Mouse Heart Transplantation Research Articles

Graft-Infiltrating Macrophages Adopt an M2 Phenotype and Are Inhibited by Purinergic Receptor P2X7 Antagonist in Chronic Rejection.

Macrophages exhibit diverse phenotypes and functions; they are also a major cell type infiltrating chronically rejected allografts. The exact phenotypes and roles of macrophages in chronic graft loss remain poorly defined. In the present study, we used a mouse heart transplant model to examine macrophages in chronic allograft rejection. We found that treatment of C57BL/6 mice with CTLA4 immunoglobulin fusion protein (CTLA4-Ig) prevented acute rejection of a Balb/c heart allograft but allowed chronic rejection to develop over time, characterized by prominent neointima formation in the graft. There was extensive macrophage infiltration in the chronically rejected allografts, and the graft-infiltrating macrophages expressed markers associated with M2 cells but not M1 cells. In an in vitro system in which macrophages were polarized into either M1 or M2 cells, we screened phenotypic differences between M1 and M2 cells and identified purinergic receptor P2X7 (P2x7r), an adenosine triphosphate (ATP)-gated ion channel protein that was preferentially expressed by M2 cells. We further showed that blocking the P2x7r using oxidized ATP (oATP) inhibited M2 induction in a dose-dependent fashion in vitro. Moreover, treatment of C57BL/6 recipients with the P2x7r antagonist oATP, in addition to CTLA4-Ig treatment, inhibited graft-infiltrating M2 cells, prevented transplant vasculopathy, and induced long-term heart allografts survival. These findings highlight the importance of the P2x7r-M2 axis in chronic rejection and establish P2x7r as a potential therapeutic target in suppression of chronic rejection.

Cardiomyocytes display low mitochondrial priming and are highly resistant toward cytotoxic T-cell killing.

Following heart transplantation, alloimmune responses can cause graft rejection by damaging donor vascular and parenchymal cells. However, it remains unclear whether cardiomyocytes are also directly killed by immune cells. Here, we used two‐photon microscopy to investigate how graft‐specific effector CD8+ T cells interact with cardiomyocytes in a mouse heart transplantation model. Surprisingly, we observed that CD8+ T cells are completely impaired in killing cardiomyocytes. Even after virus‐mediated preactivation, antigen‐specific CD8+ T cells largely fail to lyse these cells although both cell types engage in dynamic interactions. Furthermore, we established a two‐photon microscopy‐based assay using intact myocardium to determine the susceptibility of cardiomyocytes to undergo apoptosis. This feature, also known as mitochondrial priming reveals an unexpected weak predisposition of cardiomyocytes to undergo apoptosis in situ. These observations together with the early exhaustion phenotype of graft‐infiltrating specific T cells provide an explanation why cardiomyocytes are largely protected from direct CD8+ T‐cell‐mediated killing.

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice.

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models. Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

Heterotopic Cervical Heart Transplantation in Mice.

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient's common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

Heterotopic Cervical Heart Transplantation in Mice

Heterotopic Cervical Heart Transplantation in Mice

Arsenic trioxide inhibits accelerated allograft rejection mediated by alloreactive CD8+ memory T cells and prolongs allograft survival time

CD8+ memory T (Tm) cells are a significant barrier to transplant tolerance induction in alloantigen-primed recipients, and are insensitive to existing clinical immunosuppressants. Here, we studied the inhibition of CD8+ Tm cells by arsenic trioxide (As2O3) for the first time. Alloantigen-primed CD8+ Tm cells were transferred to T cell immunodeficient nude mice. The mice were subjected to heart allotransplantation, and treated with As2O3. The transplant survival time was determined, and the inhibitory effects of As2O3 on CD8+ Tm cell-mediated immune rejection were assessed through serological studies and inspection of the transplanted heart and lymphoid organs. We found that As2O3 treatment prolonged the mean survival time of the graft and reduced the number of CD8+ Tm cells in the spleen and lymph nodes. The expression of the genes encoding interleukin (IL)-2, and IFN-γ was reduced, while expression of IL-10 and transforming growth factor-β was increased in the transplant. Our findings show that As2O3 treatment inhibits allograft rejection mediated by alloreactive CD8+ Tm cells in the mouse heart transplantation model.

