IDO and regulatory T cell support are critical for cytotoxic T lymphocyte-associated Ag-4 Ig-mediated long-term solid organ allograft survival

Costimulatory blockade of CD28-B7 interaction with CTLA4Ig is a well-established strategy to induce transplantation tolerance. Although previous in vitro studies suggest that CTLA4Ig upregulates expression of the immunoregulatory enzyme IDO in dendritic cells, the relationship of CTLA4Ig and IDO in in vivo organ transplantation remains unclear. In this study, we studied whether concerted immunomodulation in vivo by CTLA4Ig depends on IDO. C57BL/6 recipients receiving a fully MHC-mismatched BALB/c heart graft treated with CTLA4Ig + donor-specific transfusion showed indefinite graft survival (>100 d) without signs of chronic rejection or donor specific Ab formation. Recipients with long-term surviving grafts had significantly higher systemic IDO activity as compared with rejectors, which markedly correlated with intragraft IDO and Foxp3 levels. IDO inhibition with 1-methyl-dl-tryptophan, either at transplant or at postoperative day 50, abrogated CTLA4Ig + DST-induced long-term graft survival. Importantly, IDO1 knockout recipients experienced acute rejection and graft survival comparable to controls. In addition, αCD25 mAb-mediated depletion of regulatory T cells (Tregs) resulted in decreased IDO activity and again prevented CTLA4Ig + DST induced indefinite graft survival. Our results suggest that CTLA4Ig-induced tolerance to murine cardiac allografts is critically dependent on synergistic cross-linked interplay of IDO and Tregs. These results have important implications for the clinical development of this costimulatory blocker.     

A link between PDL1 and T regulatory cells in fetomaternal tolerance

Acceptance of the fetus expressing allogeneic paternal Ags by the mother is a physiologic model of transplantation tolerance. Various mechanisms contribute to fetal evasion from immune attack by maternal leukocytes. We have recently demonstrated that the inhibitory costimulatory molecule PDL1 plays a critical role in fetomaternal tolerance in that PDL1 blockade or deficiency resulted in decreased allogeneic fetal survival rates. CD4(+)CD25(+) T regulatory cells (Tregs) have also been demonstrated to play an important role in fetomaternal tolerance. Since PDL1 is expressed on Tregs, we explored the interactions between PDL1 and Tregs in vivo in a mouse model of fetomaternal tolerance. Depletion of CD25(+) T cells abrogated the effect of anti-PDL1 Ab indicating that the effect of PDL1 is possibly mediated by CD25(+) Tregs. Adoptive transfer of Tregs from wild-type but not PDL1-deficient mice into PDL1-deficient recipients significantly improved fetal survival. The frequency, phenotype and placental trafficking of Tregs from PDL1-deficient mice were similar to those of wild-type controls, but were defective in inhibiting alloreactive Th1 cells in vitro. This is the first report providing evidence for a link between PDL1 and T regulatory cells in mediating fetomaternal tolerance.     

Analysis of the role of negative T cell costimulatory pathways in CD4 and CD8 T cell-mediated alloimmune responses in vivo

Negative costimulatory signals mediated via cell surface molecules such as CTLA-4 and programmed death 1 (PD-1) play a critical role in down-modulating immune responses and maintaining peripheral tolerance. However, their role in alloimmune responses remains unclear. This study examined the role of these inhibitory pathways in regulating CD28-dependent and CD28-independent CD4 and CD8 alloreactive T cells in vivo. CTLA-4 blockade accelerated graft rejection in C57BL/6 wild-type recipients and in a proportion of CD4(-/-) but not CD8(-/-) recipients of BALB/c hearts. The same treatment led to prompt rejection in CD28(-/-) and a smaller proportion of CD4(-/-)CD28(-/-) mice with no effect in CD8(-/-)CD28(-/-) recipients. These results indicate that the CTLA-4:B7 pathway provides a negative signal to alloreactive CD8(+) T cells, particularly in the presence of CD28 costimulation. In contrast, PD-1 blockade led to accelerated rejection of heart allografts only in CD28(-/-) and CD8(-/-)CD28(-/-) recipients. Interestingly, PD-1 ligand (PD-L1) blockade led to accelerated rejection in wild-type mice and in all recipients lacking CD28 costimulation. This effect was accompanied by expansion of IFN-gamma-producing alloreactive T cells and enhanced generation of effector T cells in rejecting allograft recipients. Thus, the PD-1:PD-L1 pathway down-regulates alloreactive CD4 T cells, particularly in the absence of CD28 costimulation. The differential effects of PD-1 vs PD-L1 blockade support the possible existence of a new receptor other than PD-1 for negative signaling through PD-L1. Furthermore, PD-1:PD-L1 pathway can regulate alloimmune responses independent of an intact CD28/CTLA-4:B7 pathway. Harnessing physiological mechanisms that regulate alloimmunity should lead to development of novel strategies to induce durable and reproducible transplantation tolerance.     

