Immunosuppressive Drug Rapamycin of September 25, 2021

Inhibition of Cell Cycle Progression by a Synthetic Peptide Corresponding to Residues 65− 79 of an HLA Class II Sequence: Functional Similarities but Mechanistic Differences with the This information is current as Immunosuppressive Drug Rapamycin of September 25, 2021. Michelle L. Boytim, Shu-Chen Lyu, Ron Jung, Alan M. Krensky and Carol Clayberger J Immunol 1998; 160:2215-2222; ; http://www.jimmunol.org/content/160/5/2215 Downloaded from

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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 © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Inhibition of Cell Cycle Progression by a Synthetic Peptide Corresponding to Residues 65–79 of an HLA Class II Sequence: Functional Similarities but Mechanistic Differences with the Immunosuppressive Drug Rapamycin1

Michelle L. Boytim,* Shu-Chen Lyu,* Ron Jung,* Alan M. Krensky,† and Carol Clayberger2*† ␣ ␣ A synthetic peptide corresponding to a region of the 1 -helix of DQA03011 (DQ 65–79) inhibits the proliferation of human PBL and T cells in an allele-nonspeciﬁc manner. It blocks proliferation stimulated by anti-CD3 mAb, PHA-P, and alloantigen, but not by PMA and ionomycin. Substitution of each amino acid with serine shows that residues 66, 68, 69, 71–73, and 75–79 are critical Downloaded from for function. Inhibition of proliferation is long lasting and is not reversible with exogenous IL-2. The peptide can be added 24 to 48 h after stimulation and still block proliferation. The DQ 65–79 peptide does not affect expression of IL-2 or IL-2R; however,

IL-2-stimulated proliferation is inhibited. Cell cycle progression is blocked at the G1/S transition, and the activity of cdk2 (cyclin- dependent kinase 2) kinase is impaired by the continued presence of p27. Although these results suggest a mechanism similar to that of rapamycin, the peptide inhibition is not reversed with FK-506, which indicates a distinct mechanism. The Journal of Immunology, 1998, 160: 2215–2222. http://www.jimmunol.org/

cells are crucial for the defense of the host against infec- In addition to their natural role in host defense and T cell edu- tion, and the key to T cell activation is recognition of cation, MHC molecules play a pivotal role in the process of graft T nonself. Processed peptides from both internal and exter- rejection. Both direct recognition of allogeneic MHC and indirect nal sources are bound to MHC molecules and presented to T cells recognition through presentation of allogeneic MHC molecules as by APC (1). Other regions of MHC molecules interact with T cell peptides in host MHC molecules can stimulate rejection (11, 12). coreceptors CD4 or CD8 (2–6). Not only are MHC molecules Conversely, MHC molecules have also been implicated in the pre- vention of graft rejection. Circumstantial evidence comes from critical for inducing immune responses, but they are also important by guest on September 25, 2021 in controlling them. Thymocytes are deleted if they cannot recog- studies in which concurrent donor-specific transfusions prolong nize self MHC molecules or if they strongly recognize self pep- graft survival in humans, and studies in rodents in which injection tides in the context of self MHC molecules. In the periphery, rec- of spleen cells or lymphocytes from the donor strain induce toler- ognition of self peptides presented by self MHC molecules can ance to the graft (13, 14). MHC molecules themselves are shown lead to deletion or anergy of mature T cells (7, 8). Recently, it has to induce unresponsiveness by injection of cells transfected with been demonstrated that lysis of target cells by NK cells can be class I or class II donor MHC molecules into graft recipients (14). inhibited by the specific recognition of MHC class I molecules on High doses of purified class I molecules induce tolerance in the rat the target cell by killer cell-inhibitory receptors (KIR)3 on the NK allograft model; however, this is not the case for purified class II cell (9, 10). Thus, MHC molecules are important for both initiation molecules (15, 16). In contrast, peptides corresponding to the ␤ and regulation of normal immune responses. -chain of donor rat class II MHC could initiate unresponsiveness to a transplant via oral or intrathymic administration in the recipient (17, 18). Additionally, synthetic peptides derived from nonpolymorphic regions of MHC class II have inhibitory effects (19). *Department of Cardiothoracic Surgery and †Division of Immunology and Trans- During the past decade, we have characterized the effects of plantation Biology/Department of Pediatrics, Stanford University, Stanford, CA synthetic peptides corresponding to HLA class I sequences on im- 94305 mune responses. A peptide corresponding to residues 222–235 of Received for publication August 8, 1997. Accepted for publication November the ␣ domain, the CD8 binding domain of the HLA class I mol- 17, 1997. 3 ecule, blocks the differentiation of pre-CTL into mature effector 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 cells in an allele-unrestricted manner (5). We reported that pep- with 18 U.S.C. Section 1734 solely to indicate this fact. tides corresponding to residues 98–113 or 56–69 of HLA-A2 1 This work was supported by National Institutes of Health Grants AI 35125, DK block CTL responses in an allele-specific manner (20, 21). Finally, 35008, AI 41520, and HD 34214 and by funds from the Ralph and Marion Falk a peptide derived from residues 75–84 of the ␣ ␣-helix of HLA- Memorial Research Trust. A.M.K. is the Shelagh Galligan Professor of Pediatrics at 1 Stanford University and a Burroughs Wellcome Scholar in Experimental Therapeu- B2702 inhibits lysis of target cells by CTL in an allele-nonspecific tics. M.L.B. is a Howard Hughes Medical Institute Predoctoral Fellow. manner (22). This peptide prevents graft rejection in rodent models 2 Address correspondence and reprint requests to Dr. Carol Clayberger, Department (23–25). of Cardiothoracic Surgery, Stanford University, Stanford, CA 94305-5407. E-mail We sought to determine whether synthetic peptides correspond- address: [email protected] ing to conserved regions of class II molecules could have similar 3 Abbreviations used in this paper: KIR, killer cell-inhibitory receptor; cdk, cyclin- dependent kinase; FKBP, FK-506-binding protein; PHA-P, phytohemagglutinin-P; immunoregulatory effects. After testing a panel of peptides, we TOR, target of rapamycin. found that a peptide corresponding to residues 56–80 of

