CD40-gp39 INTERACTIONS PLAY A CRITICAL ROLE DURING... : Transplantation (original) (raw)
Activated T cells play a pivotal role in the rejection of allografts(1,2). Activation of T cells to proliferate and secrete cytokines requires both recognition of major histocompatibility-peptide complexes and costimulation via molecules such as CD28, LFA-1, and CD2 (3, 4). Once activated, T cells may act as direct cytotoxic effectors or provide “help” for antibody production and macrophage-mediated cytotoxicity via cytokine release(soluble help) or contact-dependent (cognate help) mechanisms.
Upon activation, T cells express gp39, a member of the tumor necrosis factor cytokine superfamily, which serves as a ligand for CD40 on various antigen-presenting cells, including dendritic cells, B cells, macrophages, and endothelial cells (5, 6). Ligation of CD40 on these cells provides a signaling mechanism from the T cell to the antigen-presenting cells. For example, CD40 ligation induces expression of B7 molecules on both DC and B cells (7-9) and CD40 signals are required for initiation of T-dependent antibody responses(10,11). Finally, T cells provide cognate help for monocyte/macrophage differentiation and activation via the CD40-gp39 pathway(12, 13).
Studies in vivo have documented the importance of CD40-gp39 interactions in the development of humoral immune responses to foreign and auto-antigens(14-18). In contrast, the role of the CD40-gp39 pathway in predominantly cell-mediated immune responses, such as acute allograft rejection, has not been defined. In this report, we show that CD40 and gp39 transcripts are strongly induced during rejection. Further, perioperative treatment with MR1, a mAb specific for gp39, prolongs survival of cardiac allografts in both naive and sensitized recipients, whereas delaying treatment until postoperative day 5 has no effect on survival. Allografts from MR1-treated recipients have a decrease in expression of transcripts for the macrophage product, inducible nitric oxide synthase(iNOS*), but essentially unaltered expression of T cell cytokine transcripts, including interleukin (IL)-2, interferon (IFN)-γ, IL-4, and IL-10. In addition, alloantibody responses in the MR1-treated mice were significantly inhibited. These data suggest that blockade of CD40-gp39 interactions may inhibit allograft rejection primarily by interfering with T cell help for effector function, rather than by interference with T cell activation.
MATERIALS AND METHODS
Mice. Male C3H/He (H2k), BALB/c (H2d), C57BL/10(H2b), and C57BL/10-Igh-6tm1Cgn mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and used at 8-12 weeks of age.
Monoclonal antibodies. MR1, a hamster mAb specific for murine gp39, was produced as ascites and purified by ion-exchange HPLC(10).
Heart transplantation. Primarily vascularized heterotopic heart transplantation was performed using microsurgical techniques(19). Rejection was defined by the loss of palpable cardiac contractions with confirmation by laparotomy with direct visualization. Heart transplant recipients were treated intravenously with 250μg of MR1 at the time of transplantation and with intraperitoneal doses(250 μg) on days 2 and 4 after transplantation for a total of 3 treatments. Control recipients received no treatment. Prior studies in our laboratory have confirmed that survival of untreated recipients is identical to that in animals treated with control antibodies (human IgG and the L6 fusion protein, data not shown).
Sensitized recipients were treated subcutaneously with 0.25 ml of donor-specific fresh whole blood 7 days before transplantation. The MR1 treatment protocol for sensitized recipients was as described above.
Detection of alloantibody. Serum was obtained by tail bleed from normal C3H/He mice or C3H/He transplant recipients at specified times after transplantation. Anti-donor antibody titers were assayed using flow cytometry on the P815 (H2d) mastocytoma cell line. EL4 (H2b) cells were used as a third-party control. Cells were incubated with serum that had been diluted 1:10, 1:100, 1:500, and/or 1:1000 in PBS. Cells were washed twice with PBS and anti-H2d antibodies were detected with FITC-conjugated rat anti-mouse IgG2a, IgG1, or IgM (10 μg/ml, Jackson ImmunoResearch Inc., West Grove, PA).
