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Rejection of Mouse Cardiac Allografts by Costimulation in trans¹

Didier A. Mandelbrot23^*,, Koji Kishimoto^2,, Hugh Auchincloss, Jr., Arlene H. Sharpe^4,* and Mohamed H. Sayegh^4,

^* Immunology Research Division, Department of Pathology, Laboratory of Immunogenetics and Transplantation, and Transplantation Unit, Surgical Services, Brigham and Women’s Hospital, Boston, MA 02115; and Renal Division, University of Massachusetts Medical Center, Worcester, MA 01655

	Abstract

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The activation of T cells by B7 costimulation in trans has been demonstrated in vitro, but the in vivo relevance is unknown. To study costimulation in trans of CD4⁺ T cells in vivo, we performed cardiac transplants from B7-1/B7-2-deficient mice to recipients that do not express MHC class II molecules on peripheral APCs, but do have functional CD4⁺ T cells (II^-/4⁺ mice). This model restricts the B7-dependent activation of CD4⁺ T cells to costimulation in trans and excludes any contribution from indirect Ag presentation. We find that II^-/4⁺ recipients reject B7-deficient grafts as rapidly as wild-type grafts, suggesting that costimulation in trans can mediate rejection as potently as costimulation in cis. Treatment of II^-/4⁺ recipients of B7-deficient grafts with depleting Abs to CD4 or CD8 demonstrates that indirect Ag presentation to CD8⁺ cells does not significantly contribute to rejection. This is the first demonstration that costimulation in trans can mediate an immune response in vivo and has important therapeutic implications.

	Introduction

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Costimulation of T cells by B7 plays a critical role in the regulation of immune responses. The optimal activation of T cells requires two distinct signals. One is Ag specific and is transmitted through the TCR. The second is a costimulatory signal. Many different molecules can potentiate T cell proliferation, but the engagement of CD28 on T cells by B7-1 (CD80) and B7-2 (CD86) holds particular interest as a therapeutic target in transplant rejection and autoimmunity (1). Stimulation through CD28 is important for the production of IL-2 and the anti-apoptotic protein Bcl-x_L (2), and blockade of B7 costimulation by CTLA4-Ig prolongs allograft survival in rodents (3) and primates (4).

The relationship between antigenic and costimulatory signals to T cells has been extensively investigated (5). Some studies suggest that T cells are more efficiently activated when both signals are presented simultaneously on the same APC (costimulation in cis) (6). However, others have found that costimulation provided from a bystander APC (costimulation in trans, or trans-costimulation), distinct from the cell providing the Ag-specific signal, is equally effective in activating CD4⁺ T cells (7). Until now, B7 trans-costimulation of CD4⁺ T cells has only been studied in vitro. Here we report the development of a model system in which the role of costimulation in trans can be tested in vivo.

We previously reported that wild-type mice reject fully MHC mismatched cardiac allografts from mice lacking B7-1 and B7-2 (B7-1/B7-2^-/-) as rapidly as grafts from wild-type mice. This alloresponse is B7 dependent, because administration of CTLA4-Ig produces long term graft survival (D. A. Mandelbrot, A. H. Sharpe, and M. H. Sayegh, unpublished observations). In these experiments all donor cells lack B7-1 and B7-2, so B7 costimulation can only be provided by recipient APCs. This could occur by either of two mechanisms (Fig. 1). One mechanism is indirect Ag presentation, in which recipient APCs would both present allopeptides and provide B7 costimulation to responding T cells. The second mechanism is costimulation in trans, by which alloreactive T cells would recognize allogeneic MHC molecules on donor cells, but receive B7 costimulation in trans from recipient cells.

View larger version (20K): [in this window] [in a new window]   FIGURE 1. Two possible mechanisms could mediate the rejection of B7-1/B7-2-/- cardiac allografts by wild-type (WT) mice. In both, B7 costimulation is provided to recipient T cells by recipient APCs, since donor APCs lack B7. In indirect allorecognition the antigenic signal is also provided by recipient APCs in the form of self MHC/allopeptide complexes. In costimulation in trans the Ag is intact allo-MHC on donor cells.

Using this model we demonstrate that costimulation in trans can mediate a potent alloresponse, both in vitro and in vivo. We find that CD4⁺ T cells from II^-/4⁺ mice respond strongly to costimulation in trans in a modified MLR, and that II^-/4⁺ mice acutely reject B7-1/B7-2^-/- cardiac allografts. This allograft rejection is B7 dependent because CTLA4-Ig significantly prolongs survival. We also demonstrate that graft rejection is dependent on CD4⁺ T cells, but is not inhibited by Ab to CD8, suggesting that indirect allorecognition by CD8⁺ T cells does not contribute to the alloresponse in this model.