DQA03011 inhibits a variety of immune responses, and this ac- Kinase assay tivity is localized to residues 65–79. In this study, we characterize Assays were performed as described (30) with the following modifications. the inhibitory effects of this peptide and describe its mechanism of Preactivated T cells (5 ϫ 106) were stimulated with 100 U/ml rIL-2. At action. 24 h, the cells were washed in PBS and lysed in lysis buffer (0.5% Triton X-100, 1 mM DTT, 10 mM ␤-glycerophosphate, 20 mM sodium fluoride, Materials and Methods 0.2 mM sodium orthovanadate, 10 ␮g/ml leupeptin, and 5 ␮g/ml aproti- Peptides, Abs, and reagents nin). Lysates were incubated on ice for 10 min, then spun down for 3 min in a microcentrifuge. The amount of protein in each supernatant was mea- Peptides were synthesized using Fmoc chemistry on an Applied Biosys- sured using the Bio-Rad protein assay (Bio-Rad, Hercules, CA). Anti-cy- tems (Foster City, CA) peptide synthesizer, and were purified to greater clinEAb(2␮g) was added to supernatants containing equivalent amounts than 95% purity by HPLC (26), and the composition was confirmed by of protein, and the samples were rocked at 4°C for 1 h. Protein A/G beads mass spectrometry. Stock solutions of peptide were made by dissolving (15 ␮l) (Santa Cruz Biotechnology) were added, and the samples were peptide in DMSO (25 mg/ml). Cyclosporin A was a gift from Merck Re- rocked for 45 min at 4°C. The beads were washed twice in lysis buffer and search Laboratories (Rahway, NJ), and rapamycin was a gift from Wyeth twice in kinase buffer (20 mM HEPES, pH 7, 10 mM ␤-glycerophosphate, Ayerst (Philadelphia, PA). rIL-2 was obtained from Sigma Chemical Co. 5 mM magnesium chloride, 10 ␮g/ml leupeptin, and 1 mM DTT). Kinase (St. Louis, MO). Abs specific for cyclin E, p21, p27, and cdk2 were pur- activity was assayed by resuspending the beads in 40 ␮l of kinase buffer chased from Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti-mouse supplemented with 8 ␮Ci [␥-32P]ATP, 4 ␮M ATP, and 20.6 mg/ml histone horseradish peroxidase and goat anti-rabbit horseradish peroxidase were H1 type III-S. Samples were incubated at 37°C for 15 min, and the reaction obtained from Caltag (South San Francisco, CA). Abs to CD2, CD3, CD4, was stopped by adding 40 ␮l of ice-cold 20 mM EDTA, pH 8. Triplicate CD8, CD28, CD45RO, CD69, IL-2R␣,␤,␥, class I, and class II were pur- aliquots were spotted onto phosphocellulose filters (Life Technologies, chased from PharMingen (San Diego, CA). Unless specified, all other re- Grand Island, NY). The filters were soaked briefly in 1% H3PO4/10 mM 32 Downloaded from agents were obtained from Sigma Chemical Co. Na4P2O7 and washed three times for 15 min in 1% H3PO4. P incorporation was determined by liquid scintillation counting. PBL and T cell isolation, cell culture Immunoprecipitations and Western blots PBL were obtained from volunteer donors or buffy coats from Stanford Blood Center and were isolated via Ficoll-Hypaque density centrifugation. Preactivated cells (1–2 ϫ 107) were treated with peptide and 100 U/ml IL-2 T cells were isolated following adherence of macrophages on plastic petri for 24 h, and immunoprecipitations were performed as described (28). For dishes and removal of B cells on nylon wool columns (27). T cell purities Western blots, 2.5 ϫ 106 preactivated T cells were incubated with peptide were greater than 90% by staining with anti-CD3 mAb. Cells were cultured (40 ␮M) and 100 U/ml rIL-2 and prepared as described (31). Proteins were http://www.jimmunol.org/ in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin/strepto- separated on either 7.5% or 12% SDS-PAGE gels. The proteins were trans- mycin, 2 mM L-glutamine, and 1 mM HEPES. For preactivated cells, pu- ferred electrophoretically to supported nitrocellulose (Amersham, Arling- rified T cells were cultured with 0.8 ␮g/ml PHA-P for 72 h before stim- ton Heights, IL), and the membrane was blocked with a solution of 5% ulation with rIL-2 (100 U/ml), as described (28). nonfat dry milk in 5 mM Tris-buffered saline, pH 7.5, with 0.05% Tween- Proliferation assay 20. Blots were probed with the indicated Abs at 1:1000. This step was followed by a secondary Ab conjugated to horseradish peroxidase at Proliferation assays were performed essentially as described (27). For each 1:3000, and proteins were visualized by chemiluminescence. stimulation protocol/cell type, the time for the optimal proliferative response was determined empirically. For human PBL, 96-well plates were Results

