Quantification of CD4(+) T Cell Alloreactivity and Its Control by Regulatory T Cells Using Time-Lapse Microscopy and Immune Synapse Detection.

Assays designed to select transplant recipients for immunosuppression withdrawal have met with limited success, perhaps because they measure events downstream of T cell-alloantigen interactions. Using in vitro time-lapse microscopy in a mouse transplant model, we investigated whether transplant outcome would result in changes in the proportion of CD4(+) T cells forming prolonged interactions with donor dendritic cells. By blocking CD4-MHC class II and CD28-B7 interactions, we defined immunologically relevant interactions as those ≥500 s. Using this threshold, T cell-dendritic cell (T-DC) interactions were examined in rejection, tolerance and T cell control mediated by regulatory T cells. The frequency of T-DC contacts ≥500 s increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. Regulatory T cells reduced prolonged T-DC contacts. Importantly, this effect was replicated with human polyclonally expanded naturally occurring regulatory T cells, which we have previously shown can control rejection of human tissues in humanized mouse models. Finally, in a proof-of-concept translational context, we were able to visualize differential allogeneic immune synapse formation in polyclonal CD4(+) T cells using high-throughput imaging flow cytometry.

Harnessing regulatory T cells for clinical use in transplantation: the end of the beginning.

Owing to the adverse effects of immunosuppression and an inability to prevent chronic rejection, there is a pressing need for alternative strategies to control alloimmunity. In three decades, regulatory T cells (Tregs) have evolved from a hypothetical mediator of adoptively transferred tolerance to a well-defined population that can be expanded ex vivo and returned safely to patients in clinical trials. Herein, we review the historical developments that have permitted these advances and the current status of clinical trials examining Tregs as a cellular therapy in transplantation. We conclude by discussing the critical unanswered questions that face this field in the coming years.

Identification of gene markers for the prediction of allograft rejection or permanent acceptance.

The clinical success of new treatment strategies aiming on inducing permanent graft acceptance will rely on the ability to determine whether specific unresponsiveness to donor alloantigens has developed and for how long it is maintained. To identify markers for such posttransplant monitoring, genes differentially expressed by graft infiltrating leukocytes during tolerance induction or rejection after kidney transplantation in rats were compared. A subsequently performed full kinetic analysis in two different transplant models, kidney and heart, in two species, rat and mouse identified two markers (TOAG-1, alpha-1,2-mannosidase) with high specificity and reproducibility, which are highly expressed during induction and maintenance of acceptance, and downregulated during rejection. Expression level of these markers showed a strong positive correlation with graft function. In addition, expression of both genes was downregulated in the peripheral blood and the graft prior to rejection, suggesting that these markers may be useful for monitoring in clinical transplantation where peripheral blood is the most easily accessible patient sample. Interestingly, downregulation of TOAG-1 and alpha-1,2-mannosidase expression occurred in graft infiltrating cells and expression of both genes was also downregulated after T-cell activation in vitro.

GITR ligation blocks allograft protection by induced CD25+CD4+ regulatory T cells without enhancing effector T-cell function.

The induction of operational tolerance prior to transplant could provide a solution to the complications of current immunosuppression in transplantation. In rodents, operational tolerance frequently correlates with the presence of CD25(+)CD4(+) regulatory T cells (Tregs) but their function is usually demonstrated by adoptive transfer into lymphopenic hosts leading some to question their relevance to normal immunocompetent recipients. The role of these cells in primary transplant recipients has been explored using anti-CD25 antibody but specific targeting of Treg is not possible since CD25 is also up-regulated on activated effector T cells. To overcome this limitation we targeted the Treg associated molecule GITR in tolerized primary transplant recipients. This reverses regulation resulting in acute allograft rejection. This is not due to co-stimulation of effector cells since rejection mediated by isolated populations of CD4(+)CD25(-) or CD8(+)CD25(-) T cells transferred into Rag(-/-) mice was not enhanced by anti-GITR antibody. Furthermore, GITR cross-linking does not provide co-stimulation for in vitro proliferation of the same CD4(+)CD25(-) or CD8(+)CD25(-) T-cell populations in response donor-strain APC. Thus, CD4(+)CD25(+)GITR(+) Treg play an essential role in early graft protection in primary transplant recipients following tolerance induction providing further support for protocols that might generate similar populations in clinical transplantation.

