M. Sabet-Baktach

A color-coded reporter model to study the effect of immunosuppressants on CD8+ T-cell memory in antitumor and alloimmune responses.

Background Mammalian target of rapamycin (mTOR) inhibitors possess anticancer properties potentially useful in reducing posttransplantation malignancy. Besides controlling tumor-sensitive proliferative and angiogenic effects, mTOR influences transcription factors T-bet and Eomesodermin (Eomes) in CD8+ cytotoxic T cells (Tc), which are key in rejecting tumors, and allografts. Methods To study the role of mTOR in tumor and transplant immunity in an antigen-specific way, we used T-cell receptor transgenic B6.OTI recipients, B6.OVA.TG donors, and OVA-B16F10 melanoma cells. For tracking color-coded OTI-Tc cells associated with antitumor and alloimmunity in vivo, CD8-OTI transgenic reporter mice were created by crossbreeding DsRed-expressing B6.Nagy mice with B6.OTI mice. Results The role of mTOR in regulating the differentiation and function of alloreactive Tc cells in vitro was explored by stimulating OTI-Tc cells with ovalbumin-transgenic antigen-presenting cells in the presence of rapamycin or tacrolimus. Rapamycin, but not tacrolimus, induced a pro-antitumor phenotypic shift from CD62L-CD44+ effector memory Tc cells to CD62L+CD44+ central memory Tc cells, which featured up-regulated levels of T-bet and Eomes and preserved levels of interferon-&ggr; and perforin. For future investigations, an in vivo model was established whereby DsRed+OTI-Tc cells adoptively transferred into B6 mice bearing either a ovalbumin-transgenic mouse skin transplant or OVA-B16F10 tumor could be traced by fluorescence-activated cell sorting analysis as effector or memory Tc cells in transplant and tumor tissues. Conclusion mTOR, but not calcineurin, inhibition spares antitumoral memory Tc cells by distinctively regulating T-bet and Eomes. This finding is now testable in a new tumor transplant model, which incorporates DsRed+OTI-Tc cell tracing, opening the way to study the differential effects of immunosuppressants in posttransplantation malignancy.

Unconventional RORγt+ T Cells Drive Hepatic Ischemia Reperfusion Injury

An emerging body of evidence suggests a pivotal role of CD3+ T cells in mediating early ischemia reperfusion injury (IRI). However, the precise phenotype of T cells involved and the mechanisms underlying such T cell–mediated immune responses in IRI, as well as their clinical relevance, are poorly understood. In this study, we investigated early immunological events in a model of partial warm hepatic IRI in genetically targeted mice to study the precise pathomechanistic role of RORγt+ T cells. We found that unconventional CD27−γδTCR+ and CD4−CD8− double-negative T cells are the major RORγt-expressing effector cells in hepatic IRI that play a mechanistic role by being the main source of IRI-mediating IL-17A. We further show that unconventional IRI-mediating T cells are contingent on RORγt, as highlighted by the fact that a genetic deficiency for RORγt, or its therapeutic antagonization via digoxin, is protective against hepatic IRI. Therefore, identification of CD27−γδTCR+ and CD4−CD8− double-negative T cells as the major source of IL-17A via RORγt in hepatic IRI opens new therapeutic options to improve liver transplantation outcomes.

KLRG1+ NK Cells Protect T-bet–Deficient Mice from Pulmonary Metastatic Colorectal Carcinoma

We studied the developmental and functional mechanisms behind NK cell–mediated antitumor responses against metastatic colorectal carcinoma (CRC) in mice. In particular, we focused on investigating the significance of T-box transcription factors and the immunotherapeutic relevance of IL-15 in the development and function of tumor-reactive NK cells. Pulmonary CRC metastases were experimentally seeded via an adoptive i.v. transfer of luciferase-expressing CT26 CRC cells that form viewable masses via an in vivo imaging device; genetically deficient mice were used to dissect the antitumor effects of developmentally different NK cell subsets. IL-15 precomplexed to IL-15 receptor-α was used in immunotherapy experiments. We found that mice deficient for the T-box transcription factor T-bet lack terminally differentiated antitumor CD27lowKLRG1+ NK cells, leading to a terminal course of rapid-onset pulmonary CRC metastases. The importance of this NK cell subset for effective antitumor immunity was shown by adoptively transferring purified CD27lowKLRG1+ NK cells into T-bet–deficient mice and, thereby, restoring immunity against lung metastasis formation. Importantly, immunity to metastasis formation could also be restored in T-bet–deficient recipients by treating mice with IL-15 precomplexed to IL-15 receptor-α, which induced the development of eomesodermin+KLRG1+ NK cells from existing NK cell populations. Thus, contingent upon their T-bet–dependent development and activation status, NK cells can control metastatic CRC in mice, which is highly relevant for the development of immunotherapeutic approaches in the clinic.