Reverse transcriptase polymerase chain reaction (RT-PCR). At specified intervals after transplantation, the cardiac grafts were removed and total RNA was prepared from tissues using TRIzol reagent (Gibco BRL, Gaithersburg, MD). Complementary DNA was synthesized using 5 μg of total RNA template with a Superscript preamplification system (Gibco) in a final volume of 20 μl. PCR reactions contained 0.4 μl (25 mM) of dNTPs, 1.25 U/reaction Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN), 1 μM specific primers, and 2 μl of cDNA template in a final volume of 50 μl. The PCR cycles consisted of a 15-sec, 95°C denaturation step, a 30-sec annealing step (see temperatures in Table 1), and a 30-sec 72°C extension step on a Perkin-Elmer Cetus (Foster City, CA) 9600 Thermocycler. In some reactions, TaqStart antibody (0.275 μg/50 μl; Clontech Laboratories Inc., Palo Alto, CA) was also included. The specific primer sets and conditions used are summarized in Table 1. Conditions for semiquantitative analysis of each primer set were optimized by first maximizing sensitivity and specificity through Mg++ concentration and annealing temperatures and then titrating the cycle number such that the product was detectable but well below plateau. PCR products were visualized on ethidium-bromide-stained 1% agarose (Bio-Rad, Hercules, CA)/2% NuSieve GTG agarose (FMC BioProducts, Rockland, ME) gels. Gel images were stored using a UVP gel documentation system 5000. Band intensity was quantified using Gelreader analysis software (National Center for Supercomputing Applications, Urbana, IL).
RESULTS AND DISCUSSION
CD40 and gp39 are strongly expressed in rejecting cardiac allografts. The potent inhibitory effects of anti-gp39 on T-dependent antibody responses (21) and the impaired T-dependent immunity observed in patients with gp39 mutations (22) prompted us to explore the role of the CD40-gp39 pathway in allograft rejection. Primarily vascularized BALB/c or C3H/He hearts were transplanted into C3H/He recipients. Rejection of BALB/c hearts in C3H/He recipients occurs between 12 and 18 days with a median survival time (MST) of 12 days(Table 2, group 1). The expression of CD40 and gp39 transcripts in normal and transplanted heart tissue was examined on days 1, 3, 5, 8, and 12 after transplantation using RT-PCR. CD40 and gp39 transcripts were virtually undetectable in normal heart and were not significantly up-regulated in syngeneic transplants (Fig. 1). In contrast, both CD40 and gp39 were strongly expressed at posttransplant days 5, 8, and 12 (Fig. 1).
Anti-gp39 prolongs cardiac allograft survival in naive and sensitized recipients. The functional role of CD40-gp39 interactions in allograft rejection was determined by testing the effect of MR1, a mAb specific for gp39, on cardiac allograft survival. Treatment of recipient C3H/He mice with three 250-μg doses of MR1 produced marked prolongation of BALB/c cardiac allografts (MST>75 days, Table 2, group 2). Control recipients rejected BALB/c hearts at a median of 12 days(Table 2, group 1). Marked prolongation of cardiac allograft survival was also observed in the C3H/He to C57BL/10 strain combination (Table 2, groups 6 and 7). Interestingly, while MR1 alone had a minimal effect, the combination of CTLA4-Ig and MR1 produced markedly prolonged survival of primary skin grafts (manuscript in preparation).
Anti-gp39 interferes with signal transduction from activated T cells to CD40-bearing antigen-presenting cells and may thus block cognate T cell help for the effector arm of the immune response. Therefore, we hypothesized that anti-gp39 might also prolong the survival of cardiac allografts in sensitized recipients. C3H/He mice were primed subcutaneously with 0.25 ml of BALB/c blood 7 days before transplantation (Table 2). Primed recipients rejected their allografts in an accelerated fashion (MST=7 days,Table 2, groups 4) relative to unprimed C3H/He mice(MST=12 days, Table 2, groups 1). MR1 treatment markedly prolonged the survival of allografts in sensitized recipients (MST=63 days,Table 2 group 5), which suggests that the CD40-gp39 pathway plays an important role in immune responses, distinct from the initial sensitization phase of allograft rejection.