Critical role for IL-4 in the development of transplant arteriosclerosis in the absence of CD40-CD154 costimulation.

Blockade of the CD40-CD154 pathway can inhibit CD4(+) T cell activation but is unable to prevent immune responses mediated by CD8(+) T cells. However, even in the absence of CD8(+) T cells, inhibition of the CD40-CD154 pathway is insufficient to prevent the development of transplant arteriosclerosis. This study investigated the mechanisms of transplant arteriosclerosis in the absence of the CD40 pathway. C57BL/6 CD40(-/-) (H2(b)) recipients were transplanted with MHC-mismatched BALB/c (H2(d)) aortas. Transplant arteriosclerosis was evident in both CD40(-/-) and CD40(+/-) mice (intimal proliferation was 59 +/- 5% for CD40(-/-) mice vs 58 +/- 4% for CD40(+/-) mice) in the presence or absence of CD8(+) T cells (intimal proliferation was 46 +/- 7% for CD40(-/-) anti-CD8-treated mice vs 50 +/- 10% for CD40(+/-) anti-CD8-treated mice), confirming that CD8(+) T cells are not essential effector cells for the development of this disease. In CD40(-/-) recipients depleted of CD8(+) T cells, the number of eosinophils infiltrating the graft was markedly increased (109 +/- 24 eosinophils/grid for CD40(-/-) anti-CD8-treated mice vs 28 +/- 7 for CD40(+/-) anti-CD8-treated mice). The increased presence of eosinophils correlated with augmented intragraft production of IL-4. To test the hypothesis that IL-4 was responsible for the intimal proliferation, CD8 T cell-depleted CD40(-/-) recipients were treated with anti-IL-4 mAb. This resulted in significantly reduced eosinophil infiltration into the graft (12 +/- 5 eosinophils/grid for CD40(-/-) anti-CD8(+), anti-IL-4-treated mice vs 109 +/- 24 for CD40(-/-) anti-CD8-treated mice), intragraft eotaxin, CCR3 mRNA production, and the level of intimal proliferation (18 +/- 5% for CD40(-/-) anti-CD8(+)-, anti-IL-4-treated mice vs 46 +/- 7% for CD40(-/-) anti-CD8-treated mice). In conclusion, elevated intragraft IL-4 production results in an eosinophil infiltrate and is an important mechanism for CD8(+) T cell-independent transplant arteriosclerosis in the absence of CD40-CD154 costimulation.

Alloantigen-induced specific immunological unresponsiveness.

When the immune system encounters alloantigen it can respond in any one of a number of different ways. The choice that is made will take into account factors such as where, when and how the contact with the alloantigen takes place, as well as the environmental conditions that prevail at the time the alloantigen is encountered. Alloantigen administration before transplantation either alone or in combination with therapeutic agents that modulate the functional activity of the responding leucocytes can be a powerful way of inducing specific unresponsiveness to alloantigens in vivo. The molecular mechanisms that influence the way the outcome of the immune response to alloantigen develops, either activation or unresponsiveness to the triggering antigen, hold the key to our ability to manipulate the immune system effectively by exposing it to donor antigen for therapeutic purposes. This review will focus on alloantigen-induced immunological unresponsiveness and how insights into the mechanisms of unresponsiveness have driven the development of novel tolerance-induction strategies that show promise for translation into the clinic in the future.

Paper alert. Immunology.

A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in immunology.

IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo.

