Intact active bone transplantation synergizes with anti-CD40 ligand therapy to induce B cell tolerance

Blockade of T cell costimulatory pathways can result in the prolongation of allograft survival through the suppression of Th1 responses; however, late allograft rejection is usually accompanied by an emerging allograft-specific humoral response. We have recently determined that intact active bone (IAB) fragments transplanted under the kidney capsule can synergize with transient anti-CD40 ligand (CD40L) treatment to induce robust donor-specific allograft tolerance and suppress the alloantibody response. In this study, we take advantage of the ability of galactosyltransferase-deficient knockout (GT-Ko) mice to respond to the carbohydrate epitope, galactose-alpha1,3-galactose (Gal), to investigate whether IAB plus transient anti-CD40L therapy directly tolerize B cell responses. GT-Ko mice tolerized to Gal-expressing C3H hearts and IAB plus transient anti-CD40L therapy were challenged with pig kidney membranes that express high levels of Gal. The anti-Gal IgM and IgG responses were significantly suppressed in IAB-tolerant mice compared with controls, while the non-Gal anti-pig Ab responses were comparable. The anti-pig T cell cytokine response (IFN-gamma and IL-4) was comparable in IAB-tolerant and control mice. The tolerant state for the anti-Gal IgM response could be reversed with repeated immunization, whereas the tolerant state for the IgG response was robust and resisted repeated immunization. These observations provide an important proof-of-concept that adjunct therapies can synergize with anti-CD40L Abs to tolerize B cell responses independent of their effects on T cells. This model, which does not require mixed chimerism, provides a unique opportunity for investigating the mechanism of peripheral tolerance in a clinically relevant population of carbohydrate-specific B cells.     

Antibody-mediated rejection of cardiac allografts in CCR5-deficient recipients

Rejected MHC-mismatched cardiac allografts in CCR5(-/-) recipients have low T cell infiltration, but intense deposition of C3d in the large vessels and capillaries of the graft, characteristics of Ab-mediated rejection. The roles of donor-specific Ab and CD4 and CD8 T cell responses in the rejection of complete MHC-mismatched heart grafts by CCR5(-/-) recipients were directly investigated. Wild-type C57BL/6 and B6.CCR5(-/-) (H-2(b)) recipients of A/J (H-2(a)) cardiac allografts had equivalent numbers of donor-reactive CD4 T cells producing IFN-gamma, whereas CD4 T cells producing IL-4 were increased in CCR5(-/-) recipients. Numbers of donor-reactive CD8 T cells producing IFN-gamma were reduced 60% in CCR5(-/-) recipients. Day 8 posttransplant serum titers of donor-specific Ab were 15- to 25-fold higher in CCR5(-/-) allograft recipients, and transfer of this serum provoked cardiac allograft rejection in RAG-1(-/-) recipients within 14 days, whereas transfer of either serum from wild-type recipients or immune serum from CCR5-deficient recipients diluted to titers observed in wild-type recipients did not mediate this rejection. Wild-type C57BL/6 and B6.CCR5(-/-) recipients rejected A/J cardiac grafts by day 11, whereas rejection was delayed (day 12-60, mean 21 days) in muMT(-/-)/CCR5(-/-) recipients. These results indicate that the donor-specific Ab produced in CCR5(-/-) heart allograft recipients is sufficient to directly mediate graft rejection, and the absence of recipient CCR5 expression has differential effects on the priming of alloreactive CD4 and CD8 T cells.     

Tolerance to cardiac allografts via local and systemic mechanisms after adenovirus-mediated CTLA4Ig expression