Computational modeling of transplant rejection: a systems view of the dynamic roles of T cells, antigen presenting cells, and cytokines during rejection (TRAN1P.941)

Abstract The quality of life of organ transplant recipients is compromised by complications that arise from ensuing life-long immunosuppression and by limited long-term graft survival due to absence of tolerance induction. There is then an urgent need to better understand the complex interactions of the immune system leading to transplant rejection, so to identify innovative strategies to promote transplant acceptance while preserving immune competence. In this study we have pioneered a systems biology approach in which an experimentally-based mathematical model is used to predict how alterations in the immune response influence the rejection of mouse heart transplants. The model correctly predicts that in the absence of CD4 T cells the transplant survives indefinitely, while in the absence of CD8 T cells it is quickly rejected, providing also dynamics of innate cells that fit recently published data. As expected, decreasing the migration of effectors to the graft delays transplant rejection. This latter relation is not monotonic as increasing the translocation of CD8 T cells has a beneficial effect. This unexpected result derives from the interconnections with the relative concentrations of pro- and anti-inflammatory cytokines and with the ratio between Treg and T cells. This suggests possible key points of intervention for a testable novel strategy of intervention that highlights the usefulness of this innovative multidisciplinary approach.

Hyperbranched polyglycerol as a colloid in cold organ preservation solutions.

Hydroxyethyl starch (HES) is a common colloid in organ preservation solutions, such as in University of Wisconsin (UW) solution, for preventing graft interstitial edema and cell swelling during cold preservation of donor organs. However, HES has undesirable characteristics, such as high viscosity, causing kidney injury and aggregation of erythrocytes. Hyperbranched polyglycerol (HPG) is a branched compact polymer that has low intrinsic viscosity. This study investigated HPG (MW-0.5 to 119 kDa) as a potential alternative to HES for cold organ preservation. HPG was synthesized by ring-opening multibranching polymerization of glycidol. Both rat myocardiocytes and human endothelial cells were used as an in vitro model, and heart transplantation in mice as an in vivo model. Tissue damage or cell death was determined by both biochemical and histological analysis. HPG polymers were more compact with relatively low polydispersity index than HES in UW solution. Cold preservation of mouse hearts ex vivo in HPG solutions reduced organ damage in comparison to those in HES-based UW solution. Both size and concentration of HPGs contributed to the protection of the donor organs; 1 kDa HPG at 3 wt% solution was superior to HES-based UW solution and other HPGs. Heart transplants preserved with HPG solution (1 kDa, 3%) as compared with those with UW solution had a better functional recovery, less tissue injury and neutrophil infiltration in syngeneic recipients, and survived longer in allogeneic recipients. In cultured myocardiocytes or endothelial cells, significantly more cells survived after cold preservation with the HPG solution than those with the UW solution, which was positively correlated with the maintenance of intracellular adenosine triphosphate and cell membrane fluidity. In conclusion, HPG solution significantly enhanced the protection of hearts or cells during cold storage, suggesting that HPG is a promising colloid for the cold storage of donor organs and cells in transplantation.

IL-15 is decreased upon CsA and FK506 treatment of acute rejection following heart transplantation in mice.

The aim of this study was to investigate the effect of cyclosporine A (CsA) and tacrolimus (FK506) on interleukin-15 (IL-15) production during acute rejection following heart transplantation in mice. Inbred male Balb/c (H-2d) and C57BL/6 (H-2b) mice were used to establish a heterotopic intra-abdominal cardiac transplantation model. The mice were divided in four groups: syngeneic control, allogeneic acute rejection, allogeneic rejection treated with CsA, and allogeneic rejection treated with FK506. The expression of IL-15, IL-2, and tumor necrosis factor-α (TNF-α) was measured using reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. A low level of IL-15 was detected in transplanted hearts of the control group, with a significant increase observed in the allogeneic acute rejection group. Compared to the allogeneic acute rejection group, IL-15 expression was significantly decreased in the CsA-and FK506-treated allogeneic rejection groups. The TNF-α expression pattern was similar to that of IL-15 in all groups. IL-2 expression was increased in the allogeneic acute rejection group and was inhibited in mice treated with CsA and FK506. In conclusion, increased IL-15 expression in rejected murine heart grafts may be reduced by CsA and FK506 in vivo.

Timing and Dose of Mesenchymal Stem Cells Do Matter in Inhibiting Rejection After Heart Transplantation in Mice.

Timing and Dose of Mesenchymal Stem Cells Do Matter in Inhibiting Rejection After Heart Transplantation in Mice.