␮ ␮ by guest on September 25, 2021 coated with 50 l of anti-CD3 mAb (5 g/ml) in PBS for1hat37°C. PBL Synthetic peptides corresponding to HLA class II sequences were added at 4 ϫ 105 cells/well and peptide at a concentration of 40 ␮M. The cells were pulsed with [3H]TdR (1 ␮Ci/well) (DuPont NEN, Boston, inhibit proliferation of PBL and T cells 3 MA) and were harvested with a PhD cell harvester at 72 h. [ H]TdR in- HLA class II molecules are heterodimers composed of an ␣- and corporation was determined by liquid scintillation counting. Purified T ␤ ␤ ␣ cells were cultured at 2 ϫ 105 cells/well, pulsed at 72 h, and harvested at a -chain. While the -chain is highly polymorphic, the -chain is 96 h. Where indicated, rIL-2 (100 U/ml) or anti-CD28 mAb (1 ␮g/ml) was relatively conserved (32). From the hypothetical model of class II, added at the start of culture. In indicated assays, PHA-P (5 ␮g/ml) or PMA later confirmed by the crystal structure of HLA-DR (33, 34), we and ionomycin (10 ng/ml and 250 ng/ml, respectively) were used to stim- ␣ ␣ selected the 1 -helix region for study based on our previous ulate cells, and with these treatments, cells were pulsed at 24 h and har- studies with MHC class I (20–22). Peptides corresponding to the vested at 48 h. Preactivated cells were stimulated with rIL-2 (100 U/ml), ␣ pulsed at 24 h, and harvested at 48 h. Cyclosporin A (100 nM) and rapa- -helix of DRA, DQA, and DPA (32) were synthesized. mycin (100 nM) were used in indicated experiments. The peptide corresponding to residues 56–80 of DQA03011 Restimulation assay blocked proliferation of PBL to anti-CD3 mAb (data not shown). Overlapping 15-amino acid peptides were synthesized from this 6 PBL (4 ϫ 10 /ml) were incubated in the presence of peptide (40 ␮M) or sequence and were tested for function. A peptide corresponding to ␮ rapamycin (100 nM) with or without immobilized anti-CD3 mAb (5 g/ residues 65–79 (Table I), designated DQ 65–79, recapitulated the ml) for 24 h at 37°C in 2-ml wells. The cells were then washed three times with PBS and were replated at the same density. After a 7-day incubation, effects of the full-length peptide. The DQ 65–79 peptide inhibited the cells were washed, plated at equivalent densities in 96-well microtiter [3H]thymidine incorporation by PBL or purified T cells stimulated plates, and restimulated with anti-CD3 mAb (5 ␮g/ml). After 48 h, the cells with anti-CD3 mAb (Fig. 1, A and B). Furthermore, the inhibitory 3 were pulsed with [ H]TdR and were harvested at 72 h. In some experi- effect of the peptide was not reversed by the addition of anti-CD28 ments, 100 U/ml of rIL-2 was added during the restimulation. mAb to purified T cells stimulated by anti-CD3 mAb (Fig. 1C). Flow cytometry Inhibition was not limited to stimulation via anti-CD3 mAb, as Cell cycle analysis. PBL (2 ϫ 105/well) were cultured with PHA-P (5 PHA-P-stimulated (Fig. 2) or alloantigen-stimulated cells (data not ␮g/ml) in the presence of peptide (40 ␮M), cyclosporin A (100 nM), rapa- shown) were also blocked by peptide; however, DQ 65–79 did not mycin (100 nM), or medium. At 48 h, the cells were washed in PBS and block PMA- and ionomycin-stimulated proliferation (Fig. 1D). In- ␮ resuspended in 250 l of a solution containing 4 mM sodium citrate, 0.05% hibition of proliferation by peptide was observed with cells ob- Nonidet P-40, 0.45 mg/ml RNase, and 50 ␮g/ml propidium iodide. The cells were incubated on ice for 10 min, at which time 25 ␮l of 1.5 M tained from 35 donors, indicating the effects were allele unre- sodium chloride was added. The cells were analyzed on a Becton Dickin- stricted. The inhibition of proliferation was not due to cell death son (San Jose, CA) FACScan at the Stanford FACS facility. Propidium because no significant increase in staining by either trypan blue or iodide fluorescence was plotted on a linear scale vs orthogonal scatter. propidium iodide was observed over the course of the proliferation Cell surface receptor analysis. PBL (2 ϫ 105 cells) were stained as previously described (29) with the indicated FITC-conjugated Abs (1 ␮g/ assay (data not shown). A second peptide, in which aspartic acid 106 cells). Cells were also stained with propidium iodide immediately be- was substituted for asparagine at amino acid 72, did not inhibit fore analysis to exclude dead cells. proliferation like the DQ 65–79 peptide (Table I and Figs. 1 and 2). The Journal of Immunology 2217

Table I. Amino acid sequences of DQA03011-derived peptides

Amino Acid Residue Positiona

HLA-Derived Peptide 65 70 75 79

DQ 65–79 NIAVLKHNLNIVIKR DQ 65–79 65S SIAVLKHNLNIVIKR DQ 65–79 66S N SAVLKHNLNIVIKR DQ 65–79 67S N I SVLKHNLNIVIKR DQ 65–79 68S N I A SLKHNLNIVIKR DQ 65–79 69S N I A V SKHNLNIVIKR DQ 65–79 70S N I A V L SHNLNIVIKR DQ 65–79 71S N I A V L K SNLNIVIKR DQ 65–79 72S N I A V L K H SLNIVIKR DQ 65–79 73S N I A V L K H N SNIVIKR DQ 65–79 74S N I A V L K H N L SIVIKR DQ 65–79 75S N I A V L K H N L N SVIKR DQ 65–79 76S N I A V L K H N L N I SIKR DQ 65–79 77S N I A V L K H N L N I V SKR DQ 65–79 78S N I A V L K H N L N I V I SR

DQ 65–79 79S N I A V L K H N L N I V I K S Downloaded from DQ 65–79 72D N I A V L K H DLNIVIKR

a Substituted residues are outlined.