To explore the role of the CD40-gp39 pathway in ongoing rejection, unsensitized recipients were treated with MR1 beginning 5 days after transplantation. Five days after transplantation in this model corresponds to a time when T cell cytokines (IFN-γ, IL-2, IL-4, and IL-10), T cell and macrophage effector transcripts (perforin, granzyme, fas ligand, tumor necrosis factor-α, and iNOS), and CD40 and its ligand are readily detectable (Figs. 1 and 3), yet the heart remains functional as assessed by palpation. In contrast to therapy starting at the time of transplantation, delayed administration of anti-gp39 until postoperative day 5 failed to prevent or even significantly delay the rejection of BALB/c cardiac allografts in C3H/He recipients(Table 2, group 3 vs. group 1).
Anti-gp39 blocks alloantibody production and impairs intragraft expression of iNOS, while leaving T cell cytokine transcripts unaltered. Having ascertained that CD40-gp39 interactions play a pivotal role in allograft rejection, we initiated studies to explore the mechanisms by which anti-gp39 mAb might block the rejection responses. Previous in vitro and in vivo studies suggested several possible modes of action of MR1, including prevention of alloantibody formation, blockade of costimulation of CD4+ T cells, and inhibition of macrophage-mediated defense mechanisms. The crucial role of CD40-gp39 interactions in the development of antibody responses(21, 22) suggests that MR1 would prevent alloantibody development after transplantation. Alternatively, since the CD40-gp39 pathway has been reported to be an important costimulatory pathway for CD4+ T cells (23), blockade of gp39 might impair T cell activation either directly or indirectly through a decreased expression of B7 costimulatory molecules on dendritic and B cells. Decreased expression of the B7 molecules might block the positive feedback loop between activated T cells and antigen-presenting cells and thereby lead to a decreased expression of T cell cytokines such as IL-2, IL-4, and IFNγ. Finally, the CD40-gp39 pathway has been shown to be a major contributor to T cell-dependent activation of macrophage-mediated mechanisms(13). Therefore, during allograft rejection, anti-gp39 may prevent T cell-dependent macrophage activation and cytotoxicity.
The development of donor-specific alloantibodies in cardiac transplant recipients was compared using flow cytometry between untreated C3H/He recipients of BALB/c hearts and recipients treated with MR1. Normal C3H/He mice expressed no detectable anti-H2d IgM or IgG antibodies, whereas untreated cardiac allograft recipients developed IgM, IgG1, and IgG2a alloantibody responses by 30 days after transplantation (Fig. 2). In contrast, treatment with MR1 significantly inhibited the development of alloantibodies at this time point. Moreover, alloantibody responses were even blocked in the sensitized cardiac allograft recipients described above at days 60-70 after transplantation. In the two sensitized recipients with surviving allografts, there were no detectable anti-H2d IgG2a antibodies, whereas the recipient whose graft had failed had anti-H2d IgG2a antibodies at a titer of 1:100 (data not shown).
To begin to distinguish whether the dominant mechanism of action of MR1 in prolonging allograft survival involved its effect on blocking cognate T cell help for B cells, transplants were performed using B cell-deficient recipients. Untreated immunoglobulin heavy chain knock-out mice (C57BL/10 background) promptly rejected C3H hearts (MST=7 days, n=5,Table 2, group 8), whereas MR1-treated, B cell-deficient recipients experienced long-term allograft acceptance (MST>60, n=6,Table 2, group 9). These results indicate that B cells are not required for allograft rejection and that blockade of cognate help for recipient B cells is not the sole mechanism by which MR1 promotes graft acceptance. Nonetheless, the ability of MR1 to block this pathway may be of particular significance in the prevention of chronic rejection.
Next, intragraft expression of B7 costimulatory molecules, T cell cytokines, and macrophage activation/effector transcripts was assessed using RT-PCR. Allografts from treated and control recipients were removed for analysis 3, 5, 8, or 12 days after transplantation. Surprisingly, the expression of transcripts for B7 molecules and T cell cytokines (IL-2, IL-4, and IFNγ) was not appreciably altered in MR1-treated recipients relative to untreated controls (Fig. 3). In contrast, transcripts for the macrophage enzyme iNOS were reduced in allografts from MR1-treated recipients (Fig. 3).
