyclospor- trations of cyclosporine (CsA) and then stimulated for 5 min with iono- ine (CsA). Relative proliferation corresponds to [3H]thymidine incorpora- mycin. E, p38 MAPK phosphorylation in CD4ϩ T cells. Cells were pre- tion observed relative to that by the control (an identical mixture of CD4ϩ incubated with or without 40 nM cyclosporine (CsA), stimulated with T cells and DCs lacking cyclosporine). PMA and ionomycin for 20 min, and then analyzed by flow cytometry. 6034 CypA MEDIATES CYCLOSPORINE SENSITIVITY

PpiaϪ/Ϫ cells was modestly decreased by the presence of 25 nM exudate cells (Fig. 4A). In the absence of cyclosporine treatment, cyclosporine, and remained detectable at drug concentrations up to Ppiaϩ/ϩ and PpiaϪ/Ϫ mice eradicated the tumor cells with similar 750 nM. These results were confirmed by RT-PCR analysis of IL-2 kinetics (Fig. 4B). Ppiaϩ/ϩ mice treated with 30 mg/kg/day cy- mRNA (Fig. 3B). In the absence of drug, steady-state levels of closporine were unable to clear the tumor cells, which expanded IL-2 mRNA were similar in Ppiaϩ/ϩ and PpiaϪ/Ϫ cells. Addition and completely overtook the endogenous cell population by day 10 of 25 nM cyclosporine greatly reduced IL-2 mRNA in Ppiaϩ/ϩ after injection (Fig. 4, C and D). In striking contrast, PpiaϪ/Ϫ mice cells, but had only a slight effect on IL-2 mRNA levels in PpiaϪ/Ϫ receiving the same dose of cyclosporine cleared the tumor cells as cells. Higher concentrations of cyclosporine caused gradual reduc- efficiently as animals given PBS instead of drug. tions in the amounts of IL-2 mRNA detected in PpiaϪ/Ϫ cells, but Ϫ/Ϫ the levels seen were still well above those in Ppiaϩ/ϩ cells at drug Cyclosporine resistance in Ppia mice correlates with concentrations up to 250 nM. preservation of T cell responses Two other genes that are regulated by calcineurin encode the Eradication of P815 tumor cells as an allogeneic challenge requires IL-2R ␣-chain (CD25) and the very early activation Ag CD69 Ag-specific CD8ϩ T cell responses (37). Robust, specific killing ϩ (18). Cell surface expression of these proteins on CD4 T cells by CD8ϩ T cells from Ppiaϩ/ϩ and PpiaϪ/Ϫ mice primed with stimulated with anti-CD3 was therefore assessed (Fig. 3C). When P815 cells was observed (Fig. 5A). The cytotoxic activity of cells ϩ ϩ Ϫ Ϫ no cyclosporine was present, Ppia / and Ppia / cells expressed from PpiaϪ/Ϫ mice was consistently lower activity than those from similar levels of CD25 and CD69. Addition of 25 nM cyclosporine Ppiaϩ/ϩ mice, suggesting that CD8ϩ T cell responses in PpiaϪ/Ϫ was sufficient to completely block expression of both proteins on mice are slightly impaired. CD8ϩ T cells from Ppiaϩ/ϩ mice ϩ ϩ Ppia / cells, but caused only a slight decrease in the percentage treated with cyclosporine completely lacked killing activity, but of PpiaϪ/Ϫ CD4ϩ T cells expressing high level CD25 and CD69. Downloaded from Increasing doses of drug further reduced the proportion of PpiaϪ/Ϫ 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 CD4ϩ T cells (Fig. 1D) completely blocked expression of CD25 and CD69 on both Ppiaϩ/ϩ and PpiaϪ/Ϫ cells. http://www.jimmunol.org/ These results show that the expression of calcineurin-regulated target genes by PpiaϪ/Ϫ cells is cyclosporine resistant.

Calcineurin and MAPK pathways in PpiaϪ/Ϫ 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 by guest on October 2, 2021 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 Ppiaϩ/ϩ and PpiaϪ/Ϫ cells (Fig. 3D). Pretreatment with 25 nM cyclosporine completely abolished NFATc2/p/1 dephosphorylation in Ppiaϩ/ϩ cells treated with ionomycin. In contrast, dephosphorylation of NFATc2/p/1 could be detected in ionomycin-stimulated PpiaϪ/Ϫ cells pretreated with concentrations of cyclosporine as high as 750 nM. Thus, based on direct analysis of a physiologic substrate, cal- cineurin activity in PpiaϪ/Ϫ 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 Ppiaϩ/ϩ and PpiaϪ/Ϫ CD4ϩ T cells. Addition of 40 nM cyclosporine completely inhibited ϩ/ϩ phosphorylation of p38 in Ppia cells, but had no effect on the FIGURE 4. Allogeneic tumor cell clearance in PpiaϪ/Ϫ mice is not sup- Ϫ Ϫ 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 PpiaϪ/Ϫ cells (data not shown), suggesting that another cyclophi- (H2d) MHC I in peritoneal exudate cells recovered on the indicated days 7 lin can substitute for CypA, but only at high doses of drug. These after i.p. injection of 1 ϫ 10 tumor cells into wild-type mice. B, Normal Ϫ/Ϫ results show that CypA mediates inhibition of p38 MAPK activa- allotumor cell clearance in Ppia mice. The percentage of peritoneal tion by cyclosporine. exudate cells expressing P815 tumor cell MHC I was quantified by flow cytometric analysis on the indicated days after tumor cell injection, as PpiaϪ/Ϫ 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 ϫ 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- b d vivo, H2 recipient mice were challenged with allogeneic H2 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 6035