Blockade of the CD28/B7 T cell costimulatory pathway prolongs allograft survival and induces tolerance in some animal models. We analyzed the efficacy of a CTLA4Ig-expressing adenovirus in preventing cardiac allorejection in rats, the mechanisms underlying heart transplant acceptance, and whether the effects of CTLA4Ig were restricted to the graft microenvironment or were systemic. CTLA4Ig gene transfer into the myocardium allowed indefinite graft survival (>100 days vs 9 +/- 1 days for controls) in 90% of cases, whereas CTLA4Ig protein injected systemically only prolonged cardiac allograft survival (by up to 22 days). CTLA4Ig could be detected in the graft and in the serum for at least 1 year after gene transfer. CTLA4Ig gene transfer induced local intragraft immunomodulation at day 5 after transplantation, as shown by decreased expression of the IL-2R and MHC II Ags; decreased levels of mRNA encoding for IFN-gamma, inducible NO synthase, and TGF-beta; and inhibited proliferative responses of graft-infiltrating cells. Systemic immune responses were also down-modulated, as shown by the suppression of Ab production against donor alloantigens and cognate Ags, up to at least 120 days after gene transfer. Alloantigenic and mitogenic proliferative responses of graft-infiltrating cells and total splenocytes were inhibited and were not reversed by IL-2. In contrast, lymph node cells and T cells purified from splenocytes showed normal proliferation. Recipients of long-term grafts treated with adenovirus coding for CTLA4Ig showed organ and donor-specific tolerance. These data show that expression of CTLA4Ig was high and long lasting after adenovirus-mediated gene transfer. This expression resulted in down-modulation of responses against cognate Ags, efficient suppression of local and systemic allograft immune responses, and ultimate induction of donor-specific tolerance.     

IL-6 promotes cardiac graft rejection mediated by CD4+ cells

IL-6 mediates numerous immunologic effects relevant to transplant rejection; however, its specific contributions to these processes are not fully understood. To this end, we neutralized IL-6 in settings of acute cardiac allograft rejection associated with either CD8(+) or CD4(+) cell-dominant responses. In a setting of CD8(+) cell-dominant graft rejection, IL-6 neutralization delayed the onset of acute rejection while decreasing graft infiltrate and inverting anti-graft Th1/Th2 priming dominance in recipients. IL-6 neutralization markedly prolonged graft survival in the setting of CD4(+) cell-mediated acute rejection and was associated with decreased graft infiltrate, altered Th1 responses, and reduced serum alloantibody. Furthermore, in CD4(+) cell-dominated rejection, IL-6 neutralization was effective when anti-IL-6 administration was delayed by as many as 6 d posttransplant. Finally, IL-6-deficient graft recipients were protected from CD4(+) cell-dominant responses, suggesting that IL-6 production by graft recipients, rather than grafts, is necessary for this type of rejection. Collectively, these observations define IL-6 as a critical promoter of graft infiltration and a shaper of T cell lineage development in cardiac graft rejection. In light of these findings, the utility of therapeutics targeting IL-6 should be considered for preventing cardiac allograft rejection.     

The role of the CD134-CD134 ligand costimulatory pathway in alloimmune responses in vivo

The CD134-CD134 ligand (CD134L) costimulatory pathway has been shown to be critical for both T and B cell activation; however, its role in regulating the alloimmune response remains unexplored. Furthermore, its interactions with other costimulatory pathways and immunosuppressive agents are unclear. We investigated the effect of CD134-CD134L pathway blockade on allograft rejection in fully MHC-mismatched rat cardiac and skin transplantation models. CD134L blockade alone did not prolong graft survival compared with that of untreated recipients, and in combination with donor-specific transfusion, cyclosporine, or rapamycin, was less effective than B7 blockade in prolonging allograft survival. However, in combination with B7 blockade, long-term allograft survival was achieved in all recipients (>200 days). Moreover, this was synergistic in reducing the frequency of IFN-gamma-producing alloreactive lymphocytes and inhibiting the generation of activated/effector lymphocytes. Most impressively, this combination prevented rejection in a presensitized model using adoptive transfer of primed lymphocytes into athymic heart transplant recipients. In comparison to untreated recipients (mean survival time (MST): 5.3 +/- 0.5 days), anti-CD134L mAb alone modestly prolonged allograft survival (MST: 14 +/- 2.8 days) as did CTLA4Ig (MST: 21.5 +/- 1.7 days), but all grafts were rejected within 24 days. Importantly, combined blockade further and significantly prolonged allograft survival (MST: 75.3 +/- 12.7 days) and prevented the expansion and/or persistence of primed/effector alloreactive T cells. Our data suggest that CD134-CD134L is a critical pathway in alloimmune responses, especially recall/primed responses, and is synergistic with CD28-B7 in mediating T cell effector responses during allograft rejection. Understanding the mechanisms of collaboration between these different pathways is important for the development of novel strategies to promote long-term allograft survival.     

