A significant risk for transplant recipients is the development of tumors. In general, some but not all malignancies are more frequent in transplant hosts because of chronic immunosuppression caused by compromised immune surveillance. Of additional relevance, checkpoint blockade therapies (CBTs) to treat malignancies can also drive transplant rejection. In a recent study published in Nature Communications, Dunlap et al reported a case study of a patient who experienced kidney allograft rejection after CBT for melanoma. The foresight of longitudinally preserving donor splenocytes, blood samples, and graft biopsies in addition to tumor and metastatic lymph nodes enabled paired single-cell RNA sequencing (scRNA-seq) and T-cell receptor sequencing (TCR-seq) and subsequent tracking of alloreactive T cells before and after CBT. This revealed enrichment of alloreactive TCRs in the kidney transplant post-CBT but not the tumor. In addition, this approach helped identify an alloreactive CD8+ T-cell subset with a unique transcriptional profile. This study illustrates possible advances in personalized medicine and highlights a transcriptional signature that may serve as a prospective biomarker of rejection. The emergence of tumors is a great risk to transplant recipients. Moreover, using CBTs may result in the rejection of the transplanted organ1 (Figure 1). In a recent issue of Nature Communications, Dunlap et al2 reported a case study of a patient who, for 10 y post–kidney transplantation for chronic glomerulonephritis, experienced episodes of allograft rejection following the use of CBT (pembrolizumab, 200 mg every 3 wk) for metastatic melanoma. The authors hypothesized that graft rejection was because of the expansion, post-CBT, of preexisting alloreactive T cells. The authors evaluated donor-specific alloreactivity using a mixed-lymphocyte reaction (MLR) in which recipient peripheral blood mononuclear cells (PBMCs) collected post-CBT were labeled with carboxyfluorescein succinimidyl ester and stimulated with banked donor splenocytes. Proliferating T cells were then sorted and underwent subsequent paired scRNA-seq/TCR-seq to identify a population of bona fide alloreactive CD8+ T cells, defined by their proliferative transcriptional signature and clonal expansion. Next, systemic T cells were evaluated by bulk TCR-seq longitudinally pre-CBT in addition to post–first and second rounds of pembrolizumab. Similar studies were performed in biopsies from the kidney transplant, pre-CBT metastatic lymph node, and post-CBT metastatic skin samples. The authors confirmed the MLR-identified alloreactive clones in post-CBT but not pre-CBT PBMCs and observed them infiltrating the kidney transplant but not the tumor sites. This tissue restriction to graft, perhaps unexpected, is significant and novel. Others have previously shown that activated alloreactive T cells and irrelevant effector CD8+ T cells enter murine allografts3 and that bystander (nontumor reactive) effector CD8+ T cells enter human malignancies.4 Although with limitations as a case report, the current study demonstrates the feasibility of tracking alloreactive T-cell phenotypes over time and helps to understand CD8+ T-cell expansion and activation as a threat to the transplanted allograft after CBT.FIGURE 1.: CBT can promote both tumor control and transplant rejection. CBT blocks negative interactions between T cells and APCs or between T cells and target cells, unleashing restrained T cells. Those effects can result in beneficial antitumor immunity but may also activate alloreactive T cells, leading to transplant rejection. APC, antigen-presenting cell; CBT, checkpoint blockade therapy.Previous cutting-edge work had already taken advantage of MLR assays to identify and subsequently track donor-specific T-cell clones. An article published in Science Translational Medicine in 2015 by Morris et al5 tracked alloreactive T cells in patients receiving dual-donor bone marrow and kidney grafts, helping the authors establish clonal deletion as a mechanism of human transplantation tolerance. To this end, the authors defined a pretransplant fingerprint of the donor-specific TCR repertoire by leveraging high-throughput TCR-β CDR3 sequencing of T cells having expanded in an antidonor MLR. CDR3 areas were amplified for all 54 Vβ and all 13 Jβ regions before high-throughput sequencing. Posttransplant T-cell tracking in PBMCs was then verified by repeated MLR and evaluation of TCR repertoires. The findings by Dunlap et al took this method a step further; by analyzing MLR-expanded antidonor T cells with paired scRNA-seq/TCR-seq, the authors are able to not only track the identified alloreactive clones directly ex vivo but also examine their transcriptional signature, leading to the identification of a ZNF683+ (Hobit) subset of CD8+ T cells that represents proliferating alloreactive T cells. Their approach has thus revealed a potential tractable biomarker of rejection. Moreover, access to biopsies and the capacity to sequence graft- and tumor-infiltrating T cells and determine where these circulating proliferating alloreactive T cells go after CBT added unprecedented depth. Previous efforts to track alloreactive T cells have been limited to evaluation of PBMCs, whereas the direct comparison of alloreactive T cells in the PBMC compartment with alloreactive T cells in tissue biopsies provides a more complete understanding of these cells’ function. Whether this molecular method can be used to predict risk of CBT-associated rejection before treatment with a low clonal frequency of alloreactive PBMCs before CBT remains to be determined. However, Morris et al were able to use MLRs to identify donor-specific clones pretransplantation, suggesting that the approach of Dunlap et al may succeed before CBT to identify alloreactive clones and use the expansion of the ZNF683+ subset post-CBT to flag patients for careful graft function monitoring. Paired evaluation of scRNA-seq/TCR-seq from MLR-expanded T cells remains a highly skilled and onerous technique that is not yet widely accessible. Moreover, subsequent tracking of alloreactive T cells requires diligent, repeated sampling and biobanking from the recipient’s blood and graft. Additionally, access to donor cells as stimulators for the MLR is a prerequisite. For deceased donors, donor spleens as used by Dunlap et al are an excellent and large source of donor antigen that is not currently used, although some investigators have proposed injecting donor splenocytes into transplant recipients for inducing chimerism,6 T-cell dysfunction,7 or as a desensitization strategy in sensitized patients (NCT04827186).8 Alternative sources of antigens would be necessary for live donor transplants with PBMCs representing promising candidates. The expansion of alloreactive T cells may also represent a potential biomarker of rejection. Although important to treat tumors, CBT poses a rejection risk, and Dunlap et al demonstrated that alloreactive CD8+ T-cell clones expand post-CBT. The authors also identified a transcriptionally unique subset of expanded alloreactive CD8+ T cells characterized by their proliferative phenotype and expression of ZNF683. ZNF683 is a transcription factor implicated in migration, memory formation, and tissue residency of CD8+ T cells. Whether ZNF683+ CD8+ T cells that expand post-CBT and infiltrate the kidney allograft represent key drivers of rejection and whether expansion of this subset in the blood can be used as an early biomarker of rejection will require additional studies. If confirmed, tracking of this subset before and after CBT may inform ongoing clinical trials (NCT03816332, NCT04339062)9,10 aimed at preventing rejection after CBT. Overall, the study by Dunlap et al illustrated new possibilities in personalized medicine using the currently available molecular technologies in an innovative way while demonstrating the usefulness of longitudinal biobanking in clinical transplantation.