The DQ 65–79 peptide and its derivatives were used to character- the unresponsiveness of the DQ 65–79 peptide-treated cells (Fig. ize the immunoregulatory effects and mechanism of action. 3D). Interestingly, cells cultured with the DQ 65–79 peptide alone http://www.jimmunol.org/ were not unresponsive upon restimulation (Fig. 3B). These cells, The inhibitory effects of the DQ 65–79 peptide are sequence after washing and resting, were indistinguishable from control speciﬁc and long lasting cells in their response to anti-CD3 mAb. Thus, simultaneous sig- To determine which amino acids were necessary for peptide inhi- nals provided by peptide and anti-CD3 mAb are required to induce bition, peptides were synthesized in which each amino acid of the long term unresponsiveness. sequence was substituted individually with serine (Fig. 2). Pep- tides with serine substitutions at residues 65 and 67 maintained The DQ 65–79 peptide does not inhibit early activation events activity, while serine substitutions at 70 or 74 increased activity. We examined the effect of the DQ 65–79 peptide on two key

All remaining substituted peptides lost functional activity. events involved in T cell activation: early gene expression and cell by guest on September 25, 2021 We next examined whether peptide-induced unresponsiveness surface receptor expression. In some experiments, the effects of the was transitory or long-lived. PBL treated with peptide and/or anti- DQ 65–79 peptide were compared with the immunosuppressive CD3 mAb were washed, rested 7 days, and restimulated with anti- drugs cyclosporin A or rapamycin, agents that have divergent CD3 mAb alone. Cells initially treated with both the DQ 65–79 mechanisms of action (35). peptide and anti-CD3 mAb were unresponsive to restimulation The hallmark of cyclosporin A activity is its block of IL-2 gene when compared with cells initially treated with anti-CD3 mAb expression, a critical early event in T cell proliferation (35). Cy- with either DMSO or the DQ 65–79 72D peptide (Fig. 3C). Ad- closporin A treatment also decreases expression of other early dition of exogenous IL-2 during the restimulation did not reverse

FIGURE 1. The DQ 65–79 peptide inhibits PBL and T cell proliferation. PBL (A and D) or puriﬁed T cells (B and C) were stimulated with anti-CD3 mAb (A and B), anti-CD3 plus anti-CD28 mAb (C), or PMA and FIGURE 2. Serine substitutions affect the function of the DQ 65–79 ionomycin (D) in the presence of 40 ␮M peptide. Cells were pulsed with peptide. Peptide sequences are as shown in Table I. The proliferation assay [3H]TdR at 24 (D), 48 (A and C), or 72 (B) h and harvested 24 h later. was conducted using PBL stimulated with PHA-P in the presence of 40 Each point is a mean of six replicates with the SD. Results are represen- ␮M peptide. Cells were pulsed at 24 h and harvested 24 h later. Each point tative of at least three experiments with PBL from different donors. DMSO is a mean of six replicates with the SD. Results are representative of at least indicates the media control. three experiments. 2218 A SYNTHETIC HLA PEPTIDE INHIBITS CELL CYCLE PROGRESSION

Table II. The DQ 65–79 peptide inhibits cell cycle progression

Proliferation % Cells in (cpm) S/G2/M No stimulation 578 1 DMSOa 81,833 29 DQ 65–79b 16,066 14 DQ 65–79 72Db 68,783 30 Cyclosporin Ac 35,314 19 Rapamycinc 19,248 12

a Cells were stimulated with PHA-P in the presence of the indicated reagents for 48 h prior to analysis of DNA content. Results are representative of three experiments. b 40 ␮M. c 100 nM.

FIGURE 3. DQ 65–79 induces a long term unresponsiveness that is not reversed with IL-2. PBL were cultured with anti-CD3 mAb in the presence These ﬁndings indicate that, like rapamycin, the functional effects of the indicated agent and were pulsed with [3H]TdR at 72 h (A). In B, C, of the DQ 65–79 peptide are most likely downstream of the IL-2R. and D, PBL were preincubated for 24 h with the indicated agent, washed at 24 h, and then cultured in medium for 7 days. The cells were then The DQ 65–79 peptide impedes cell cycle progression Downloaded from restimulated with anti-CD3 mAb and pulsed at 24 h with [3H]TdR. In B PBL were preincubated with peptide alone, while in C and D PBL were To further examine the effect of the DQ 65–79 peptide on prolif- preincubated with peptide plus anti-CD3 mAb. rIL-2 (100 U/ml) was added eration, cell cycle and kinetics studies were performed. Analysis of to the restimulation in D. Each point is the mean of six replicates with the DNA content showed that the peptide, along with cyclosporin A SD. Results are representative of four similar experiments with PBL from and rapamycin, prevented DNA replication by blocking the G1 to different donors. S transition (Table II). We examined the kinetics of peptide activity by adding it at various time points after activation. [3H]Thy- http://www.jimmunol.org/ midine incorporation of PBL stimulated with anti-CD3 mAb was genes such as IFN-␥ and IL-2R␣ (36). In contrast, rapamycin does inhibited when the DQ 65–79 peptide was added up to 24 h after not affect the expression of these early genes, but instead blocks stimulation (Fig. 5). The kinetics data concur with the block at signaling events downstream of the IL-2R (29). No decrease in G1/S of the cell cycle. The ability of the DQ 65–79 peptide to IL-2, IL-2R␣, or IFN-␥ gene expression was detected by Northern block T cell proliferation at a relatively late time was similar to blot analysis of PHA-P-activated PBL treated with the DQ 65–79 that of rapamycin, but contrasted with cyclosporin A, which lost its peptide as compared with DMSO-treated cells (data not shown). In effect by 24 h. agreement with these data, both the DQ 65–79 peptide and rapa- The DQ 65–79 peptide inhibits cdk2 kinase activity by guest on September 25, 2021 mycin blocked anti-CD3- or rIL-2-induced proliferation of preactivated PBL, while cyclosporin A blocked only anti-CD3-induced Because the DQ 65–79 peptide functioned until relatively late after proliferation (Figs. 4, A and B). activation and blocked the G1/S cell cycle transition, we focused Because the DQ 65–79 peptide inhibited IL-2-mediated proliferation, cell surface expression of proximal signaling molecules was examined. The levels of CD2, CD4, CD8, MHC class I, and MHC class II were identical in anti-CD3 mAb-stimulated PBL in the presence or absence of the DQ 65–79 peptide (data not shown). Additionally, there was no difference between the expression of the activation markers IL-2R␣, CD69, and CD45RO in peptide- treated or untreated cells (data not shown). Because the peptide inhibited IL-2-mediated proliferation (Fig. 4B), the expression of the IL-2R␤ and IL-2R␥ chains also was examined; however, DQ 65–79 had no effect on either of these molecules (data not shown).