A role for fractalkine and its receptor (CX3CR1) in cardiac allograft rejection

The hallmark of acute allograft rejection is infiltration of the inflamed graft by circulating leukocytes. We studied the role of fractalkine (FKN) and its receptor, CX(3)CR1, in allograft rejection. FKN expression was negligible in nonrejecting cardiac isografts but was significantly enhanced in rejecting allografts. At early time points, FKN expression was particularly prominent on vascular tissues and endothelium. As rejection progressed, FKN expression was further increased, with prominent anti-FKN staining seen around vessels and on cardiac myocytes. To determine the capacity of FKN on endothelial cells to promote leukocyte adhesion, we performed adhesion assays with PBMC and monolayers of TNF-alpha-activated murine endothelial cells under low-shear conditions. Treatment with either anti-FKN or anti-CX(3)CR1-blocking Ab significantly inhibited PBMC binding, indicating that a large proportion of leukocyte binding to murine endothelium occurs via the FKN and CX(3)CR1 adhesion receptors. To determine the functional significance of FKN in rejection, we treated cardiac allograft recipients with daily injections of anti-CX(3)CR1 Ab. Treatment with the anti-CX(3)CR1 Ab significantly prolonged allograft survival from 7 +/- 1 to 49 +/- 30 days (p < 0.0008). These studies identify a critical role for FKN in the pathogenesis of acute rejection and suggest that FKN may be a useful therapeutic target in rejection.     

Modulation of tissue-specific immune response to cardiac myosin can prolong survival of allogeneic heart transplants

The role of immune response to tissue-specific Ags in transplant rejection is poorly defined. We have previously reported that transplantation of cardiac allografts triggers a CD4(+) Th1 cell response to cardiac myosin (CM), a major contractile protein of the heart, and that pretransplant activation of proinflammatory CM-specific T cells accelerates rejection. In this study, we show that administration of CM together with IFA (CM/IFA) can prevent acute rejection of an allogeneic heart transplant. Prolongation of cardiac graft survival is associated with activation of CM- and allo-specific T cells secreting type 2 cytokines (IL-4, IL-5) and reduction of the frequency of proinflammatory IFN-gamma-secreting (type 1) alloreactive T cells. Blocking of IL-4 cytokine with Abs abrogates the prolongation. CM/IFA treatment prevents acute rejection of MHC class I-mismatched, but not fully mismatched grafts. However, if donor heart is devoid of MHC class II expression, CM-IFA administration delays rejection of fully allogeneic cardiac transplants. This finding suggests that the effect of CM modulation depends on the type (direct vs indirect) and strength of recipient's CD4(+) T cell alloresponse. Our results underscore the important role of host immunity to tissue-specific Ags in the rejection of an allograft. This study demonstrates that modulation of the immune response to a tissue-specific Ag can significantly prolong cardiac allograft survival, an observation that may have important implications for the development of novel selective immune therapies in transplantation.     

Anti-MHC class I antibody activation of proliferation and survival signaling in murine cardiac allografts

Anti-MHC class I alloantibodies have been implicated in the process of acute and chronic rejection because these Abs can bind to endothelial cells and transduce signals leading to the activation of cell survival and proliferation pathways. To characterize the role of the MHC class I-signaling pathway in the pathogenesis of Ab-mediated rejection, we developed a mouse vascularized heterotopic cardiac allograft model in which B6.RAG1 KO hosts (H-2K(b)/D(b)) received a fully MHC-incompatible BALB/c (H-2K(d)/D(d)) heart transplant and were passively transfused with anti-donor MHC class I Ab. We demonstrate that cardiac allografts of mice treated with anti-MHC class I Abs show characteristic features of Ab-mediated rejection including microvascular changes accompanied by C4d deposition. Phosphoproteomic analysis of signaling molecules involved in the MHC class I cell proliferation and survival pathways were elevated in anti-class I-treated mice compared with the isotype control-treated group. Pairwise correlations, hierarchical clustering, and multidimensional scaling algorithms were used to dissect the class I-signaling pathway in vivo. Treatment with anti-H-2K(d) Ab was highly correlated with the activation of Akt and p70S6Kinase (S6K). When measuring distance as a marker of interrelatedness, multidimensional scaling analysis revealed a close association between members of the mammalian target of rapamycin pathway including mammalian target of rapamycin, S6K, and S6 ribosomal protein. These results provide the first analysis of the interrelationships between these signaling molecules in vivo that reflects our knowledge of the signaling pathway derived from in vitro experiments.     