While advances have been made in therapies that achieve tolerance in mouse models of transplantation, successful tolerance remains vulnerable to inflammatory insults, which can trigger graft rejection. To identify strategies for improving the robustness of tolerance, we aim to better understand the mechanisms by which grafts are rejected after donor-specific tolerance is established. In a mouse model of cardiac allograft tolerance in which late infection with Listeria monocytogenes (Lm) can trigger graft rejection, we previously showed that host CD4 +T cells reactive to host MHC-II presenting a donor MHC-I-derived peptide (TCR75 cells) became intrinsically dysfunctional if the alloantigen persisted for 3 weeks or greater. Intriguingly, infection-dependent transplant rejection was not associated with transcriptional or functional reinvigoration of these cells. We hypothesized that there might be heterogeneity in the level of dysfunction of alloreactive T cells depending on duration of their cognate alloantigen persistence. Unlike TCR75 cells, CD4 +T cells specific for host MHC-II presenting a peptide derived from donor MHC Class II (TEa cells), a graft antigen that declines post-transplantation, retained functionality during tolerance induction. Consequently, TEa but not TCR75 cells expanded following Lm-infection, supporting a role for TEa-like cells in driving infection-triggered rejection. Prolonging T cell exposure to cognate alloantigens aggravated TEa dysfunction and rendered grafts resistant to Lm-dependent rejection. These findings demonstrate that inducing dysfunction in a broader array of allospecific T cells, by prolonging exposure to more alloantigens, lessens graft vulnerability to infections. Supported by the UChicago Growth Development and Disabilities Training Program (T32 HD007009). AHA predoctoral fellowships (20PRE35210946, 3PRE14550022 and 15PRE22180007). NIH T32-AI007090. UChicago Cardiovascular Pathophysiology and Biochemistry Training Grant (T32 HL07237). HHMI Med-into-Grad Program training grant (56006772). NIAID Grant P01AI-97113.

Long-term transplantation tolerance depends on the control of graft-reactive T cells. In particular, the alloreactivity of CD4+ T cells needs to be suppressed as these cells are both necessary and sufficient for cardiac allograft rejection. Although experimental protocols such as anti-CD154-mediated co-stimulation blockade can stably induce donor-specific tolerance in MHC-mismatched cardiac allografts, other more immunogenic organs such as skin are less susceptible to tolerogenic treatments, in part due to additional reactivity of the host against donor commensals that accompany the graft. The transcriptional mechanisms that enable CD4+ T cells to drive rejection and whose targeting can promote tolerance are not well understood. RNAseq comparison of the transcriptome in graft-reactive T cells from rejecting versus tolerant heart allograft recipients revealed several transcription factors differentially expressed between rejected and tolerant mice in both spleen and graft. Here, we report that the transcription factor Runx3 in alloreactive CD4+ T cells promotes Th1 and cytotoxic response against the allograft and resistance to tolerance induction. Conversely, ablating Runx3 in alloreactive CD4+ T cells augments the efficacy of anti-CD154 blockade treatment enabling skin graft acceptance.