FIGURE 4. The DQ 65–79 peptide blocks proliferation of preactivated FIGURE 5. The DQ 65–79 peptide blocks PBL proliferation when T cells to both anti-CD3 mAb and IL-2. Preactivated T cells (see Materials added 24 h after activation. PBL were stimulated with immobilized anti- and Methods) were stimulated with immobilized anti-CD3 mAb (A)or100 CD3 mAb. Peptide (40 ␮M), cyclosporin (100 nM), or rapamycin (100 U/ml IL-2 (B) in the presence of 40 ␮M peptide, 100 nM cyclosporin, or nM) was added at the indicated times after the start of culture. Cells were 100 nM rapamycin. Cells were pulsed with [3H]TdR at 24 h (B)or48h(A) pulsed with [3H]TdR at 48 h and harvested at 72 h. Each point is the mean and harvested 24 h later. Results are representative of at least three of six replicates with the SD. Results are representative of at least three experiments. experiments with different donors. The Journal of Immunology 2219 on events that occur during this time period. The cyclin-dependent kinases (cdks) are key regulators of cell cycle progression. Acti- vation of the cdks is a complex process, requiring both the recruit- ment of positive and the release of negative regulators. These kinases are regulated positively by association with cyclin proteins and by threonine phosphorylation, and they are regulated negatively by tyrosine phosphorylation and the association with either the Ink, Cip, or Kip families of inhibitory proteins. The negative signals from these inhibitory proteins overwhelm positive signals. Thus, an inhibitory protein must be removed from the cyclin-cdk complex before the kinase can become active (37, 38).

Cdk2 is critical for the transition from G1 into S phase of the cell cycle. It must associate with cyclin E and be phosphorylated on threonine 160 for activation. Cdk2 can be regulated negatively by phosphorylation of tyrosine 15 and by the association of either p21Cip1 or p27Kip1 (28). Because the DQ 65–79 peptide inhibited cell cycle progression at G1/S, we examined its effects on cdk2 kinase activity. T cells stimulated with IL-2 in the presence or Downloaded from absence of the DQ 65–79 peptide were assayed for cdk2 kinase activity. Phosphorylation of the cdk2 kinase substrate histone H1 was decreased with DQ 65–79 peptide treatment (Fig. 6A). This effect was not due to a change in the expression of cdk2 or cyclin E because the amount of these proteins was essentially equivalent between cells treated with the DQ 65–79 peptide and controls over a 2-day time course (Fig. 6C). Nor was inhibition due to a detect- http://www.jimmunol.org/ able change in phosphorylation, as the phosphorylation pattern was equivalent between treatment conditions, as shown by the doublet bands (Fig. 6B). Cdk2 in DQ 65–79 peptide-treated cells was able to form a complex with cyclin E, as shown by the coimmunopre- cipitation of cdk2 with Abs specific for cyclin E (Fig. 6B). There was no significant change in complex formation in cells treated with the DQ 65–79 peptide as compared with controls (Fig. 6, B and C). However, the steady state level of the inhibitory protein FIGURE 6. The DQ 65–79 peptide inhibits cdk2 kinase activity and by guest on September 25, 2021 p27 was higher, while the level of p21 was lower, in the cells prolongs p27 expression. Incorporation of 32P into histone H1 by cdk2 treated with the DQ 65–79 peptide as compared with cells treated kinase immunoprecipitated from IL-2-stimulated preactivated T cells with the control 72D peptide (Fig. 6C). Rapamycin retards G1/S treated with the indicated agents at 24 h after stimulation (A). Each point cell cycle progression by the inhibition of the cdk2 kinase via a is the mean of triplicates with the SD. Lysates from IL-2-stimulated pre- block in p27 degradation, and also blocks the up-regulation of p21 activated T cells treated with the indicated agents (D ϭ medium control; (Fig. 6C) (30, 39, 40). Thus, DQ 65–79 affected events down- Q ϭ DQ 65–79; 72 ϭ DQ 65–79 72D; and R ϭ rapamycin) were immu- stream of the IL-2R in a manner similar to rapamycin, resulting in noprecipitated with anti-cyclin E mAb, separated on a 12% SDS-PAGE the inhibition of cell cycle progression and subsequent gel, and probed with anti-cdk2 mAb (B). Preactivated T cells were activated for 0, 1, or 2 days with IL-2 plus the indicated agent. Lysates were proliferation. separated on 7.5% SDS-PAGE gels. Western blots were probed with the The DQ 65–79 peptide is not antagonistic with FK-506 indicated Abs (C). ␤-Tubulin was the control for loading. Analysis by densitometry from left to right: p27 (1026, 954, 617; 950, 651, 203; 1064, Because of the multiple functional similarities between DQ 65–79 877, 481); ␤-tubulin (904, 870, 614; 926, 1007, 1054; 774, 829, 658); cdk2 and rapamycin, we asked whether the DQ 65–79 peptide bound to (997, 1101, 941; 1117, 1154, 1156; 877, 852, 764); cyclin E (1096, 1101, the immunophilin FK-506-binding protein (FKBP). Rapamycin 815; 1159, 1175, 1273; 915, 1087, 860); p21 (360, 140, 104; 284, 255, 892; and FK-506 bind to FKBP, and this complex formation is critical 251, 429, 189). for their function (41). Rapamycin and FK-506 have different effects and are mutually antagonistic. Excess FK-506 prevents ra- not bind to FKBP at all. Thus, while DQ 65–79 is functionally pamycin-mediated inhibition of IL-2-mediated proliferation, and similar to rapamycin, it acts via a distinct mechanism. likewise, an excess of rapamycin prevents FK-506-mediated inhibition of PMA- and ionomycin-stimulated proliferation (41). Since the DQ 65–79 peptide blocked IL-2-mediated proliferation, but not Discussion PMA- and ionomycin-mediated proliferation, we conducted sim- A synthetic peptide corresponding to residues 65–79 of the ilar experiments in which the DQ 65–79 peptide was substituted ␣-chain of DQA03011 inhibits the proliferation of PBL and puri- for rapamycin. The DQ 65–79 peptide had no effect on FK-506- fied T cells in an allele-nonspecific manner. This peptide induces mediated inhibition of PBL proliferation stimulated with PMA and long-term unresponsiveness that is not reversible with IL-2 and is ionomycin in a range of 10 to 10,000 molar excess (Fig. 7A). dependent upon concomitant activation through the TCR or IL-2R. Likewise, no concentration of FK-506 tested blocked the effect of The DQ 65–79 peptide does not inhibit early mRNA accumulation the DQ 65–79 peptide on IL-2-mediated proliferation, although or cell surface receptor expression, but instead blocks the G1 to S FK-506 did reverse the inhibition of proliferation by rapamycin transition of the cell cycle. Cells treated with the DQ 65–79 pep- (Fig. 7B). Therefore, the DQ 65–79 peptide does not compete for tide exhibit repressed cdk2 kinase activity and prolonged expres- binding to the FK-506 binding site on FKBP, and probably does sion of the p27 inhibitor. 2220 A SYNTHETIC HLA PEPTIDE INHIBITS CELL CYCLE PROGRESSION