Immunomodulation Induced By a Novel Immunosuppressant, NK026680 Plus Donor Specific Transfusion Permits a Long-Term Cardiac Allograft Survival in Mice.

Background: We have recently demonstrated that a novel triazolopylimidine derivative, NK026680 (NK), inhibits T cell proliferation and prolongs rat cardiac allograft survival. In this study, we examined the immunomodulatory effect of NK in combination with donor specific transfusion (DST) in mice. Methods: A fully MHC incompatible BALB/c-to-C57BL/6 mouse heart transplantation was performed on day 0. DST, 20x106 donor-splenocytes, was given intravenously on day -7. NK (40 mg/kg/day) was orally administered from day -7 to day +6. Recipient-splenocytes were cocultured with irradiated donor-splenocytes, and T cell proliferation (3H-thymidine incorporation), IFN-g production (ELISPOT) and cell phenotypes (FACS) were assessed. Results: Untreated mice promptly rejected cardiac allograft at a graft median survival time (gMST) of 6.5 days. DST or NK monotherapy prolonged gMST to 24.5 and 25.5 days, respectively. In a sharp contrast, NK plus DST treatment (NK+DST) markedly prolonged gMST to 75.5 days. (A). In the NK+DST group, both proliferation and IFN-g production against donorantigens transiently increased on day 0, while they were both significantly suppressed on day 7 (p<0.05, vs. Untreated). Following in vivo antigen-challenge by infusing 20x106 donor-splenocytes on day 0, the splenic Foxp3+CD4+ T cells increased[Figure 1](B); however, CD69+ effector T cells were abolished.Figure: No Caption available.(C and D) in the NK+DST group. Conclusion:NK together with DST induces a potent immunomodulatory effect by shifting immune-balance towards a regulatory milieu, and markedly prolongs cardiac allograft survival in mice. DISCLOSURES:Saiga, K.: Employee, salary.

Role of Macrophage Subsets in Chronic Allograft Rejection and Novel Strategies to Target Them.

chronic rejection or transplant vasculopathy is a major problem in the clinic hindering long-term transplant survival. Chronic rejection primarily targets the graft vasculature, but the exact mechanism underlying the slow and progressive vasculature damage in transplanted organs remains poorly defined. In the present study, we addressed the role of macrophages in chronic allograft rejection in a mouse heart transplant model. We found that treatment of B6 recipients with a single dose of CTLA-4Ig prolonged the survival of Balb/c heart allografts(MST=35d) as compared to untreated controls (MST=8 days). Histologically, the CTLA-4Ig treated grafts showed signs of extensive chronic rejection characterized by heavy leukocyte infiltration and prominent neointima formation. Phenotypically, macrophages were a key infiltrating cell type in the vascular lesions in the grafts. Further studies revealed a M2 (F4/80+CD206+) phenotype for grafting infiltrating macrophages. An increased number of M2 macrophages were also detected in the spleen of recipient mice that experienced chronic rejection. We polarized macrophages in vitro to M1 and M2 subsets and systemically screened for differences and specific pathways that could be targeted for inhibition of M1 and M2 macrophages. We showed that when macrophages were polarized to a M2 phenotype, they preferentially expressedP2x7 receptor, a member of P2x family receptors that sense metabolites. We also found that oxidized ATP (P2x7R inhibitor)could effectively inhibit M2 induction in vitro. Importantly, in the heart transplant model, CTLA-4Ig plus oxidized ATP induced long-term heart allograft survival(MST>100 days), which was associated with markedly reduced neointima generation in the grafts, and prevention of chronic rejection was associated with significant inhibition of M2 macrophages in situ. Our studies identified a previously unknown mechanism of transplant vasculopathy and pinpointed as a molecular target in the inhibition of infiltrating M2 macrophages and chronic rejection.

Identification of Novel Mechanisms of Tolerance Resistance Induced By OX40.