Even when successfully induced, immunological tolerance to solid organs remains vulnerable to inflammatory insults, which can trigger rejection. In a mouse model of cardiac allograft tolerance in which infection with Listeria monocytogenes (Lm) precipitates rejection of previously accepted grafts, we showed that recipient CD4+ TCR75 cells reactive to a donor MHC class I–derived peptide become hypofunctional if the allograft is accepted for more than 3 weeks. Paradoxically, infection-induced transplant rejection was not associated with transcriptional or functional reinvigoration of TCR75 cells. We hypothesized that there is heterogeneity in the level of dysfunction of different allospecific T cells, depending on duration of their cognate antigen expression. Unlike CD4+ TCR75 cells, CD4+ TEa cells specific for a peptide derived from donor MHC class II, an alloantigen whose expression declines after transplantation but remains inducible in settings of inflammation, retained function in tolerant mice and expanded during Lm-induced rejection. Repeated injections of alloantigens drove hypofunction in TEa cells and rendered grafts resistant to Lm-dependent rejection. Our results uncover a functional heterogeneity in allospecific T cells of distinct specificities after tolerance induction and reveal a strategy to defunctionalize a greater repertoire of allospecific T cells, thereby mitigating a critical vulnerability of tolerance.

Limiting CD4 + T cell responses is important to prevent solid organ transplant rejection. In a mouse model of costimulation blockade-dependent cardiac allograft tolerance, we previously reported that alloreactive CD4 + conventional T cells (Tconvs) develop dysfunction, losing proliferative capacity. In parallel, induction of transplantation tolerance is dependent on the presence of regulatory T cells (Tregs). Whether susceptibility of CD4 + Tconvs to Treg suppression is modulated during tolerance induction is unknown. We found that alloreactive Tconvs from transplant tolerant mice had augmented sensitivity to Treg suppression when compared with memory T cells from rejector mice and expressed a transcriptional profile distinct from these memory T cells, including down-regulated expression of the transcription factor Special AT-rich sequence-binding protein 1 (Satb1). Mechanistically, Satb1 deficiency in CD4 + T cells limited their expression of CD25 and IL-2, and addition of Tregs, which express higher levels of CD25 than Satb1-deficient Tconvs and successfully competed for IL-2, resulted in greater suppression of Satb1-deficient than wild-type Tconvs in vitro. In vivo, Satb1-deficient Tconvs were more susceptible to Treg suppression, resulting in significantly prolonged skin allograft survival. Overall, our study reveals that transplantation tolerance is associated with Tconvs’ susceptibility to Treg suppression, via modulated expression of Tconv-intrinsic Satb1. Targeting Satb1 in the context of Treg-sparing immunosuppressive therapies might be exploited to improve transplant outcomes.

Cellular metabolism is central to T cell function and proliferation, with most of the research to date focusing on cancer and autoimmunity. Cellular metabolism is associated with a host of physiological phenomena, from epigenetic changes, to cellular function and fate. For the purpose of this review, we will discuss the metabolism of T cells relating to their differentiation and function. We will cover a variety of metabolic processes, ranging from glycolysis to amino acid metabolism. Understanding how T cell metabolism informs T cell function may be useful to understand alloimmune responses and design novel therapies to improve graft outcome.

Patients weaned from immunosuppressive drugs can reject their graft after years of stability, often after an infection. We have modeled this in mice: infection with Listeria monocytogenes (Lm) at the maintenance phase of tolerance can precipitate rejection of a cardiac allograft. Here, we investigated which alloreactive Tconvs may mediate rejection after Lm infection. To track the function of allospecific Tconvs, we seeded tracer TCR75 cells, which recognize I-Ab-restricted donor Kd peptide, into B6 mice prior to Balb/c cardiac transplantation with or without tolerance induction by anti-CD154 + Balb/c splenocytes. ≥35 days post-transplantation, TCR75 cells persisting in tolerant mice developed a PD-1hi CD127lo exhausted-like phenotype and expressed the anergy markers FR4 and CD73. TCR75 cells were dysfunctional, exhibiting 10-fold less recall expansion compared to memory cells and lacked IFNγ and TNF production upon re-stimulation. Intriguingly, TCR75 cells in tolerant hosts remained dysfunctional after Lm infection, suggesting that they may not participate in infection-triggered rejection. Having found that programming T cell dysfunction in tolerance required persistence of the allograft for ~3 weeks, we tested whether T cells specific for alloantigens expressed transiently after transplantation, such as donor MHC Class II, remain functional during tolerance and may mediate rejection after Lm infection. We used tracer TEa cells specific for a donor Class II Eα peptide presented on I-Ab. During tolerance, TEa cells remained PD-1lo and CD73lo, and retained recall expansion and TNF production. We conclude that functional allospecific Tconvs present during tolerance may pose a previously unappreciated risk to the graft.