Role of STAT4 and STAT6 signaling in allograft rejection and CTLA4-Ig-mediated tolerance

STAT4(-/-) mice have impaired type 1 T cell differentiation, whereas STAT6(-/-) mice fail to generate type 2 responses. The role of type 1 and type 2 T cell differentiation in acute cardiac allograft rejection and in the induction of tolerance was examined in wild-type, STAT4(-/-), and STAT6(-/-) recipients. All recipients rejected the grafts promptly. Analysis of in situ cytokine gene expression in the allografts confirmed decreased levels of IFN-gamma in STAT4(-/-) recipients and undetectable levels of IL-4 and IL-5 in STAT6(-/-) mice. Blockade of the CD28/B7 costimulatory pathway prolonged cardiac graft survival for >100 days in 100% of wild-type and STAT4(-/-) mice. However, 14% of CTLA4-Ig-treated STAT6(-/-) mice rejected their grafts between 20 and 100 days. Moreover, of those animals followed past 100 days, 60% of the STAT6(-/-) mice rejected their grafts. Splenocytes harvested on day 145 posttransplant from CTLA4-Ig-treated rejecting STAT6(-/-) recipients were transfused into syngeneic SCID mice transplanted with donor or third party cardiac allografts. Both donor and third party grafts were rejected, indicating that the initial graft loss may be due to an immunological rejection. In contrast, when splenocytes from CTLA4-Ig-treated wild-type or nonrejecting STAT6(-/-) mice were transferred into SCID recipients, donor allografts were accepted, but third party hearts were rejected. Thus, long-term prolongation of cardiac allograft survival by CTLA4-Ig is STAT4-independent but, at least in part, STAT6-dependent. These data suggest that the balance of type 1 and type 2 T lymphocyte differentiation is not critical for acute rejection but influences the robust tolerance induced by CD28/B7 blockade in this model.     

The novel costimulatory programmed death ligand 1/B7.1 pathway is functional in inhibiting alloimmune responses in vivo

The programmed death ligand 1 (PDL1)/programmed death 1 (PD1) costimulatory pathway plays an important role in the inhibition of alloimmune responses as well as in the induction and maintenance of peripheral tolerance. It has been demonstrated recently that PDL1 also can bind B7.1 to inhibit T cell responses in vitro. Using the bm12 into B6 heart transplant model, we investigated the functional significance of this interaction in alloimmune responses in vivo. PD1 blockade unlike PDL1 blockade failed to accelerate bm12 allograft rejection, suggesting a role for an additional binding partner for PDL1 other than PD1 in transplant rejection. PDL1 blockade was able to accelerate allograft rejection in B7.2-deficient recipients but not B7.1-deficient recipients, indicating that PDL1 interaction with B7.1 was important in inhibiting rejection. Administration of the novel 2H11 anti-PDL1 mAb, which only blocks the PDL1-B7.1 interaction, aggravated chronic injury of bm12 allografts in B6 recipients. Aggravated chronic injury was associated with an increased frequency of alloreactive IFN-γ-, IL-4-, and IL-6-producing splenocytes and a decreased percentage of regulatory T cells in the recipients. Using an in vitro cell culture assay, blockade of the interaction of PDL1 on dendritic cells with B7.1 on T cells increased IFN-γ production from alloreactive CD4(+) T cells, whereas blockade of dendritic cell B7.1 interaction with T cell PDL1 did not. These data indicate that PDL1 interaction with B7.1 plays an important role in the inhibition of alloimmune responses in vivo and suggests a dominant direction for PDL1 and B7.1 interaction.     