peptide is important for its function. This contrasts with our previous findings with the synthetic peptide corresponding to residues 75–84 of the class I HLA-B2702 sequence. This peptide blocks lysis of target cells by CTL, and this effect is completely dependent on the presence of an isoleucine at position 80 (22). Substitution of the other residues had little, if any, effect on the function of the class I peptide. A potential mechanism for DQ 65–79 peptide function is that it interferes with Ag presentation by binding to the peptide groove of MHC molecules. Peptides derived from this conserved region of HLA DQ do bind to HLA class II molecules (43, 44). However, the DQ 65–79 peptide blocks proliferation of all T cells tested, indicating an allele-unrestricted effect. Furthermore, the DQ 65–79 peptide inhibits proliferation of purified T cells stimulated by anti- CD3 mAb or PHA-P, both of which bypass APC. These findings suggest that the peptide-mediated inhibition is independent of Ag presentation.

It has been postulated that soluble whole or fragmented MHC Downloaded from molecules modulate immune responses through interaction with the TCR, potentially by steric inhibition of the TCR interaction with membrane-bound MHC/Ag complexes (3, 5). It is possible that the DQ 65–79 peptide bypasses allele speciﬁcity by binding to conserved regions of the TCR. It is also possible that the DQ

65–79 peptide could interfere with costimulation, causing cells to http://www.jimmunol.org/ become anergic (45). This type of unresponsiveness can often be overcome by the addition of exogenous IL-2. However, neither exogenous IL-2 nor anti-CD28 mAb reversed the inhibitory effect of the peptide. Therefore, the DQ 65–79 peptide functions through a mechanism not involving TCR/MHC interactions or the induction of classical anergy. The DQ 65–79 peptide described in this work and the class