Our data indicate that CD40-gp39 interactions play a critical role in the T-dependent immune response, allograft rejection. The importance of the CD40-gp39 pathway appears to be distinct from the sensitization phase of allograft rejection, as anti-gp39 was able to significantly prolong the survival of allografts in sensitized recipients. Furthermore, T cell cytokine transcripts for IL-2, IL-4, and IFNγ were essentially unaltered by MR1, as assessed by semiquantitative RT-PCR. More subtle differences in transcript expression or differences in protein expression due to posttranscriptional regulation cannot be excluded. Additionally, MR1 was unable to rescue allografts when treatment was delayed until post-operative day 5, which suggests that MR1 is not able to block effector function once initiated. Taken together, our data suggest that gp39 is a pivotal molecule in the transition between the afferent and efferent arms of the immune system. Blockade of CD40-gp39 interactions interrupts T cell help for alloantibody production and for iNOS induction. The later finding is of particular interest in light of recent reports implicating nitric oxide as an important mediator in the rejection process (24). Moreover, in vitro data suggest that CD40-gp39 signals are important for the induction of iNOS activity in macrophages (13). The absence of alloantibody may also indirectly inhibit the role of macrophage-mediated effector function by preventing antibody-dependent, cell-mediated cytotoxicity.
Why anti-gp39, which has failed to promote T cell unresponsiveness in vivo in other models (15), should produce long-term allograft survival is unclear. This may be due in part to the long serum half-life of the MR1 mAb (15). Indeed, 3 of 4 MR1-treated transplant recipients with surviving heart transplants had low (0.15-7.9 μg/ml) but detectable serum levels of MR1 when analyzed at between 87 and 95 days after transplantation. The functional significance of this concentration of mAb in vivo is unknown. Alternatively, perioperative treatment with anti-gp39, like a variety of immunosuppressive agents, may prevent early rejection, and by allowing prolonged exposure of the recipient immune system to donor antigen in the absence of intact costimulatory pathways, it may promote the development of long-term graft acceptance (25, 26).
Inhibition of the gp39 signaling mechanism provides a potent means of interrupting the T-dependent process of allograft rejection. Unlike existing agents, such as cyclosporine, blockade of the CD40-gp39 pathway may be an effective treatment modality for preventing activated T cells from providing cognate help for the effector arm of both nascent and ongoing immune responses. Moreover, the suppression of alloantibody responses might retard the development of chronic rejection. Anti-gp39 may, therefore, be an attractive adjunct to existing agents that block T cell activation, such as cyclosporine, and new agents, such as CTLA4-Ig.
Acknowledgments. We thank Kathryn J. Wood and Peter Jensen for their critical review of the manuscript.
CD40 and gp39 are strongly expressed in rejecting cardiac allografts. Total RNA was prepared from normal heart tissue and cardiac grafts at 1, 3, 5, 8, and 12 days after transplant and analyzed for CD40 and gp39 transcripts by RT-PCR. CD40 and gp39 transcripts were virtually undetectable in normal heart (N) and were not significantly up-regulated in syngeneic transplants. In contrast, both CD40 and gp39 were detectable at low levels as early as 3 days (visible on the original gel) and were strongly expressed at days 5, 8, and 12 after transplantation in allografts. RT-PCR reactions without cDNA template (-) yielded no specific products.
MR1 treatment prevents the development of alloantibodies in cardiac allograft recipients. Serum from normal C3H/He mice or C3H/He recipients of BALB/c cardiac allografts (at 32 days after transplantation) was assayed for anti-H2d antibodies using flow cytometry on the P815(H2d) mastocytoma cell line or EL4 (H2b) cells as a third-party control. Cells were incubated with serum from normal mice diluted 1:10 in PBS(―) (a-f) or serum from transplanted mice diluted 1:10 (• • • •), 1:100(----), or 1:500 (••••••) in PBS. Normal C3H/He mice expressed no detectable anti-H2d IgM or IgG donor-specific alloantibodies (solid lines, a -f),whereas untreated C3H/He recipients of BALB/c hearts (a-c) developed IgM (a), IgG1 (b), and IgG2a (c) alloantibodies by 30-35 days after transplantation. Treatment with MR1 completely abrogated the development of both IgM and IgG alloantibodies (d-f). A representative experiment is shown; similar results were obtained in 4 individual MR1-treated recipients. Labeling of EL4 cells was uniformly absent, except for low level cross-reactivity of untreated allograft recipient serum at 1:10 only (data not shown).