Absence of recipient CCR5 promotes early and increased allospecific antibody responses to cardiac allografts

Acute rejection is mediated by T cell infiltration of allografts, but mechanisms mediating the delayed rejection of allografts in chemokine receptor-deficient recipients remain unclear. The rejection of vascularized, MHC-mismatched cardiac allografts by CCR5(-/-) recipients was investigated. Heart grafts from A/J (H-2(a)) donors were rejected by wild-type C57BL/6 (H-2(b)) recipients on day 8-10 posttransplant vs day 8-11 by CCR5(-/-) recipients. When compared with grafts from wild-type recipients, however, significant decreases in CD4(+) and CD8(+) T cells and macrophages were observed in rejecting allografts from CCR5-deficient recipients. These decreases were accompanied by significantly lower numbers of alloreactive T cells developing to IFN-gamma-, but not IL-4-producing cells in the CCR5(-/-) recipients, suggesting suboptimal priming of T cells in the knockout recipients. CCR5 was more prominently expressed on activated CD4(+) than CD8(+) T cells in the spleens of allograft wild-type recipients and on CD4(+) T cells infiltrating the cardiac allografts. Rejecting cardiac allografts from wild-type recipients had low level deposition of C3d that was restricted to the graft vessels. Rejecting allografts from CCR5(-/-) recipients had intense C3d deposition in the vessels as well as on capillaries throughout the graft parenchyma similar to that observed during rejection in donor-sensitized recipients. Titers of donor-reactive Abs in the serum of CCR5(-/-) recipients were almost 20-fold higher than those induced in wild-type recipients, and the high titers appeared as early as day 6 posttransplant. These results suggest dysregulation of alloreactive Ab responses and Ab-mediated cardiac allograft rejection in the absence of recipient CCR5.     

Programmed death-1 targeting can promote allograft survival

The recently identified CD28 homolog and costimulatory molecule programmed death-1 (PD-1) and its ligands, PD-L1 and PD-L2, which are homologs of B7, constitute an inhibitory regulatory pathway of potential therapeutic use in immune-mediated diseases. We examined the expression and functions of PD-1 and its ligands in experimental cardiac allograft rejection. In initial studies, we found that most normal tissues and cardiac isografts had minimal expression of PD-1, PD-L1, or PD-L2, but intragraft induction of all three molecules occurred during development of cardiac allograft rejection. Intragraft expression of all three genes was maintained despite therapy with cyclosporin A or rapamycin, but was prevented in the early posttransplant period by costimulation blockade using CD154 or anti-inducible costimulator mAb. We prepared PD-L1.Ig and PD-L2.Ig fusion proteins and showed that each bound to activated PD-1(+) T cells and inhibited T cell functions in vitro, thereby allowing us to test the effects of PD-1 targeting on allograft survival in vivo. Neither agent alone modulated allograft rejection in wild-type recipients. However, use of PD-L1.Ig administration in CD28(-/-) recipients, or in conjunction with immunosuppression in fully MHC-disparate combinations, markedly prolonged cardiac allograft survival, in some cases causing permanent engraftment, and was accompanied by reduced intragraft expression of IFN-gamma and IFN-gamma-induced chemokines. PD-L1.Ig use also prevented development of transplant arteriosclerosis post-CD154 mAb therapy. These data show that when combined with limited immunosuppression, or in the context of submaximal TCR or costimulatory signals, targeting of PD-1 can block allograft rejection and modulate T and B cell-dependent pathologic immune responses in vivo.     