FIGURE 5. Allotumor cell-specific T cell responses in PpiaϪ/Ϫ mice are resistant to cyclosporine. A, Cyto- toxic activity from P815 tumor cell-primed mice. Kill- ing by CD8ϩ T cells purified from mice primed with P815 tumor cells 10 days earlier was assayed using 51Cr-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 CD8ϩ T cells purified from treated mice was assayed using 51Cr-labeled P815 ϩ ϩ

cells as target. C, CD4 and CD8 T cell activation in Downloaded from 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). http://www.jimmunol.org/

cells from PpiaϪ/Ϫ mice given the same dose of drug were po- not shown). In contrast, cyclosporine doses of at least 70 mg/kg/ by guest on October 2, 2021 tently and specifically cytotoxic (Fig. 5B). Relative to PpiaϪ/Ϫ day were required to inhibit tumor cell clearance in PpiaϪ/Ϫ mice, mice given PBS, the specific killing activity generated in cyclo- which were able to tolerate drug doses up to 90 mg/kg/day and sporine-treated PpiaϪ/Ϫ mice was greater, indicating that cyclo- showed no overt signs of tumor growth in the liver. sporine can paradoxically augment CD8ϩ T cell responses in these The killing activity of CD8ϩ T cells from PpiaϪ/Ϫ mice treated animals. with different doses of cyclosporine was also analyzed (Fig. 6B). The cell surface marker CD62L is down-regulated when naive T Cells from mice given 50 mg/kg/day cyclosporine were potently cells are stimulated (38). Low level expression of CD62L is main- cytotoxic, while those from animals treated with higher doses of tained on effector and memory T cell populations and correlates drug had reduced activity. Still, the killing activity by CD8ϩ T with cytolytic activity by CD8ϩ T cells (39). Following injection cells from PpiaϪ/Ϫ mice receiving the 90 mg/kg/day drug was of tumor cells, CD62L expression was decreased on CD4ϩ cells greater than that by cells from Ppiaϩ/ϩ mice given 3 times less and CD8ϩ cells from the spleens of Ppiaϩ/ϩ and PpiaϪ/Ϫ mice to drug, which had essentially no activity. a similar extent (Fig. 5C). Down-regulation of CD62L on CD4ϩ T Ϫ/Ϫ cells most likely reflects a specific response to the injected cells, Cyclosporine resistance is transferred with Ppia splenocytes because Th cell can accelerate the clearance of P815 cells (37, 40). Two mechanisms could explain the cyclosporine resistance of When 30 mg/kg/day cyclosporine was administered during the tu- PpiaϪ/Ϫ mice. Cyclosporine might be unable to inhibit immune mor cell challenge, CD62L expression remained high on either responses because the necessary receptor is not expressed in target ϩ ϩ ϩ ϩ CD4 or CD8 cells from Ppia / mice, but was low on both cell cells. Alternatively, cyclosporine resistance might be secondary to Ϫ Ϫ types from Ppia / mice. These results indicate that T cell re- abnormal of the drug due to the absence of CypA in Ϫ Ϫ sponses to allograft challenge in Ppia / mice are resistant to other nonimmune tissues. To distinguish these possibilities, the cyclosporine. effects of cyclosporine on Rag2Ϫ/Ϫ mice reconstituted with Ppiaϩ/ϩ or PpiaϪ/Ϫ splenocytes were evaluated. Ppiaϩ/ϩ and Assessment of the magnitude of cyclosporine resistance in Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ Ppia splenocytes reconstituted the of Rag2 Ppia mice recipients with similar efficiencies (Fig. 7A). In the absence of To roughly determine the difference in cyclosporine sensitivity be- cyclosporine treatment, mice reconstituted with either Ppiaϩ/ϩ or tween Ppiaϩ/ϩ and PpiaϪ/Ϫ mice, tumor cell clearance in animals PpiaϪ/Ϫ cells eradicated the tumor cells with similar efficiency treated with different doses of cyclosporine was assessed (Fig. 6A). (Fig. 7B). When mice were treated with cyclosporine, the tumor A dose of 30 mg/kg/day was sufficient to block tumor cell clear- cells expanded in mice reconstituted with Ppiaϩ/ϩ cells, but were ance in Ppiaϩ/ϩ mice, while administration of 70 mg/kg/day com- cleared in those that received PpiaϪ/Ϫ cells. These results dem- bined with the tumor cell challenge led to premature sacrifice, with onstrate that the lack of CypA expression in immune cells results evidence that the injected tumor cells had infiltrated the liver (data in cyclosporine resistance. 6036 CypA MEDIATES CYCLOSPORINE SENSITIVITY Downloaded from