Expression of immune mediator transcripts in MR1-treated cardiac allografts. Intragraft expression of immune mediator transcripts was assessed using RT-PCR. Allografts from treated and control animals were analyzed at 1, 3, 5, 8, and 12 days after transplantation. Normal heart tissue (N) and a syngeneic heart graft (Syn) at 8 days after transplantation were included for comparison. No consistent differences in the expression of T cell cytokine transcripts for IL-2, IL-4, IL-10, and IFNγ were detectable between the control allografts and MR1-treated allografts. Similarly, B7-1 and B7-2 transcripts were not different as assessed with this technique. In contrast, transcripts for the macrophage effector molecule, iNOS, were consistently decreased at days 5, 8, and 12 after transplantation. RT-PCR reactions for all primer sets yielded no products in the absence of cDNA template (-). Moreover, PCR reactions using template prepared without reverse transcriptase yielded no products, even for the intron-less GADPH gene, confirming the absence of contaminating genomic DNA. Similar results were obtained on two sets of transplants each analyzed on two independent sets of cDNA reactions.
Footnotes
This study was supported by NIH grants 1R29 AI33588-01A1, AR42687, and 1F32 HL09226-01A1, an American Heart Association grant-in-aid, and an Emory University research committee grant.
Abbreviations: IFN, interferon; IL, interleukin; iNOS, inducible nitric oxide synthase; MST, median survival time; RT-PCR, reverse transcriptase polymerase chain reaction.
REFERENCES
1. Dallman MJ, Mason DW, Webb M. The roles of host and donor cells in the rejection of skin allografts by T cell-deprived rats injected with syngeneic T cells. Eur J Immunol 1982; 12: 511-518.
2. Bolton EM, Gracie JA, Briggs JD, Kampinga J, Bradley JA. Cellular requirements for renal allograft rejection in the athymic nude rat. J Exp Med 1989; 169: 1931-1946.
3. June CH, Ledbetter JA, Linsley PS, Thompson CB. Role of the CD28 receptor in T-cell activation. Immunol Today 1990; 11(6): 211-216.
4. Jenkins MK, Taylor PS, Norton SD, Urdahl KB. CD28 delivers a costimulatory signal involved in antigen-specific IL-2 production by human T cells. J Immunol 1991; 147(8): 2461-2466.
5. Hollenbaugh D, Grosmaire LS, Kullas CD, et al. The human T cell antigen gp39, a member of the TNF gene family, is a ligand for the CD40 receptor: expression of a soluble form of gp39 with B cell co-stimulatory activity. Embo J 1992; 11(12): 4313-4321.
6. Armitage RJ, Fanslow WC, Strockbine L, et al. Molecular and biological characterization of a murine ligand for CD40. Nature 1992; 357(6373): 80-82.
7. Caux C, Massacrier C, Vanbervliet B, et al. Activation of human dendritic cells through CD40 cross-linking. J Exp Med 1994; 180(4): 1263-1272.
8. Pinchuk L, Polacino P, Agy M, Klaus S, Clark E. The role of CD40 and CD80 accessory cell molecules in dendritic cell-dependent HIV-1 infection. Immunity 1994; 1: 317-325.
9. Ranheim EA, Kipps TJ. Activated T cells induce expression of B7/BB1 on normal or leukemic B cells through a CD40-dependent signal. J Exp Med 1993; 177(4): 925-935.
10. Noelle R, Roy M, Shepherd D, Stamenkovic I, Ledbetter J, Aruffo A. A 39-kDa protein on activated helper T cells binds CD40 and transduces the signal for cognate activation of B cells. Proc Natl Acad Sci U S A 1992; 89: 6550-6554.
11. Lane P, Brocker T, Hubele S, Padovan E, Lanzavecchia A, Mc-Connell F. Soluble CD40 ligand can replace the normal T cell-derived CD40 ligand signal to B cells in T cell-dependent activation. J Exp Med 1993; 177(4): 1209-1213.
12. Alderson M, Armitage R, Tough T, Strockbine L, Fanslow W, Spriggs M. CD40 expression by human monocytes: regulation by cytokines and activation of monocytes by the ligand for CD40. J Exp Med 1993; 178: 669-674.