Immune Depletion in Combination with Allogeneic Islets Permanently Restores Tolerance to Self-Antigens in Diabetic NOD Mice

The destruction of beta cells in type 1 diabetes (T1D) results in loss of insulin production and glucose homeostasis. Treatment of non-obese diabetic (NOD) mice with immune-depleting/modulating agents (e.g., anti-CD3, murine anti-thymocyte-globulin (mATG)) can lead to diabetes reversal. However, for preclinical studies with these and other agents seeking to reverse disease at onset, the necessity for exogenous insulin administration is debated. Spontaneously diabetic NOD mice were treated with a short-course of mATG and insulin provided as drug therapy or by way of allogeneic islet implants. Herein we demonstrate that exogenous insulin administration is required to achieve disease reversal with mATG in NOD mice. Unexpectedly, we also observed that provision of insulin by way of allogeneic islet implantation in combination with mATG leads to a pronounced reversal of diabetes as well as restoration of tolerance to self-islets. Expansion/induction of regulatory cells was observed in NOD mice stably cured with mATG and allogeneic islets. These data suggest that transient provision of allogeneic insulin-producing islets might provide a temporary window for immune depletion to be more effective and instilling stable tolerance to endogenous beta cells. These findings support the use of a never before explored approach for preserving beta cell function in patients with recent onset T1D.     

CCR5 blockade modulates inflammation and alloimmunity in primates

Pharmacologic antagonism of CCR5, a chemokine receptor expressed on macrophages and activated T cells, is an effective antiviral therapy in patients with macrophage-tropic HIV infection, but its efficacy in modulating inflammation and immunity is only just beginning to be investigated. In this regard, the recruitment of CCR5-bearing cells into clinical allografts is a hallmark of acute rejection and may anticipate chronic rejection, whereas conventionally immunosuppressed renal transplant patients homozygous for a nonfunctional Delta32 CCR5 receptor rarely exhibit late graft loss. Therefore, we explored the effects of a potent, highly selective CCR5 antagonist, Merck's compound 167 (CMPD 167), in an established cynomolgus monkey cardiac allograft model. Although perioperative stress responses (fever, diminished activity) and the recruitment of CCR5-bearing leukocytes into the graft were markedly attenuated, anti-CCR5 monotherapy only marginally prolonged allograft survival. In contrast, relative to cyclosporine A monotherapy, CMPD 167 with cyclosporine A delayed alloantibody production, suppressed cardiac allograft vasculopathy, and tended to further prolong graft survival. CCR5 therefore represents an attractive therapeutic target for attenuating postsurgical stress responses and favorably modulating pathogenic alloimmunity in primates, including man.     

B lymphocyte-directed immunotherapy promotes long-term islet allograft survival in nonhuman primates

We found that an induction immunotherapy regimen consisting of rabbit anti-thymocyte globulin (Thymoglobulin) and the monoclonal antibody to CD20 rituximab (Rituxan) promoted long-term islet allograft survival in cynomolgus macaques maintained on rapamycin monotherapy. B lymphocyte reconstitution after rituximab-mediated depletion was characterized by a preponderance of immature and transitional cells, whose persistence was associated with long-term islet allograft survival. Development of donor-specific alloantibodies was abrogated only in the setting of continued rapamycin monotherapy.     

Blockade of Notch ligand δ1 promotes allograft survival by inhibiting alloreactive Th1 cells and cytotoxic T cell generation

The Notch signaling pathway has been recently shown to contribute to T cell differentiation in vitro. However, the in vivo function of Notch signaling in transplantation remains unknown. In this study, we investigated the importance of Delta1 in regulating the alloimmune response in vivo. Delta1 expression was upregulated on dendritic cells and monocytes/macrophages upon transplantation in a BALB/c into B6 vascularized cardiac transplant model. Whereas administration of anti-Delta1 mAb only slightly delayed survival of cardiac allografts in this fully MHC-mismatched model, it significantly prolonged graft survival in combination with single-dose CTLA4-Ig or in CD28 knockout recipients. The prolongation of allograft survival was associated with Th2 polarization and a decrease in Th1 and granzyme B-producing cytotoxic T cells. The survival benefit of Delta1 blockade was abrogated after IL-4 neutralization and in STAT6KO recipients, but was maintained in STAT4KO recipients, reinforcing the key role of Th2 cell development in its graft-prolonging effects. To our knowledge, these data demonstrate for the first time an important role of Delta1 in alloimmunity, identifying Delta1 ligand as a potential novel target for immunomodulation in transplantation.     