CTLA4 signals are required to optimally induce allograft tolerance with combined donor-specific transfusion and anti-CD154 monoclonal antibody treatment

Sensitization to donor Ags is an enormous problem in clinical transplantation. In an islet allograft model, presensitization of recipients through donor-specific transfusion (DST) 4 wk before transplantation results in accelerated rejection. We demonstrate that combined DST with anti-CD154 (CD40L) therapy not only prevents the deleterious presensitization produced by pretransplant DST in the islet allograft model, it also induces broad alloantigen-specific tolerance and permits subsequent engraftment of donor islet or cardiac grafts without further treatment. In addition, our data strongly indicate that CTLA4-negative T cell signals are required to achieve prolonged engraftment of skin allograft or tolerance to islet allograft in recipients treated with a combination of pretransplant DST and anti-CD154 mAb. We provide direct evidence that a CD28-independent CTLA4 signal delivers a strong negative signal to CD4+ T cells that can block alloimmune MLR responses. In this study immune deviation into a Th2 (IL-4) response was associated with, but did not insure, graft tolerance, as the inopportune timing of B7 blockade with CTLA4/Ig therapy prevented uniform tolerance but did not prevent Th2-type immune deviation. While CTLA4-negative signals are necessary for tolerance induction, Th1 to Th2 immune deviation cannot be sufficient for tolerance induction. Combined pretransplant DST with anti-CD154 mAb treatment may be attractive for clinical deployment, and strategies aimed to selectively block CD28 without interrupting CTLA4/B7 interaction might prove highly effective in the induction of tolerance.     

CD4+ T regulatory cell induction and function in transplant recipients after CD154 blockade is TLR4 independent

Although the role of CD4(+) T regulatory cells (Treg) in transplantation tolerance has been established, putative mechanisms of Treg induction and function in vivo remain unclear. TLR4 signaling has been implicated in the regulation of CD4(+)CD25(+) Treg functions recently. In this study, we first examined the role of recipient TLR4 in the acquisition of operational CD4(+) Treg following CD154 blockade in a murine cardiac transplant model. Then, we determined whether TLR4 activation in allograft tolerant recipients would reverse alloimmune suppression mediated by CD4(+) Treg. We document that donor-specific immune tolerance was readily induced in TLR4-deficient recipients by a single dose of anti-CD154 mAb, similar to wild-type counterparts. The function and phenotype of CD4(+) Treg in both wild-type and TLR4 knockout long-term hosts was demonstrated by a series of depletion experiments examining their ability to suppress the rejection of secondary donor-type test skin grafts and to inhibit alloreactive CD8(+) T cell activation in vivo. Furthermore, TLR4 activation in tolerant recipients following exogenous LPS infusion in conjunction with donor-type skin graft challenge, failed to break Treg-mediated immune suppression. In conclusion, our data reveals a distinctive property of CD4(+) Treg in tolerant allograft recipients, whose induction and function are independent of TLR4 signaling.     

A novel alloantigen-specific CD8+PD1+ regulatory T cell induced by ICOS-B7h blockade in vivo

Delayed ICOS-B7h signal blockade promotes significant prolongation of cardiac allograft survival in wild-type but not in CD8-deficient C57BL/6 recipients of fully MHC-mismatched BALB/c heart allografts, suggesting the possible generation of CD8(+) regulatory T cells in vivo. We now show that the administration of a blocking anti-ICOS mAb results in the generation of regulatory CD8(+) T cells. These cells can transfer protection and prolong the survival of donor-specific BALB/c, but not third party C3H, heart grafts in CD8-deficient C57BL/6 recipients. This is unique to ICOS-B7h blockade, because B7 blockade by CTLA4-Ig prolongs graft survival in CD8-deficient mice and does not result in the generation of regulatory CD8(+) T cells. Those cells localize to the graft, produce both IFN-gamma and IL-4 after allostimulation in vitro, prohibit the expansion of alloreactive CD4(+) T cells, and appear to mediate a Th2 switch of recipient CD4(+) T cells after adoptive transfer in vivo. Finally, these cells are not confined to the CD28-negative population but express programmed death 1, a molecule required for their regulatory function in vivo. CD8(+)PD1(+) T cells suppress alloreactive CD4(+) T cells but do not inhibit the functions by alloreactive CD8(+) T cells in vitro. These results describe a novel allospecific regulatory CD8(+)PD1(+) T cell induced by ICOS-B7h blockade in vivo.     