FIGURE 7. Cyclosporine resistance is transferred with PpiaϪ/Ϫ spleno- cytes. A, Ppiaϩ/ϩ and PpiaϪ/Ϫ splenocytes reconstitute Rag2Ϫ/Ϫ mice with similar efficiencies. Flow cytometric analysis of splenocytes from Rag2Ϫ/Ϫ http://www.jimmunol.org/ mice injected i.v. with either PBS (None) or 3 ϫ 107 splenocytes 10 days earlier. B, Surface expression of host and allogeneic tumor cell MHC I on peritoneal exudate cells from reconstituted Rag2Ϫ/Ϫ mice. Animals were primed with tumor cells on day 0 and given daily injections of PBS or 30 FIGURE 6. Assessment of the magnitude of cyclosporine resistance in mg/kg cyclosporine (CsA) for 10 days. PpiaϪ/Ϫ mice. A, Allotumor cell growth in Ppiaϩ/ϩ and PpiaϪ/Ϫ mice treated with different doses of cyclosporine (CsA). The absolute number of tumor cells recovered 10 days after i.p. injection of 1 ϫ 107 cells was derived from flow cytometric analysis by quantifying the percentage of studies, we cannot rule out the possibility that ligands other than peritoneal exudate cells expressing allogeneic MHC I (see Fig. 4, C and D). by guest on October 2, 2021 †, Signifies that tumor cell number was not determined because the mice CypA contribute to the immunosuppressive effects of cyclospor- were sacrificed prematurely. B, Cytotoxic activity elicited from PpiaϪ/Ϫ ine. Nevertheless, our results clearly demonstrate that CypA is mice treated with different doses of cyclosporine. Mice were primed with required for immunosuppression by cyclosporine at physiologi- tumor cells on day 0 and given the indicated amounts of cyclosporine cally relevant concentrations of the drug. ϩ (CsA) for 10 days. Killing by CD8 cells from treated mice was assayed It is widely accepted that a complex formed between cyclospor- 51 using Cr-labeled P815 cells as target. 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 Discussion strong support for this model (22, 28, 29, 42). Our data show a Our results demonstrate that, despite the fact that mammalian cells strong correlation between the maintenance of calcineurin activity express multiple cyclophilins with demonstrated or predicted af- and cyclosporine resistance in lymphocytes. finity for cyclosporine, CypA is the predominant, if not the sole, CypA, B, and C all form complexes with cyclosporine that in- mediator of the drug’s immunosuppressive effects. Although cells hibit calcineurin activity in vitro (30, 43). Moreover, CypB is sig- and mice lacking Ppia remain partially sensitive to high doses of nificantly more efficient than CypA at mediating calcineurin inhi- cyclosporine, the concentrations of drug required for these effects bition in vitro (21). The unique importance of CypA for are well above that required to bring about immunosuppression in calcineurin inhibition demonstrated in this study must therefore the wild-type counterparts. Our findings also confirm that the mo- reflect factors other than affinity for cyclosporine or calcineurin. lecular basis for immunosuppression by cyclosporine is the for- CypA is found at high concentrations in cells, and a pool of CypA mation of a drug-CypA complex that inhibits calcineurin. must be accessible for interaction with both cyclosporine and cal- Determination of the crystal and solution structure of CypA cineurin. CypB is localized to membrane components of the se- bound to cyclosporine has identified 15 aa within CypA that me- cretory pathway and may be sequestered due to spatial distribution diate contacts with the drug (15, 20). Alignment of the 15 cyclo- or competing interactions with other cellular factors. An analogous philins encoded in the mouse genome (Table I) reveals that CypB, situation seems to apply to the FKBP family of PPIases and the C, and F share identity with CypA at all of these residues, while 4 calcineurin inhibitor FK506. Although at least three FKBPs can other cyclophilins, CypD, E, Ppil1, and Riken clone J08, contain form complexes with FK506 that inhibit calcineurin, studies using single substitutions. CypA, B, and C all bind with high affinity to mutant mice have shown that FKBP12 is the sole mediator of the cyclosporine in vitro (30), whereas secreted CypB augments cel- drug’s effect on T cells in vitro (44). lular uptake of cyclosporine via interaction with cell surface bind- Our data clearly show that CypA is the predominant cyclospor- ing sites (41). These observations argue that multiple cyclophilins ine receptor. Yet, cells lacking CypA are not completely resistant have the potential to interact with cyclosporine in cells. From our to the drug, because high levels of cyclosporine can inhibit T cell The Journal of Immunology 6037 responses. Although likely to be nonphysiologic, these effects sug- References gest that another cyclophilin can mediate calcineurin inhibition 1. Borel, J. F., C. Feurer, H. U. Gubler, and H. Stahelin. 1976. Biologic effects of when high concentrations of cyclosporine are present. Previous cyclosporin A: a new anti-lymphocyte agent. 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