B lymphocytes differentially influence acute and chronic allograft rejection in mice

The relative contributions of B lymphocytes and plasma cells during allograft rejection remain unclear. Therefore, the effects of B cell depletion on acute cardiac rejection, chronic renal rejection, and skin graft rejection were compared using CD20 or CD19 mAbs. Both CD20 and CD19 mAbs effectively depleted mature B cells, and CD19 mAb treatment depleted plasmablasts and some plasma cells. B cell depletion did not affect acute cardiac allograft rejection, although CD19 mAb treatment prevented allograft-specific IgG production. Strikingly, CD19 mAb treatment significantly reduced renal allograft rejection and abrogated allograft-specific IgG development, whereas CD20 mAb treatment did not. By contrast, B cell depletion exacerbated skin allograft rejection and augmented the proliferation of adoptively transferred alloantigen-specific CD4(+) T cells, demonstrating that B cells can also negatively regulate allograft rejection. Thereby, B cells can either positively or negatively regulate allograft rejection depending on the nature of the allograft and the intensity of the rejection response. Moreover, CD19 mAb may represent a new approach for depleting both B cells and plasma cells to concomitantly impair T cell activation, inhibit the generation of new allograft-specific Abs, or reduce preexisting allograft-specific Ab levels in transplant patients.     

Early chemokine cascades in murine cardiac grafts regulate T cell recruitment and progression of acute allograft rejection

The identification of early inflammatory events after transplant in solid tissue organ grafts that may direct T cell recruitment and promote acute allograft rejection remain largely unknown. To better understand temporal aspects of early inflammatory events in vascularized organ grafts, we tested the intragraft expression of four different chemokines in heterotopically transplanted A/J (H-2(a)) and syngeneic heart grafts in C57BL/6 (H-2(b)) recipient mice from 1.5 to 48 h after transplant. Similar temporal expression patterns and equivalent levels of chemokine expression were observed in both syngeneic and allogeneic cardiac allografts during this time period. Expression of the neutrophil chemoattractant growth-related oncogene alpha (KC) was observed first and reached peak levels by 6 h after transplant and was followed by the monocyte/macrophage chemoattractant protein-1 (JE) and then macrophage inflammatory proteins 1beta and 1alpha. Administration of rabbit KC antiserum to allograft recipients within 30 min of cardiac transplantation attenuated downstream events including intra-allograft expression of the T cell chemoattractants IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma, cellular infiltration into the allograft, and graft rejection. Similarly, depletion of recipient neutrophils at the time of transplantation significantly extended allograft survival from day 8 to 10 in control-treated recipients up to day 21 after transplant. These results indicate the induction of highly organized cascades of neutrophil and macrophage chemoattractants in cardiac grafts and support the proposal that early inflammatory events are required for optimal recruitment of T cells into allografts during the progression of acute rejection of cardiac allografts.     

The generation of CD25+ CD4+ regulatory T cells that prevent allograft rejection does not compromise immunity to a viral pathogen