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.     

Critical role of CD4 help in CD154 blockade-resistant memory CD8 T cell activation and allograft rejection in sensitized recipients

Allograft rejection in sensitized recipients remains the major problem in clinical organ transplantation. We have developed a donor-type skin-sensitized mouse cardiac allograft model (BALB/c-->C57BL/6) in which both rejection (<5 days) and alloreactive CD8 activation are resistant to CD154 blockade. First, we attempted to elucidate why CD154 blockade fails to protect cardiac grafts in sensitized recipients. The gene array analysis has revealed that treatment with anti-CD154 mAb (MR1) had distinctive impact on host immunity in naive vs sensitized animals. Unlike in naive counterparts, host sensitization mitigated the impact of CD154 blockade on critical immune signaling pathways. Indeed, we identified 3234 genes in cardiac grafts that were down-regulated by MR1 in naive (at least 5-fold), but remained unaffected in sensitized hosts. Moreover, MR1 treatment failed to prevent accumulation of CD4 T cells in cardiac allografts of sensitized recipients. Then, to determine the role of CD4 help in CD154 blockade-resistant immune response, we used CD4-depleting and CD4-blocking Ab, in conjunction with MR1 treatment. Our data revealed that CD154 blockade-resistant CD8 activation in sensitized mice was dependent on CD4 T cells. In the absence of CD4 help, CD154 blockade prevented differentiation of alloreactive CD8 T cells into CTL effector/memory cells and abrogated acute rejection (cardiac graft survival for >30 days), paralleled by selective target gene depression at the graft site. These results provide the rationale to probe potential synergy of adjunctive therapy targeting CD4 and CD154 to overcome graft rejection in sensitized recipients.     

Activation of alloreactive CD8+ T cells operates via CD4-dependent and CD4-independent mechanisms and is CD154 blockade sensitive

CD154, one of the most extensively studied T cell costimulation molecules, represents a promising therapeutic target in organ transplantation. However, the immunological mechanisms of CD154 blockade that result in allograft protection, particularly in the context of alloreactive CD4/CD8 T cell activation, remain to be elucidated. We now report on the profound inhibition of alloreactive CD8(+) T cells by CD154 blockade via both CD4-dependent and CD4-independent activation pathways. Using CD154 KO recipients that are defective in alloreactive CD8(+) T cell activation and unable to reject cardiac allografts, we were able to restore CD8 activation and graft rejection by adoptively transferring CD4(+) or CD8(+) T cells from wild-type syngeneic donor mice. CD4-independent activation of alloreactive CD8(+) T cells was confirmed following treatment of wild-type recipients with CD4-depleting mAb, and by using CD4 KO mice. Comparable levels of alloreactive CD8(+) T cell activation was induced by allogenic skin engraftment in both animal groups. CD154 blockade inhibited CD4-independent alloreactive CD8(+) T cell activation. Furthermore, we analyzed whether disruption of CD154 signaling affects cardiac allograft survival in skin-sensitized CD4 KO and CD8 KO recipients. A better survival rate was observed consistently in CD4 KO, as compared with CD8 KO recipients. Our results document CD4-dependent and CD4-independent activation pathways for alloreactive CD8(+) T cells that are both sensitive to CD154 blockade. Indeed, CD154 blockade was effective in preventing CD8(+) T cell-mediated cardiac allograft rejection.     

Inhibition of NF-kappaB-dependent T cell activation abrogates acute allograft rejection.