Double Deficiency for RORγt and T-bet Drives Th2-Mediated Allograft Rejection in Mice

Although Th1, Th2, and Th17 cells are thought to be major effector cells in adaptive alloimmune responses, their respective contribution to allograft rejection remains unclear. To precisely address this, we used mice genetically modified for the Th1 and Th17 hallmark transcription factors T-bet and RORγt, respectively, which allowed us to study the alloreactive role of each subset in an experimental transplant setting. We found that in a fully mismatched heterotopic mouse heart transplantation model, T cells deficient for T-bet (prone to Th17 differentiation) versus RORγt (prone to Th1 differentiation) rejected allografts at a more accelerated rate, indicating a predominance of Th17- over Th1-driven alloimmunity. Importantly, T cells doubly deficient for both T-bet and RORγt differentiated into alloreactive GATA-3–expressing Th2 cells, which promptly induced allograft rejection characterized by a Th2-type intragraft expression profile and eosinophilic infiltration. Mechanistically, Th2-mediated allograft rejection was contingent on IL-4, as its neutralization significantly prolonged allograft survival by reducing intragraft expression of Th2 effector molecules and eosinophilic allograft infiltration. Moreover, under IL-4 neutralizing conditions, alloreactive double-deficient T cells upregulated Eomesodermin (Eomes) and IFN-γ, but not GATA-3. Thus, in the absence of T-bet and RORγt, Eomes may salvage Th1-mediated alloimmunity that underlies IL-4 neutralization-resistant allograft rejection. We summarize that, whereas Th17 cells predictably promote allograft rejection, IL-4–producing GATA-3+ Th2 cells, which are generally thought to protect allogeneic transplants, may actually be potent facilitators of organ transplant rejection in the absence of T-bet and RORγt. Moreover, Eomes may rescue Th1-mediated allograft rejection in the absence of IL-4, T-bet, and RORγt.

Cyclosporine A Inhibits the T-bet–Dependent Antitumor Response of CD8+ T Cells

Transplant recipients face an increased risk of cancer compared with the healthy population. Although several studies have examined the direct effects of immunosuppressive drugs on cancer cells, little is known about the interactions between pharmacological immunosuppression and cancer immunosurveillance. We investigated the different effects of rapamycin (Rapa) versus cyclosporine A (CsA) on tumor‐reactive CD8+ T cells. After adoptive transfer of CD8+ T cell receptor–transgenic OTI T cells, recipient mice received either skin grafts expressing ovalbumin (OVA) or OVA‐expressing B16F10 melanoma cells. Animals were treated daily with Rapa or CsA. Skin graft rejection and tumor growth as well as molecular and cellular analyses of skin‐ and tumor‐infiltrating lymphocytes were performed. Both Rapa and CsA were equally efficient in prolonging skin graft survival when applied at clinically relevant doses. In contrast to Rapa‐treated animals, CsA led to accelerated tumor growth in the presence of adoptively transferred tumor‐reactive CD8+ OTI T cells. Further analyses showed that T‐bet was downregulated by CsA (but not Rapa) in CD8+ T cells and that cancer cytotoxicity was profoundly inhibited in the absence of T‐bet. CsA reduces T‐bet–dependent cancer immunosurveillance by CD8+ T cells. This may contribute to the increased cancer risk in transplant recipients receiving calcineurin inhibitors.

CD27low natural killer cells prolong allograft survival in mice by controlling alloreactive CD8+ T cells in a T-bet-dependent manner.

Background Natural killer (NK) cells play a dichotomous role in alloimmune responses because they are known to promote both allograft survival and rejection. The aim of this study was to investigate the role of functionally distinct NK cell subsets in alloimmunity with the hypothesis that this dichotomy is explained by the functional heterogeneity of distinct NK cell subsets. Methods Because T-bet controls the maturation of NK cells from CD27high to terminally differentiated CD27low NK cells, we used Rag−/−T-bet−/− mice that lack mature CD27low NK cells to study the distinct roles of CD27low versus CD27high NK cells in a model of T cell–mediated skin transplant rejection under costimulatory blockade conditions. Results We found that T cell–reconstituted Rag1−/− recipients (possessing CD27low NK cells) show significantly prolonged allograft survival on costimulatory blockade when compared to Rag1−/−T-bet−/− mice (lacking CD27low NK cells), indicating that CD27low but not CD27high NK cells enhance allograft survival. Critically, Rag1−/−T-bet−/− recipients showed strikingly increased alloreactive memory CD8+ T cell responses, as indicated by increased CD8+ T cell proliferation and interferon-&ggr; production. Therefore, we speculated that CD27low NK cells directly regulate alloreactive CD8+ T cell responses under costimulatory blockade conditions. To test this, we adoptively transferred CD27low NK cells into Rag1−/−T-bet−/− skin transplant recipients and found that the CD27low NK cells restore better allograft survival by inhibiting the proliferation of alloreactive interferon-&ggr;+CD8+ T cells. Conclusions In summary, mature CD27low NK cells promote allograft survival under costimulatory blockade conditions by regulating alloreactive memory CD8+ T-cell responses.