I-derived B2702 75–84 peptide identified previously are derived by guest on September 25, 2021 from analogous regions of MHC class II and MHC class I, respectively. This region of native MHC class I is important in binding the p58 and p70 KIR of NK cells (9). There is evidence to suggest that functionally similar inhibitory receptors for MHC class II exist, although they have not yet been identified (46, 47). Although the B2702.75–84 peptide blocks NK-mediated killing, peptide function is not dependent on any defined KIR (J. E. Goldberg and FIGURE 7. The DQ 65–79 peptide and FK-506 are not mutually an- C. Clayberger, unpublished observations). Various KIRs are ex- tagonistic. PBL were stimulated with 10 ng/ml PMA and 250 ng/ml iono- pressed on subsets of NK cells and a small percentage of T cells mycin in the presence of the indicated concentrations of FK-506 and DQ 65–79 (A). Preactivated T cells were stimulated with 25 U/ml rIL-2 in the (9). Unlike KIR, the allele-nonspecific effects of the DQ 65–79 presence of the indicated concentrations of FK-506, rapamycin, and DQ peptide suggest that a potential inhibitory ligand would have to be 65–79 (B). broadly expressed. The expression of cytokines and signaling receptors is critical for the initiation of proliferation. Unlike cyclosporin A (36), no We and others have demonstrated that either intact MHC mol- inhibition of IL-2 or IFN-␥ expression was detected in DQ 65–79 ecules or synthetic peptides corresponding to polymorphic regions peptide-treated T cells stimulated with PHA-P. Expression of all of MHC molecules inhibit target cell lysis of CTL specific for cell surface receptors tested, including all three chains of the IL- those MHC Ags (17, 21). We later demonstrated that synthetic peptides corresponding to residues 75–84 of the class I molecule 2R, was normal in peptide-treated cells stimulated with anti-CD3 HLA-B2702 block lysis of target cells by CTL in an allele-non- mAb or PHA-P. Instead, the peptide interfered with signals down- restricted manner (22). Peptides derived from this region of class stream of the IL-2R. Although cdk2 activity was reduced, we I, either alone or in combination with a subtherapeutic dose of could not detect any differences in the phosphorylation pattern of cyclosporin A, prevent rejection of heterotopic heart allografts in cdk2 or its association with cyclin E in lysates from cells activated rodents (23–25, 42). Lymphocytes isolated from tolerant animals in the presence of the DQ 65–79 peptide. Instead, the peptide pro- differentiate into donor-specific CTL in vitro, consistent with the longed the presence of the inhibitor molecule p27 (39). Although notion that the cells are functionally inactivated in vivo (42). it is not known how rapamycin or the DQ 65–79 peptide modulates The inhibitory effect of the DQ 65–79 peptide is sequence spe- p27 expression, it may involve the ubiquitin degradation cific, but not MHC restricted. Peptides synthesized with single pathway (48). amino acid substitutions at individual residues 66, 68, 69, 71–73, The DQ 65–79 peptide and rapamycin share functional and and 75–79 lost function. These residues are located throughout the mechanistic activities, but they differ in several important ways. length of the peptide, indicating that the overall sequence of the First, rapamycin, in a complex with FKBP, blocks Saccharomyces The Journal of Immunology 2221 cerevisiae yeast cell division by binding to and inhibiting the ac- 6. Salter, R. D., R. J. Benjamin, P. K. Wesley, S. E. Buxton, T. P. Garrett, tivity of TOR (target of rapamycin), a phosphoinositol kinase fam- C. Clayberger, A. M. Krensky, A. M. Norment, D. R. Littman, and P. Parham. 1990. A binding site for the T-cell co-receptor CD8 on the alpha 3 domain of ily member required for growth (49). In contrast, the DQ 65–79 HLA-A2. Nature 345:41. peptide did not affect division of S. cerevisiae yeast cells at any 7. Miller, J. F., and A. Basten. 1996. Mechanisms of tolerance to self. Curr. Opin. concentration tested (S.-C. Lyu and C. Clayberger, unpublished Immunol. 8:815. 8. Kruisbeek, A. M., and D. Amsen. 1996. Mechanisms underlying T-cell tolerance. observations). Furthermore, our experiments show that DQ 65–79 Curr. Opin. Immunol. 8:233. peptide and rapamycin do not bind to the same site on FKBP, and 9. Lanier, L. L. 1997. Natural killer cell receptors and MHC class I interactions. it is likely that the DQ 65–79 peptide does not bind FKBP at all. Curr. Opin. Immunol. 9:126. 10. Lobo, P. I., M. Y. Chang, and E. Mellins. 1996. Mechanisms by which HLA-class Rapamycin has pleiotropic effects on cell proliferation down- II molecules protect human B lymphoid tumour cells against NK- and LAK- stream of the IL-2R. As a complex with FKBP, rapamycin blocks mediated cytolysis. Immunology 88:625. the activity of mTOR, the mammalian homologue of TOR, which 11. Shoskes, D. A., and K. J. Wood. 1994. Indirect presentation of MHC antigens in transplantation. Immunol. Today 15:32. is critical for cap-dependent translation. and for the activation of 12. Watschinger, B., L. Gallon, C. B. Carpenter, and M. H. Sayegh. 1994. Mecha- p70 S6 kinase (50, 51). The p70 S6 kinase has also been implicated nisms of allo-recognition: recognition by in vivo-primed T cells of specific major in the regulation of CREB/ATF transcription factors, which are histocompatibility complex polymorphisms presented as peptides by responder antigen-presenting cells. Transplantation 57:572. involved in the expression of proliferating cell nuclear Ag, a pro- 13. Cochrum, K., D. Hanes, D. Potter, H. Perkins, W. Amend, F. Vincenti, Y. Iwaki, tein that is required for entry into the S phase of the cell cycle (52). G. Opelz, P. Terasaki, B. Levin, D. Sampson, N. Feduska, and O. Salvatierra. We are currently investigating whether the DQ 65–79 peptide af- 1981. Improved graft survival following donor-specific blood transfusions. Transplant. Proc. 13:1657. fects these and other signaling events downstream of the IL-2R.

14. Madsen, J. C., K. J. Wood, R. A. Superina, and P. J. Morris. 1989. Induction of Downloaded from Do analogous peptides corresponding to regions of MHC mol- immunological unresponsiveness using recipient cells transfected with donor ecules exist in vivo, and do they mediate similar functions? Al- class I or class II MHC genes. Transplant. Proc. 21:477. 15. Foster, S., K. J. Wood, and P. J. Morris. 1989. Comparison of the effect of protein though there are no definitive answers to these questions, several micelles containing purified class I MHC antigen and a cytosolic preparation lines of evidence suggest that soluble peptides of MHC molecules containing water soluble class I molecules on rat renal allograft survival. Trans- are present and can mediate immunoregulatory functions in vivo. plant. Proc. 21:375. 16. Pouteil, N. C., K. J. Wood, and P. J. Morris. 1993. The effect of purified class II It has been recognized for some time that class I MHC molecules major histocompatibility complex antigen on the survival of vascularized organ exist in both membrane-bound and soluble forms, the latter result- allografts in the rat. Transplantation 55:656. http://www.jimmunol.org/ ing from alternative splicing (53). Soluble class II molecules also 17. Sayegh, M. H., S. J. Khoury, W. W. Hancock, H. L. Weiner, and C. B. Carpenter. 1993. Induction of immunity and oral tolerance to alloantigen by polymorphic exist, and activated lymphocytes produce large quantities of solu- class II major histocompatibility complex allopeptides in the rat. Transplant. ble MHC molecules (54–56). Soluble MHC molecules inhibit im- Proc. 25:357. mune responses in both allele-restricted and unrestricted ways, and 18. Sayegh, M. H., N. Perico, O. Imberti, W. W. Hancock, C. B. Carpenter, and G. Remuzzi. 1993. Thymic recognition of class II major histocompatibility com- they are likely to be responsible for part or all of the nonspecific plex allopeptides induces donor-specific unresponsiveness to renal allografts. immunosuppression observed following blood transfusion in a Transplantation 56:461. number of clinical settings (57, 58). 19. Murphy, B., E. Akalin, B. Watschinger, C. B. Carpenter, and M. H. Sayegh. 1995. Inhibition of the alloimmune response with synthetic nonpolymorphic class