In all but a small minority of cases, continued survival of solid organ grafts after transplantation depends on lifelong, nonselective immunosuppression that, although effective, results in increased rates of infection, cancer, and vascular disease. Therapeutic strategies that engage or mimic self-tolerance may allow prolonged allograft survival without the disadvantages of nonspecific immunotherapy. Pretreatment of recipient mice with donor alloantigen combined with transient modulation of the peripheral T cell pool with anti-CD4 Ab leads to the indefinite survival of MHC-incompatible cardiac allografts without further therapy. Tolerance is dependent on CD25+ CD4+ regulatory T cells that arise from naive CD25- precursors and regulate rejection via both IL-10 and CTLA-4. Although these cells are clearly effective at controlling rejection, the proven ability of recently activated CD25+ cells to mediate bystander regulation raises the possibility that tolerized individuals might also have a reduced capacity to respond to environmental pathogens. We have examined anti-influenza responses in tolerized primary heart recipients, secondary recipients following adoptive transfer of regulatory populations, and tolerized mice in which bystander regulation has been deliberately induced. Neither virus-specific CTL activity in vitro nor the clearance of virus in vivo was significantly diminished in any of these treatment groups compared with infected unmanipulated controls. The data suggest that the induction of dominant allograft tolerance dependent on regulatory T cells does not necessarily result in attenuated responses to pathogens providing further support for the development of tolerance induction protocols in clinical transplantation.     

Prolonged allograft survival in TNF receptor 1-deficient recipients is due to immunoregulatory effects, not to inhibition of direct antigraft cytotoxicity.

TNF-alpha and lymphotoxin (LT)alpha have been shown to be important mediators of allograft rejection. TNF-R1 is the principal receptor for both molecules. Mice with targeted genetic deletions of TNF-R1 demonstrate normal development of T and B lymphocytes but exhibit functional defects in immune responses. However, the role of TNF-R1-mediated signaling in solid organ transplant rejection has not been defined. To investigate this question, we performed vascularized heterotopic allogeneic cardiac transplants in TNF-R1-deficient (TNF-R1(-/-)) and wild-type mice. Because all allografts in our protocol expressed TNF-R1, direct antigraft effects of TNF-alpha and LTalpha were not prevented. However, immunoregulatory effects on recipient inflammatory cells by TNF-R1 engagement was eliminated in TNF-R1(-/-) recipients. In our study, cardiac allograft survival was significantly prolonged in TNF-R1(-/-) recipients. Despite this prolonged allograft survival, we detected increased levels of CD8 T cell markers in allografts from TNF-R1(-/-) recipients, suggesting that effector functions, but not T cell recruitment, were blocked. We also demonstrated the inhibition of multiple chemokines and cytokines in allografts from TNF-R1(-/-) recipients including RANTES, IFN-inducible protein-10, lymphotactin, and IL-1R antagonist, as well as altered levels of chemokine receptors. We correlated gene expression with the physiologic process of allograft rejection using self-organizing maps and identified distinct patterns of gene expression in allografts from TNF-R1(-/-) recipients. These findings indicate that in our experimental system TNF-alpha and LTalpha exert profound immunoregulatory effects through TNF-R1.     

A critical role for protein kinase C-theta-mediated T cell survival in cardiac allograft rejection

Protein kinase C (PKC)-theta mediates the critical TCR signals required for T cell activation. Previously, we have shown that in response to TCR stimulation, PKC-theta-/- T cells undergo apoptosis due to greatly reduced levels of the anti-apoptotic molecule, Bcl-xL. In this study, we demonstrate that PKC-theta-regulated expression of Bcl-xL is essential for T cell-mediated cardiac allograft rejection. Rag1-/- mice reconstituted with wild-type T cells readily rejected fully mismatched cardiac allografts, whereas Rag1-/- mice reconstituted with PKC-theta-/- T cells failed to promote rejection. Transgenic expression of Bcl-xL in PKC-theta-/- T cells was sufficient to restore cardiac allograft rejection, suggesting that PKC-theta-regulated survival is required for T cell-mediated cardiac allograft rejection in this adoptive transfer model. In contrast to adoptive transfer experiments, intact PKC-theta-/- mice displayed delayed, but successful cardiac allograft rejection, suggesting the potential compensation for PKC-theta function. Finally, a subtherapeutic dose of anti-CD154 Ab or CTLA4-Ig, which was not sufficient to prevent cardiac allograft rejection in the wild-type mice, prevented heart rejection in the PKC-theta-/- mice. Thus, in combination with other treatments, inhibition of PKC-theta may facilitate achieving long-term survival of allografts.