Induction of donor specific tolerance remains an important goal in transplantation and to identify and overcome barriers to tolerance will significantly improve transplant outcomes in the clinic. In the present study, we examined the effector programs that evade tolerance induction by costimulatory blockade in a mouse heart transplant model. Blocking CD40-CD40L costimulation using an anti-CD40L monoclonal antibody (MR1) induced prolonged survival of C57BL/6 heart allografts in BALB/c recipients (MST>60 days). However, we found that MR1 completely failed to prolong survival of heart allografts that express the OX40L transgene (OX40Ltg grafts) (MST=9 days), indicating that OX40 signaling evades tolerance induction by costimulatory blockade. Histologically, rejection of OX40Ltg heart allografts was associated with extensive lymphocyte infiltration, and a significant proportion of graft infiltrating T cells produced IL-9 (40%), while few of them produced IL-4 or IFN-γ, suggesting that OX40 may drive a new mechanism of tolerance resistance. In an in vitro T cell polarization assay, we showed that OX40 engagement inhibited the induction of TH17 cells by TGF-β/IL-6 and that of inducible Treg cells by TGF- β/IL-2. Instead, OX40 diverted CD4+ T cells to IL-9 producing TH9 cells. Mechanistically, OX40 signaling mediated the induction of TH9 cells by triggering noncanonical NF-κB activation in activated T cells. In an in vivo CFSE model in which CD4+ T cells were stimulated by alloantigens, blocking CD40-CD40L costimulation induced TH9 cells only in the presence of OX40 signaling. These data suggest that induction of TH9 cells may constitute a novel mechanism of tolerance resistance. Interestingly, blocking OX40 costimulation induced long-term heart allograft survival (MST>100 days) and markedly reduced transplant vasculopathy (chronic allograft rejection) in CD40L-/- recipient mice. In conclusion, we identified that OX40 dramatically induces the generation of TH9 cells both in vitro and in vivo, and TH9 cells are closely involved in tolerance resistance. These findings may lead to the development of new therapeutics to promote graft tolerance in transplantation.

A modified method for heterotopic mouse heart transplantion.

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents available. A difficulty is presented due to the small size of the animal and the considerable technical challenges of the microsurgery involved in heart transplantation. In particular, a high rate of technical failure early after transplantation may result from recipient death and post-operative complications such as hind limb paralysis or a non-beating heart. Here, the complete technique for heterotopic mouse heart transplantation is demonstrated, involving harvesting the donor heart and its subsequent implantation into a recipient mouse. The donor heart is harvested immediately following in situ perfusion with cold heparinized saline and transection of the ascending aorta and pulmonary artery. The recipient operation involves preparation of the abdominal aorta and inferior vena cava (IVC), followed by end-to-side anastomosis of the donor aorta with the recipient aorta using a single running 10-0 microsuture and a similar anastomosis of the donor pulmonary artery with the recipient IVC. Following the operation the animal is injected with 0.6 ml normal saline subcutaneously and allowed to recover on a 37 ° C heating pad. The results from 227 mouse heart transplants are summarized with a success rate at 48 hr of 86.8%. Of the 13.2% failures within 48 hr, 5 (2.2%) experienced hind limb paralysis, 10 (4.4%) had a non-beating heart due to graft ischemic injury and/or thrombosis, while 15 (6.6%) died within 48 hr.

Pretreatment of Transfused Donor Splenocytes and Allografts With Mitomycin C Attenuates Acute Rejection in Heart Transplantation in Mice

ObjectiveThe aim of this study was to investigate the effect of pretreatment of donor splenocytes and grafts with mitomycin C (MMC) on heart allograft survival, as well as to demonstrate the mechanism of function. MethodsDonor splenocytes from Balb/C mice were incubated with MMC (40 μg/mL) in vitro and then transfused into recipients (C57BL/6 mice). The heart allograft was perfused with MMC before harvest. Graft survival and histopathology were examined. Lymphocyte activation, regulatory T cells, and donor splenocyte apoptosis were examined with the use of flow cytometry. ResultsMMC incubation in vitro induced apoptosis of donor splenocytes (15.5 ± 2.3% vs 23.2 ± 4.2%; P < .01). Either intravenous injection of MMC-treated donor splenocytes or transplantation of allograft pretreated with MMC prolonged heart allograft mean survival time from 7 ± 0.8 days to 20.5 ± 1.9 days or 10 ± 0.9 days, respectively (both P < .01). A combination of MMC-pretreated donor splenocyte transfusion and allografts showed the best effect on prolongation of graft survival (28.5 ± 1.8 days). Activation of CD4+ T cells in spleen and peripheral lymph nodes of recipients was significantly inhibited by either MMC-splenocyte transfusion or the combination treatment. Meanwhile, the percentage of CD4+CD25+Foxp3+ regulatory T cells in the spleen was increased in the MMC-splenocyte transfusion group (15.5 ± 1.1% vs 18.2 ± 0.9%; P < .05). ConclusionsBoth injection of MMC-conditioned donor splenocytes and MMC-conditioned allograft have effects on prolongation of heart allograft survival in mice, and MMC-conditioned donor splenocytes might play an essential role. MMC pretreatment induced regulatory T cells likely through induction of donor splenocyte apoptosis, and thus it inhibited T-cell activation.