	Materials and Methods

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Mice

Wild-type mice on the BALB/c and C57BL/6 (B6) backgrounds were obtained from The Jackson Laboratory (Bar Harbor, ME). B7-1/B7-2^-/- mice (10) were backcrossed for 10 generations onto the BALB/c background. II^-/4⁺ mice (9) were generated on the B6 background and were provided by L. Glimcher (Harvard Medical School, Boston, MA). Brigham and Women’s Hospital and Harvard Medical School are accredited by the American Association of Accreditation of Laboratory Animal Care, and mice were cared for in accordance with institutional guidelines in a pathogen-free animal facility.

Flow cytometry

To determine B7-1, B7-2, and CD40 expression, splenocytes were stimulated with LPS (20 µg/ml) and dextran sulfate (10 µg/ml) for 3 days. Cells were stained using directly conjugated Abs obtained from PharMingen (San Diego, CA).

Mixed lymphocyte reactions

CD4⁺ responder T cells were purified from II^-/4⁺ spleens by magnetic beads (MACS) from Miltenyi Biotec (Auburn, CA), with purity >95% confirmed by flow cytometry. CD4⁺ T cells were plated in round-bottom wells at 5 x 10⁴ cells/well in complete medium as previously described (11). To generate APCs, splenocytes were depleted of T cells by coating with Abs to Thy1.2 (ascites from Serotec, Kidlington, U.K.), CD4 (RL1724 from American Type Culture Collection, Manassas, VA), and CD8 (3155 from American Type Culture Collection); treated with complement (Low-Tx-M Rabbit Complement; Cedarlane Laboratories, Hornby, Ontario, Canada); and inactivated using mitomycin (Bristol Laboratories, Princeton, NJ; 50 µg/ml for 40 min). Stimulator APCs from BALB/c mice were added at 5 x 10⁵ cells/well. Cells were cultured with or without 5 x 10⁵ cells/well of T-depleted II^-/4⁺ splenocytes. Proliferation on days 3–6 was assessed in triplicate by pulsing with 1 µCi [³H]thymidine for the last 8 h of the indicated day. Student’s t test was used to assess statistical significance, and p > 0.05 was considered not significant.

Mouse heart transplantation

Allografts from male donors were placed in male recipients as previously described (12). Graft function was assessed daily by palpation, with rejection defined as the absence of detectable beating. Allografts failing or graft recipients dying within 48 h of surgery were considered technical failures and were excluded from the analysis. Donor hearts were from BALB/c wild-type mice, and recipients were B6 wild-type or II^-/4⁺ mice. Purified CTLA4-Ig was provided by Bristol Myers Squibb (R. Peach, Princeton, NJ), and administered (200 µg i.p.) on days 0, 2, 4, and 6 after transplantation. The anti-CD4 mAb GK1.5 and anti-CD8 mAb 2.43 ascites were prepared from hybridomas obtained from American Type Culture Collection and administered (0.1 ml i.p.) on days 6, 3, and 1 before transplantation. This regimen insures >95% depletion of the respective cell types (13).

	Results

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APCs from II^-/4⁺ mice have unimpaired expression of costimulatory molecules

The II^-/4⁺ mice have previously been used to study the production of alloantibodies (9) and the rejection of skin grafts (14) in the absence of indirect Ag presentation to CD4⁺ T cells. We confirmed the phenotype of the II^-/4⁺ mice by staining splenocytes for CD4 and MHC class II (I-A^b). The percentage of CD4⁺ cells was similar to that of wild-type controls, and MHC class II was absent (data not shown). To determine whether APCs from the II^-/4⁺ mice regulate major costimulatory molecules in a manner comparable to wild-type mice, we used flow cytometry to measure the expression of B7-1, B7-2, and CD40 on resting and activated splenocytes. Splenocytes from B6 wild-type or II^-/4⁺ mice were stimulated for 3 days with LPS/dextran sulfate. Similar levels of costimulatory molecule expression were found on II^-/4⁺ and wild-type cells both before and after activation (Fig. 2).