MHC-derived peptides may also mediate some of the phenom- II MHC peptides. Transplant. Proc. 27:409. by guest on September 25, 2021 ena attributed to suppressor cells. There are many studies that doc- 20. Parham, P., C. Clayberger, S. L. Zorn, D. S. Ludwig, G. K. Schoolnik, and ument a potent suppressor effect of T lymphocytes or their prod- A. M. Krensky. 1987. Inhibition of alloreactive cytotoxic T lymphocytes by peptides from the alpha 2 domain of HLA-A2. Nature 325:625. ucts, and many of these effects map to the MHC. However, when 21. Clayberger, C., P. Parham, J. Rothbard, D. S. Ludwig, G. K. Schoolnik, and a thorough analysis of the MHC was conducted, no suppressor A. M. Krensky. 1987. HLA-A2 peptides can regulate cytolysis by human allo- genes were identified (59). Our findings suggest that peptides, de- geneic T lymphocytes. Nature 330:763. 22. Nossner, E., J. E. Goldberg, C. Naftzger, S. C. Lyu, C. Clayberger, and rived from intact MHC molecules by either pre- or post-transla- A. M. Krensky. 1996. HLA-derived peptides which inhibit T cell function bind tional events, can mediate immunoregulatory effects. The genera- to members of the heat-shock protein 70 family. J. Exp. Med. 183:339. tion of peptides from MHC molecules may occur only in restricted 23. Buelow, R., P. Veyron, C. Clayberger, P. Pouletty, and J. L. Touraine. 1995. Prolongation of skin allograft survival in mice following administration of sites or in response to particular stimuli, and secretion and/or pro- ALLOTRAP. Transplantation 59:455. cessing of these molecules by activated lymphocytes may be im- 24. Woo, J., L. Gao, M. C. Cornejo, and R. Buelow. 1995. A synthetic dimeric HLA portant in turning off immune responses. Characterization of such class I peptide inhibits T cell activity in vitro and prolongs allogeneic heart graft survival in a mouse model. Transplantation 60:1156. peptides in vitro should prove useful in the design of novel ther- 25. Hanaway, M. J., E. K. Geissler, J. Wang, J. J. Fechner, R. Buelow, and apies to dampen the in vivo immune response in transplant rejec- S. J. Knechtle. 1996. Immunosuppressive effects of an HLA class I-derived pep- tion and autoimmune disease. tide in a rat cardiac allograft model. Transplantation 61:1222. 26. Clayberger, C., S. C. Lyu, P. Pouletty, and A. M. Krensky. 1993. Peptides corresponding to T-cell receptor-HLA contact regions inhibit class I-restricted im- Acknowledgments mune responses. Transplant. Proc. 25:477. 27. Marshall, J. D., Y. Wen, J. S. Abrams, and D. T. Umetsu. 1993. In vitro synthesis We thank Elizabeth Mellins and Jodi Goldberg for helpful discussions and of IL-4 by human CD4ϩ T cells requires repeated antigenic stimulation. Cell. critical reading of the manuscript. Immunol. 152:18. 28. Firpo, E. J., A. Koff, M. J. Solomon, and J. M. Roberts. 1994. Inactivation of a Cdk2 inhibitor during interleukin 2-induced proliferation of human T lympho- References cytes. Mol. Cell. Biol. 14:4889. 1. Germain, R. N. 1994. MHC-dependent antigen processing and peptide presenta- 29. Dumont, F. J., M. J. Staruch, S. L. Koprak, M. R. Melino, and N. H. Sigal. 1990. tion: providing ligands for T lymphocyte activation. Cell 76:287. Distinct mechanisms of suppression of murine T cell activation by the related 2. Cammarota, G., A. Scheirle, B. Takacs, D. M. Doran, R. Knorr, W. Bannwarth, macrolides FK-506 and rapamycin. J. Immunol. 144:251. J. Guardiola, and F. Sinigaglia. 1992. Identification of a CD4 binding site on the 30. Morice, W. G., G. Wiederrecht, G. J. Brunn, J. J. Siekierka, and R. T. Abraham. beta 2 domain of HLA-DR molecules. Nature 356:799. 1993. Rapamycin inhibition of interleukin-2-dependent p33cdk2 and p34cdc2 3. Konig, R., L. Y. Huang, and R. N. Germain. 1992. MHC class II interaction with kinase activation in T lymphocytes. J. Biol. Chem. 268:22737. CD4 mediated by a region analogous to the MHC class I binding site for CD8. 31. Morice, W. G., G. J. Brunn, G. Wiederrecht, J. J. Siekierka, and R. T. Abraham. Nature 356:796. 1993. Rapamycin-induced inhibition of p34cdc2 kinase activation is associated 4. Nag, B., H. G. Wada, D. Passmore, B. R. Clark, S. D. Sharma, and with G1/S-phase growth arrest in T lymphocytes. J. Biol. Chem. 268:3734. H. M. McConnell. 1993. Purified ␤-chain of MHC class II binds to CD4 mole- 32. Marsh, S. G., and J. G. Bodmer. 1993. HLA class II nucleotide sequences, 1992. cules on transfected HeLa cells. J. Immunol. 150:1358. Immunogenetics 37:79. 5. Clayberger, C., S. C. Lyu, R. DeKruyff, P. Parham, and A. M. Krensky. 1994. 33. Brown, J. H., T. Jardetzky, M. A. Saper, B. Samraoui, P. J. Bjorkman, and Peptides corresponding to the CD8 and CD4 binding domains of HLA molecules D. C. Wiley. 1988. A hypothetical model of the foreign antigen binding site of block T lymphocyte immune responses in vitro. J. Immunol. 153:946. class II histocompatibility molecules. Nature 332:845. 2222 A SYNTHETIC HLA PEPTIDE INHIBITS CELL CYCLE PROGRESSION

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