Using a heterotopic model of transplantation, we investigated the role of T cell activation in vivo during allograft rejection in I-kappaB(DeltaN)-transgenic mice that express a transdominant inhibitor of NF-kappaB in T cells. Our results show indefinite prolongation of graft survival in the I-kappaB(DeltaN)-transgenic recipients. Interestingly, at the time of rejection of grafts in wild-type recipients, histology of grafts in the I-kappaB(DeltaN)-transgenic recipients showed moderate rejection; nevertheless, grafts in the I-kappaB(DeltaN) recipients survived >100 days. Analysis of acute phase cytokines, chemokine, chemokine receptors, and immune responses shows that the blockade of NF-kappaB activation in T cells inhibits up-regulation of many of these parameters. Interestingly, our data also suggest that the T cell component of the immune response exerted positive feedback regulation on the expression of multiple chemokines that are produced predominantly by non-T cells. In conclusion, our studies indicate NF-kappaB activation in T cells is necessary for acute allograft rejection.     

Prevention of chronic rejection in murine cardiac allografts: a comparison of chimerism- and nonchimerism-inducing costimulation blockade-based tolerance induction regimens

We have previously described a nonirradiation-based regimen combining costimulation blockade, busulfan, and donor bone marrow cells that promotes stable, high level chimerism, deletion of donor-reactive T cells, and indefinite survival of skin allografts in mice. The purpose of the current study is to determine the efficacy of this tolerance regimen in preventing acute and chronic rejection in a vascularized heart graft model and to compare this regimen with other putative tolerance protocols. Mice receiving costimulation blockade (CTLA4-Ig and anti-CD40 ligand) alone or in combination with donor cells enjoyed markedly prolonged heart graft survival and initially preserved histological structure. However, tolerance was not achieved, as evidenced by the eventual onset of chronic rejection characterized by obliterative vasculopathy and the rejection of secondary skin grafts. In contrast, following treatment with costimulation blockade, busulfan, and bone marrow, heart grafts survived indefinitely without detectable signs of chronic rejection or structural damage, even 100 days after placement of a secondary donor skin graft. We detected multilineage chimerism in peripheral blood, spleen, lymph nodes, and thymus, and peripheral deletion of donor-reactive cells was complete by day 90. These findings indicate that only the CD40/CD28 blockade chimerism induction regimen prevents both acute and chronic rejection of vascularized organ transplants. Further testing of these strategies in a preclinical large animal model is warranted.     

The CD154-CD40 T cell costimulation pathway is required for host sensitization of CD8(+) T cells by skin grafts via direct antigen presentation

Although the CD154-CD40 T cell costimulation pathway has been shown to mediate alloimmune responses in normal recipients, little is known about its role in sensitized hosts. In this work, by using novel models of cardiac allograft rejection in skin-sensitized CD154- and CD40-deficient mice, we reaffirm the key role of CD154-CD40 signaling in host sensitization to alloantigen in vivo. First, we identified CD8(+) T cells as principal effectors in executing accelerated rejection in our model. Disruption of CD154-CD40 signaling in recipients at the T cell side (CD154-deficient) but not at the APC side (CD40-deficient) abrogated accelerated (<2 days) rejection and resulted in long-term (>100 days) graft survival. This suggests that the CD154-dependent mechanism in host CD8(+) T cell sensitization operates via the direct Ag presentation. Then, in comparative studies of alloimmune responses in CD154-deficient and wild-type recipients, we showed that, although alloreactive B cell responses were inhibited, alloreactive T cell responses were down-regulated selectively in the CD8(+) T cell compartment, leaving CD4(+) T cells largely unaffected. This unique alteration in host alloreactivity, seen not only in peripheral lymphocytes but also in allograft infiltrate, may represent the key mechanism by which disruption of CD154-CD40 signaling prevents sensitization to alloantigen in vivo and leads to long-term allograft survival.     

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.     

CD28-independent costimulation of T cells in alloimmune responses.