DCs sensitized with mPD-L1-Ig fusion protein improve the effect of heart transplantation in mice by promoting the generation of T-reg cells

PurposeTo detect the effects of DCs sensitized by mPD-L1-Ig fusion protein in heart transplantation in mice as well as its mechanisms. MethodThe mPD-L1-IgG1 construct was used to build a yeast expression system, and the fusion protein was expressed by secretion after the transfection of the GS115 yeast strain, purified by affinity chromatography and ion exchange chromatography, and assayed by SDS–PAGE and Western blot. The ability of the fusion protein to bind to the acceptor PD-1 was tested by ELISA, and the ability of the fusion protein to inhibit the function of T cells was tested by mixed lymphocyte reaction (MLR). ResultsWe used the new PD-L1-IgG1 fusion protein to sensitize imDCs and maintained the immature state of DCs, so as to induce stable and effective immune tolerance to heart transplantation. After the treatment of DCs by mPD-L1-Ig in vitro, the levels of CD80, CD40 and I-Ab expression on DCs are relatively weaker, the ability of DCs to stimulates the proliferation of allogeneic spleen T cells was significantly decreased (P<0.01), and the levels of Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-10) secreted by induced allogeneic T cells were significantly decreased (P<0.01). An in vivo experiment also revealed that DCs sensitized by mPD-L1-IgG1 could prolong the survival time of a transplanted heart to 17.8±1.12days, and alleviate the pathological change of the cardiac allografts compared with other three groups. ConclusionDCs sensitized by the yeast-expressed mPD-L1-Ig fusion protein are shown to alleviate the cardiac allograft rejection in mice.

MicroRNA and mRNA Signatures in Ischemia Reperfusion Injury in Heart Transplantation

Ischemia reperfusion (I/R) injury is an unavoidable event occurring during heart transplantation, leading to graft failures and lower long-term survival rate of the recipient. Several studies have demonstrated that microRNAs (miRNAs) are vital regulators of signalling pathways involved in I/R injury. The present study aims to quantify the altered expression levels of miRNA and mRNA upon I/R injury in a mouse heart transplantation model, and to investigate whether these miRNA can regulate genes involved in I/R injury. We performed heterotopic heart transplantation on mouse models to generate heart tissue samples with I/R and non-I/R (control). The expression levels of miRNAs as well as genes were measured in heart grafts by microarray and real time RT-PCR. miRNA alteration in cardiomyocytes exposed to hypoxia was also detected by qRT-PCR. We observed significant alterations in miRNA and gene expression profile after I/R injury. There were 39 miRNAs significantly downregulated and 20 upregulated up to 1.5 fold in heart grafts with I/R injury compared with the grafts without I/R. 48 genes were observed with 3 fold change and p<0.05 and 18 signalling pathways were enriched using Keggs pathway library. Additionally, hypoxia/reperfusion induced primary cardiomyocyte apoptosis and altered miRNA expression profiles. In conclusion, this is the first report on miRNA expression profile for heart transplantation associated with I/R injury. These findings provide us with an insight into the role of miRNA in I/R injury in heart transplantation.

Delayed Anti-CD3 Therapy in a Mouse Heart Transplant Model Induced Tolerance and Long-Term Survival of Allograft: Achieving Tolerance

Goto R, You S, Zaitsu M, Chatenoud L, Wood KJ. Delayed anti-CD3 therapy results in depletion of alloreactive T cells and the dominance of Foxp3(+) CD4(+) graft infiltrating cells. Am. J. Transplant. 13(7), 1655-1664 (2013). Humanized Fc receptor nonbinding anti-CD3 monoclonal antibodies have been tested in patients with autoimmune diseases with the goal of inducing immune tolerance. However, the timing of drug administration may be an important determinant of the biologic effects, since not all T cells are equally affected, and there may be different subsets of cells involved during the evolution of immune responses. The study by Goto et al. showed that delayed administration of anti-CD3 therapy was more effective in depleting alloreactive T cells than administration at the time of transplant, and resulted in long-term survival of the graft by promoting infiltration of CD4 Tregs into the graft.

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.