View larger version (20K): [in this window] [in a new window]   FIGURE 2. Expression of B7-1, B7-2, and CD40 is intact in II-/4+ APCs. Splenocytes were stained with directly conjugated Abs before (data not shown) and 3 days after activation with LPS/dextran sulfate. The dotted line indicates isotype-matched control Ab; the thin line indicates wild-type mice; and the thick line indicates II-/4+ mice. Data are representative of three experiments. FL1-H, Fluorescence.

To determine the ability of CD4⁺ T cells from II^-/4⁺ mice to respond in vitro to costimulation in trans, we developed a modified MLR. Purified CD4⁺ responder cells from the II^-/4⁺ mouse (H-2^b) were stimulated with fully MHC mismatched BALB/c (H-2^d) wild-type or B7-1/B7-2^-/- APCs in the presence or the absence of syngeneic II^-/4⁺ APCs. Background thymidine incorporation by APCs alone, from either strain, was negligible. In the absence of II^-/4⁺ APCs, CD4⁺ T cell responders from II^-/4⁺ mice showed low proliferation in response to B7-1/B7-2^-/- APC stimulators (Fig. 3, top bar). This low response is due to the absence of B7 costimulation from APCs, since the stimulator APCs lack B7, and II^-/4⁺ APCs are not present. If II^-/4⁺ APCs are added (Fig. 3, second bar from top), proliferation is significantly enhanced (p = 0.016). Under these conditions, the II^-/4⁺ APCs cannot stimulate the CD4⁺ T cell responders by indirect Ag presentation, because these APCs lack MHC class II. Therefore, the antigenic signal is provided by B7-1/B7-2^-/- stimulator APCs, and B7 costimulation is provided in trans by the II^-/4⁺ APCs. In the presence of wild-type APCs, which provide both Ag and B7 costimulation in cis, the proliferative response of CD4⁺ T cells (Fig. 3, third bar from top) is not significantly different from that induced by costimulation in trans (Fig. 3, second bar). The addition of II^-/4⁺ APCs does not significantly increase the response to wild-type APCs (Fig. 3, fourth bar), suggesting that providing costimulation in trans does not potentiate proliferation if costimulation in cis is already present. Thus, under these conditions of MLR, costimulation in trans is of similar potency to costimulation in cis.

View larger version (19K): [in this window] [in a new window]   FIGURE 3. CD4+ T cells from II-/4+ mice respond to costimulation in trans in an MLR. CD4+ cells from II-/4+ mice were stimulated with BALB/c APCs from wild-type (WT) or B7-1/B7-2 mice in the absence or the presence of II-/4+ APCs. The addition of II-/4+ APCs significantly increases the proliferation of II-/4+ cells in response to B7-1/B7-2-/- stimulators. Data are shown from day 5 after stimulation and are representative of four experiments. The results were similar on days 4 and 6. Error bars represent the SD.

To examine the role of costimulation in trans in mediating immune responses in vivo, we performed heterotopic cardiac transplants using fully MHC mismatched BALB/c donors and B6 recipients. II^-/4⁺ recipients reject wild-type allografts as quickly as wild-type recipients (Fig. 4). With II^-/4⁺ recipients, indirect Ag presentation to CD4⁺ cells cannot contribute to rejection because recipient APCs lack MHC class II. II^-/4⁺ recipients also acutely reject grafts from B7-1/B7-2^-/- mice. In this strain combination, B7 costimulation to CD4⁺ T cells can only be provided in trans, since donor cells express allo-MHC molecules (but not B7), and recipient APCs express B7 (but not MHC class II). Thus, graft rejection by II^-/4⁺ mice of a B7-deficient allograft by costimulation in trans has similar kinetics to rejection by costimulation in cis of a wild-type graft.

View larger version (13K): [in this window] [in a new window]   FIGURE 4. II-/4+ mice reject B7-1/B7-2-/- cardiac allografts by B7 costimulation in trans. Kaplan-Meier survival curves are shown for each indicated strain combination. WT, Wild type.

Cardiac allograft rejection by II^-/4⁺ mice is dependent on CD4⁺ T cells, but not CD8 T cells

Although CD4⁺ T cells from II^-/4⁺ mice cannot respond to indirect Ag presentation, CD8⁺ T cells from these mice potentially could be activated by the indirect pathway. To investigate whether rejection of B7-1/B7-2^-/- allografts by II^-/4⁺ recipients is mediated by costimulation in trans without any contribution of the indirect pathway, we selectively depleted CD4 or CD8 T cells by a short course of Ab treatment. Ab to CD4 significantly prolonged allograft survival compared with that in untreated recipients (mean survival time, 22 vs 7.8 days; p < 0.002), while Ab to CD8 did not significantly prolong graft survival (mean survival time, 9.0 vs 7.8 days; p > 0.05; Fig. 5). Thus, CD8 T cells, potentially activated by indirect Ag presentation, do not significantly contribute to rejection of B7-1/B7-2^-/- allografts by II^-/4⁺ mice. Therefore, costimulation in trans is sufficient to mediate cardiac allograft rejection in these mice.