13. Tian L, Noelle R, Lawrence D. Activated T cell enhance nitric oxide production by murine splenic macrophages through gp39 and LFA-1. Eur J Immunol 1995; 25: 306-309.
14. Durie FH, Fava RA, Foy TM, Aruffo A, Ledbetter JA, Noelle RJ. Prevention of collagen-induced arthritis with an antibody to gp39, the ligand for CD40. Science 1993; 261(5126): 1328-1330.
15. Foy TM, Shepherd DM, Durie FH, Aruffo A, Ledbetter JA, Noelle RJ. In vivo CD40-gp39 interactions are essential for thymus-dependent humoral immunity. II. Prolonged suppression of the humoral immune response by an antibody to the ligand for CD40, gp39. J Exp Med 1993; 178(5): 1567-1575.
16. Van den Eertwegh AJM, Noelle RJ, Roy M, et al. In vivo CD40-gp39 interactions are essential for thymus-dependent humoral immunity. I. In vivo expression of CD40 ligand, cytokines, and antibody production delineates sites of cognate T-B cell interactions. J Exp Med 1993; 178(5): 1555-1565.
17. Renshaw BR, Fanslow WC, Armitage RJ, et al. Humoral immune responses in CD40 ligand-deficient mice. J Exp Med 1994; 180(5): 1889-1900.
18. Mohan C, Shi Y, Laman JD, Datta SK. Interaction between CD40 and its ligand gp39 in the development of murine lupus nephritis. J Immunol 1995; 154(3): 1470-1480.
19. Corry RJ, Winn HJ, Russell PS. Primarily vascularized allografts of hearts in mice: the role of H-2D, H-2K, and non H-2 antigens in rejection. Transplantation 1973; 16(4): 343-350.
20. Dallman MJ, Larsen CP, Morris PJ. Cytokine gene transcription in vascularized organ grafts: analysis using semi-quantitative polymerase chain reaction. J Exp Med 1991; 174: 493-496.
21. Kawabe T., Naka T, Yoshida K, et al. The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal center formation. Immunity 1994; 1: 167-178.
22. Aruffo A., Farrington M, Hollenbaugh D, et al. The CD40 ligand, gp39, is defective in activated T cells from patients with X-linked hyper-IgM syndrome. Cell 1993; 72(2): 291-300.
23. Cayabyab M, Phillips JH, Lanier LL. CD40 preferentially costimulates activation of CD4+ T lymphocytes. J Immunol 1994; 152(4): 1523-1531.
24. Worrall NK, Lazenby WD, Misko TP, et al. Modulation of in vivo alloreactivity by inhibition of inducible nitric oxide synthase. J Exp Med 1995; 181(1): 63-70.
25. Ramsdell F, Fowlkes BJ. Maintenance of in vivo tolerance by persistence of antigen. Science 1992; 257(5073): 1130-1134.
26. Matzinger P. Tolerance, danger, and the extended family. Annu Rev Immunol 1994; 12: 991-1045.
27. Ju ST, Panka DJ, Cui H. Fas(CD95)/FasL interactions required for programmed cell death after T-cell activation. Nature 1995; 373: 444-448.
28. Torres R, Clark E. Differential increase of an alternatively polyadenylated mRNA species of murine CD40 upon B lymphocyte activation. J Immunol 1992; 148: 620-626.
29. Takeuchi T, Lowry R, Konieczny B. Heart allografts in murine systems: the differential activation of Th2-line effector cells in peripheral tolerance. Transplantation 1992; 53: 1281-1294.
30. Stenger S, Thuring H, Rollinghoff M, Bogdan C. Tissue expression of inducible nitric oxide synthase is closely associated with resistance to Leishmania major. J Exp Med 1994; 180(3): 783-793.
31. Freeman GJ, Gray GS, Gimmi CD, et al. Structure, expression, and T cell costimulatory activity of the murine homologue of the human B lymphocyte activation antigen B7. J Exp Med 1991; 174: 625-631.
32. Freeman G, Borriello F, Hodes R, et al. Murine B7-2, an alternative CTLA4 counter-receptor that costimulates T cell proliferation and Interleukin 2 production. J Exp Med 1993; 178: 2185-2192.
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