We present evidence that donor-reactive CD4(+) T cells present in mice tolerant to donor alloantigens are phenotypically and functionally heterogeneous. CD4(+) T cells contained within the CD45RB(high) fraction remained capable of mediating graft rejection when transferred to donor alloantigen-grafted T cell-depleted mice. In contrast, the CD45RB(low) CD4(+) and CD25(+)CD4(+) populations failed to induce rejection, but rather, were able to inhibit rejection initiated by naive CD45RB(high) CD4(+) T cells. Analysis of the mechanism of immunoregulation transferred by CD45RB(low) CD4(+) T cells in vivo revealed that it was donor Ag specific and could be inhibited by neutralizing Abs reactive with IL-10, but not IL-4. CD45RB(low) CD4(+) T cells from tolerant mice were also immune suppressive in vitro, as coculture of these cells with naive CD45RB(high) CD4(+) T cells inhibited proliferation and Th1 cytokine production in response to donor alloantigens presented via the indirect pathway. These results demonstrate that alloantigen-specific regulatory T cells contained within the CD45RB(low) CD4(+) T cell population are responsible for the maintenance of tolerance to donor alloantigens in vivo and require IL-10 for functional activity.

Nondepleting anti-CD4 monoclonal antibody enhances the ability of oral alloantigen delivery to induce indefinite survival of cardiac allografts: oral tolerance to alloantigen.

BACKGROUND: We examined whether oral administration of alloantigen could induce the prolonged survival of cardiac allografts. METHODS: Hearts from CBK (H2k+Kb) transgenic or (C57BL/10xCBA)F1 (H2bxH2k) mice were transplanted into CBA (H2k) recipients pretreated orally with 1 x 10(7) donor splenocytes in the presence or absence of a nondepleting anti-CD4 (YTS 177, 200 microg/dose). RESULTS: Modest prolongation of CBK cardiac grafts was induced in CBA mice fed with multiple doses of CBK splenocytes (MST 42 days compared with controls fed with syngeneic CBA splenocytes, 12 days). When the CD4 monoclonal antibody, YTS177, was administered for 2 days before the first oral delivery of CBK splenocytes, all mice accepted their grafts indefinitely (MST > 100 days versus mice treated with anti-CD4 alone, 11.5 days). To determine if feeding multiple doses of alloantigen was essential, CBA mice were given CBK splenocytes orally on a single occasion in combination with the anti-CD4. The majority of the grafts survived indefinitely (MST >100 days). This oral treatment regimen also induced indefinite prolongation of (C57BL/10xCBA)F1 cardiac grafts. CONCLUSION: The induction of unresponsiveness by oral administration of alloantigen can be augmented by a nondepleting anti-CD4, YTS177, when given before the first oral delivery of allogeneic cells.

Differential role for competitive reverse transcriptase-polymerase chain reaction and intracellular cytokine staining as diagnostic tools for the assessment of intragraft cytokine profiles in rejecting and nonrejecting heart allografts.

The early and reliable diagnosis of allograft rejection is a difficult task and the assessment of cytokine expression in the grafts can be a helpful parameter. We have compared competitive reverse transcriptase-polymerase chain reaction (RT-PCR) with intracellular cytokine staining by flow cytometry as tools to measure cytokine expression in rejecting and nonrejecting murine cardiac allografts. Both techniques gave comparable results for cytokine expression in rejecting allografts and syngeneic controls. Grafts from mice pretreated with anti-CD4 antibody and donor-specific blood transfusion showed a marked reduction in cytokine expression, as assessed by competitive RT-PCR, even though a cellular infiltrate was present in the graft. In contrast, the cytokine production measured by intracellular cytokine staining of the isolated graft-infiltrating cells was high and exceeded even that of the rejecting allografts. We conclude that intracellular cytokine staining of graft-infiltrating leukocytes by flow cytometry does not necessarily reflect accurately the cytokine milieu in the graft. This technique might therefore have a limited clinical application in contrast to competitive RT-PCR for the differentiation between graft acceptance and graft rejection.

Evidence for immune regulation in the induction of transplantation tolerance: a conditional but limited role for IL-4.