View larger version (17K): [in this window] [in a new window]   FIGURE 5. Survival of B7-1/B7-2-/- cardiac allografts in II-/4+ recipients treated with depleting Abs. Kaplan-Meier survival curves are shown for each treatment group.

	Discussion

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Little progress has been made understanding costimulation in trans in recent years because of the difficulty of extending the initial studies to an in vivo system. Here we are able to use a combination of genetically deficient mice to determine whether costimulation in trans is physiologically important in a well-described model of cardiac transplantation. By using B7-1/B7-2^-/- donor hearts and II^-/4⁺ recipient mice, we restrict the mechanism of activation of CD4⁺ T cells to costimulation in trans. We find that II^-/4⁺ recipients reject B7-deficient grafts as rapidly as wild-type grafts, in which costimulation in cis is present. In addition, CD4⁺ T cells from II^-/4⁺ mice respond as strongly to B7-1/B7-2^-/- stimulators with B7 provided in trans as to wild-type stimulators. Thus, in both graft rejection and MLRs, the potency of costimulation in trans for stimulating an alloresponse is comparable to costimulation in cis.

One question that arises from these results is how can tolerance to autoantigens be maintained if costimulation in trans is sufficient to activate T cells. It has been postulated that circulating autoreactive T cells usually are not activated because they recognize self Ags on tissues such as epithelial cells, which lack B7 costimulators (17). Costimulation in trans may, in fact, be a mechanism by which self tolerance is broken, but usually tolerance is maintained because of several factors. First, the trans costimulatory signal must be delivered soon after the antigenic signal to be effective. In vitro, a delay of greater than several hours significantly limited the effectiveness of costimulation in trans (7, 18). Thus, self tolerance would be maintained as long as the trans costimulatory signal were provided after a delay. In contrast, during the immune response to an allograft, activated APCs are present simultaneously with alloantigen, so T cells could be expected to be activated even if the APC providing B7 is different from the APC providing Ag. Self tolerance is also likely to be more resistant to trans costimulation than the alloresponse, because the antigenic signal during an alloresponse is several orders of magnitude more potent. All graft cells are coated with high concentrations of foreign histocompatibility Ags, while autoantigens are likely to be present at low levels. Autoantigens expressed at high levels are likely to cause deletion of self-reactive T cells during thymic development by negative selection. Finally, the magnitude of the T cell response would be much greater in the response to an allograft, because 1 to 10% of an individual’s T cells respond to a given alloantigen (19), while the percentage of circulating autoreactive T cells is probably <0.01% (20).

In reporting that wild-type mice rapidly reject fully MHC-mismatched cardiac allografts lacking B7-1 and B7-2, we postulated that rejection might be mediated by either indirect Ag presentation or costimulation in trans (Fig. 1). Since indirect Ag presentation has been previously shown to mediate graft rejection, and we demonstrate here that costimulation in trans can also rejects grafts, it is likely that both mechanisms contribute to the rejection of B7-deficient grafts.

This study is the first to demonstrate that costimulation in trans can mediate an immune response in vivo. Our finding that costimulation in trans as well as indirect Ag presentation can mediate acute allograft rejection has important therapeutic implications. For example, attempts to prolong cardiac graft survival by pretreating grafts with reagents to block B7 before transplantation are unlikely to be successful, since recipient cells can provide sufficient B7 to cause rejection by each of these mechanisms. Our results contribute to the basic understanding of mechanisms of graft rejection and should help guide the development of therapeutic strategies to induce tolerance to allografts.

	Footnotes

 
¹ This work was supported by a grant from the American Heart Association (to D.A.M.) and National Institutes of Health Grants AI01212 (to D.A.M.), AI38310 and AI/GF41584 (to A.H.S.), and AI41521 and AI34965 (to M.H.S.).

² D.A.M. and K.K. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Didier A. Mandelbrot, Renal Division, University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, MA 01655. E-mail address: mandelbd{at}ummhc.org

⁴ A.H.S. and M.H.S. are co-senior authors.

Received for publication February 27, 2001. Accepted for publication May 16, 2001.

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