T cell costimulation by B7 molecules plays an important role in the regulation of alloimmune responses. Although both B7-1 and B7-2 bind CD28 and CTLA-4 on T cells, the role of B7-1 and B7-2 signaling through CTLA-4 in regulating alloimmune responses is incompletely understood. To address this question, we transplanted CD28-deficient mice with fully allogeneic vascularized cardiac allografts and studied the effect of selective blockade of B7-1 or B7-2. These mice reject their grafts by a mechanism that involves both CD4(+) and CD8(+) T cells. Blockade of CTLA-4 or B7-1 significantly accelerated graft rejection. In contrast, B7-2 blockade significantly prolonged allograft survival and, unexpectedly, reversed the acceleration of graft rejection caused by CTLA-4 blockade. Furthermore, B7-2 blockade prolonged graft survival in recipients that were both CD28 and CTLA-4 deficient. Our data indicate that B7-1 is the dominant ligand for CTLA-4-mediated down-regulation of alloimmune responses in vivo and suggest that B7-2 has an additional receptor other than CD28 and CTLA-4 to provide a positive costimulatory signal for T cells.     

TIM-3: a novel regulatory molecule of alloimmune activation

T cell Ig domain and mucin domain (TIM)-3 has previously been established as a central regulator of Th1 responses and immune tolerance. In this study, we examined its functions in allograft rejection in a murine model of vascularized cardiac transplantation. TIM-3 was constitutively expressed on dendritic cells and natural regulatory T cells (Tregs) but only detected on CD4(+)FoxP3(-) and CD8(+) T cells in acutely rejecting graft recipients. A blocking anti-TIM-3 mAb accelerated allograft rejection only in the presence of host CD4(+) T cells. Accelerated rejection was accompanied by increased frequencies of alloreactive IFN-γ-, IL-6-, and IL-17-producing splenocytes, enhanced CD8(+) cytotoxicity against alloantigen, increased alloantibody production, and a decline in peripheral and intragraft Treg/effector T cell ratio. Enhanced IL-6 production by CD4(+) T cells after TIM-3 blockade plays a central role in acceleration of rejection. Using an established alloreactivity TCR transgenic model, blockade of TIM-3 increased allospecific effector T cells, enhanced Th1 and Th17 polarization, and resulted in a decreased frequency of overall number of allospecific Tregs. The latter is due to inhibition in induction of adaptive Tregs rather than prevention of expansion of allospecific natural Tregs. In vitro, targeting TIM-3 did not inhibit nTreg-mediated suppression of Th1 alloreactive cells but increased IL-17 production by effector T cells. In summary, TIM-3 is a key regulatory molecule of alloimmunity through its ability to broadly modulate CD4(+) T cell differentiation, thus recalibrating the effector and regulatory arms of the alloimmune response.     

Pancreatic islets engineered with SA-FasL protein establish robust localized tolerance by inducing regulatory T cells in mice

Allogeneic islet transplantation is an important therapeutic approach for the treatment of type 1 diabetes. Clinical application of this approach, however, is severely curtailed by allograft rejection primarily initiated by pathogenic effector T cells regardless of chronic use of immunosuppression. Given the role of Fas-mediated signaling in regulating effector T cell responses, we tested if pancreatic islets can be engineered ex vivo to display on their surface an apoptotic form of Fas ligand protein chimeric with streptavidin (SA-FasL) and whether such engineered islets induce tolerance in allogeneic hosts. Islets were modified with biotin following efficient engineering with SA-FasL protein that persisted on the surface of islets for >1 wk in vitro. SA-FasL-engineered islet grafts established euglycemia in chemically diabetic syngeneic mice indefinitely, demonstrating functionality and lack of acute toxicity. Most importantly, the transplantation of SA-FasL-engineered BALB/c islet grafts in conjunction with a short course of rapamycin treatment resulted in robust localized tolerance in 100% of C57BL/6 recipients. Tolerance was initiated and maintained by CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells, as their depletion early during tolerance induction or late after established tolerance resulted in prompt graft rejection. Furthermore, Treg cells sorted from graft-draining lymph nodes, but not spleen, of long-term graft recipients prevented the rejection of unmodified allogeneic islets in an adoptive transfer model, further confirming the Treg role in established tolerance. Engineering islets ex vivo in a rapid and efficient manner to display on their surface immunomodulatory proteins represents a novel, safe, and clinically applicable approach with important implications for the treatment of type 1 diabetes.