Most experimental models of allograft tolerance depend on manipulation of immune responses at the time of transplant. In such systems, the graft itself probably plays an important role in the induction of unresponsiveness but as a consequence may suffer immune mediated damage. Ideally, recipients would be made specifically unresponsive before transplant such that the graft is protected from the outset. In this report, we demonstrate that CBA mice pretreated with donor-specific transfusion plus anti-CD4 Ab 28 days before transplant accept cardiac allografts indefinitely without further intervention. Adoptive transfer of spleen cells from mice with long term surviving grafts results in donor-specific graft acceptance in naive secondary recipients, indicating that tolerance in this system involves immuneregulation. Regulation develops as a result of the pretreatment protocol alone, since transfer of cells from pretreated but untransplanted mice to naive recipients also leads to prolonged allograft survival without additional therapy. Neutralizing IL-4 at the time of tolerance induction had no effect on graft outcome in primary recipients. However, removal of IL-4 from the adoptive transfer donors at the time of tolerance induction prevented long term engraftment in the majority of secondary recipients. Our data demonstrate that pretreatment of transplant recipients can establish immune regulation powerful enough to override the responses of an intact immune repertoire and that under stringent conditions at least, development of this regulatory population may in part be dependent on IL-4.

Immunoregulation by CD4 T cells in the induction of specific immunological unresponsiveness to alloantigens in vivo: evidence for a reduction in the frequency of alloantigen-specific cytotoxic T cells in vitro.

Donor-specific unresponsiveness to allogeneic cardiac allografts in mice can be induced by the combined pretreatment with donor alloantigen and anti-CD4 antibody (anti-CD4+DST). We have investigated whether the induction of unresponsiveness in this model is due to the presence of T cells that regulate immune responsiveness towards the allograft. First, we analysed the functional characteristics of splenocytes from pretreated mice at the time of transplantation. A significant reduction in the frequency of donor specific cytotoxic precursor was found only after the anti-CD4+DST treatment. Next, we designed an in vitro assay to identify the phenotype of the splenocyte population responsible. CD4+ and CD4- fractions were purified from mice treated with anti-CD4+DST or anti-CD4 alone (controls) by cell sorting. Interestingly, only the addition of CD4+ cells from anti-CD4+DST treated mice resulted in a selective reduction and a bimodal distribution in the donor specific CTLp response, indicating the presence of a regulatory population. CD4+ cells from controls did not have this effect. These in vitro findings were substantiated by adoptive transfer experiments in vivo. These data demonstrate that CD4+ cells with the ability to regulate immune responsiveness to a cardiac allograft are present at the time of transplantation following pretreatment with donor alloantigen in combination with anti-CD4.

The induction of operational tolerance is not prevented by simultaneous administration of cyclosporin A1.

In this study, the effect of combining anti-CD4 monoclonal antibody (mAb) and cyclosporin (CyA) therapy at the time of transplantation was examined. A mouse cardiac allograft model was used. Anti-CD4 mAb administered perioperatively induces long-term survival. The addition of a short course of CyA given subcutaneously in a regimen of either a high-dose treatment or a standard dose treatment to the anti-CD4 mAb treatment protocol did not have a detrimental effect on graft survival. Despite having no significant effect on graft survival, the addition of CyA to the treatment protocol did result in a significant decrease in the level of IL-2 present in the hearts 7 days after transplantation. The decrease in IL-2 production was directly related to the presence of CyA in vivo. When CyA treatment was continued throughout the period during which unresponsiveness to the graft is induced by anti-CD4 mAb therapy, 50% of the grafted hearts were rejected once the CyA was discontinued. In conclusion, the combined use of anti-CD4 mAb therapy and CyA did not have a negative effect on graft survival in this model when the two agents were used concurrently at the time of transplantation.

The Th1/Th2 paradigm and the allograft response.

Although Th1- and Th2-type cytokine profiles have been associated with rejection and tolerance respectively, this paradigm may not be completely accurate. Instead, recent studies suggest that there could be a hierarchy of T-cell growth factors with regard to their ability to block tolerance and induce rejection (rather than a polarized Th1/Th2 demarcation).