CD8(+) T cells activated in vivo display different metabolic profiles than those stimulated in vitro, underscoring the key role of the environment in dictating metabolic pathways downstream of T cell receptor stimulation.

Improved mouse models for type 1 diabetes (T1D) therapy development are needed. T1D susceptibility is restored to normally resistant NOD.β2m−/− mice transgenically expressing human disease–associated HLA-A*02:01 or HLA-B*39:06 class I molecules in place of their murine counterparts. T1D is dependent on pathogenic CD8+ T-cell responses mediated by these human class I variants. NOD.β2m−/−-A2.1 mice were previously used to identify β-cell autoantigens presented by this human class I variant to pathogenic CD8+ T cells and for testing therapies to attenuate such effectors. However, NOD.β2m−/− mice also lack nonclassical MHC I family members, including FcRn, required for antigen presentation, and maintenance of serum IgG and albumin, precluding therapies dependent on these molecules. Hence, we used CRISPR/Cas9 to directly ablate the NOD H2-Kd and H2-Db classical class I variants either individually or in tandem (cMHCI−/−). Ablation of the H2-Ag7 class II variant in the latter stock created NOD mice totally lacking in classical murine MHC expression (cMHCI/II−/−). NOD-cMHCI−/− mice retained nonclassical MHC I molecule expression and FcRn activity. Transgenic expression of HLA-A2 or -B39 restored pathogenic CD8+ T-cell development and T1D susceptibility to NOD-cMHCI−/− mice. These next-generation HLA-humanized NOD models may provide improved platforms for T1D therapy development.

In both NOD mice and humans, the development of type 1 diabetes (T1D) is dependent in part on autoreactive CD8+ T-cells recognizing pancreatic ß-cell peptides presented by often quite common MHC class I variants. Studies in NOD mice previously revealed the common H2-Kd and/or H2-Db class I molecules expressed by this strain acquire an aberrant ability to mediate pathogenic CD8+ T-cell responses through interactions with T1D susceptibility ( Idd ) genes outside the MHC. A gene(s) mapping to the Idd7 locus on proximal Chromosome 7 was previously shown to be an important contributor to the failure of the common class I molecules expressed by NOD mice to mediate the normal thymic negative selection of diabetogenic CD8+ T-cells. Using an inducible model of thymic negative selection and mRNA transcript analyses we initially identified an elevated Nfkbid expression variant is likely an NOD Idd7 region gene contributing to impaired thymic deletion of diabetogenic CD8+ T-cells. CRISPR/Cas9-mediated genetic attenuation of Nfkbid expression in NOD mice resulted in improved negative selection of autoreactive diabetogenic AI4 and NY8.3 CD8+ T-cells. These results indicated allelic variants of Nfkbid represent an Idd7 gene contributing to the efficiency of intrathymic deletion of diabetogenic CD8+ T-cells. However, while enhancing thymic deletion of pathogenic CD8+ T-cells, ablation of Nfkbid expression surprisingly accelerated T1D onset in NOD mice likely at least in part by numerically decreasing regulatory T- and B-lymphocytes (Tregs/Bregs), thereby reducing their peripheral immunosuppressive effects.

In both NOD mice and humans, the development of type 1 diabetes (T1D) is dependent in part on autoreactive CD8(+) T cells recognizing pancreatic beta cell peptides presented by often quite common MHC class I variants. Studies in NOD mice previously revealed that the common H2-K-d and/or H2-D-b class I molecules expressed by this strain aberrantly lose the ability to mediate the thymic deletion of pathogenic CD8(+) T cell responses through interactions with T1D susceptibility genes outside the MHC. A gene (s) mapping to proximal chromosome 7 was previously shown to be an important contributor to the failure of the common class I molecules expressed by NOD mice to mediate the normal thymic negative selection of diabetogenic CD8(+) T cells. Using an inducible model of thymic negative selection and mRNA transcript analyses, we initially identified an elevated Nfkbid expression variant as a likely NOD-proximal chromosome 7 region gene contributing to impaired thymic deletion of diabetogenic CD8(+) T cells. CRISPR/Cas9-mediated genetic attenuation of Nfkbid expression in NOD mice resulted in improved negative selection of autoreactive diabetogenic AI4 and NY8.3 CD8(+) T cells. These results indicated that allelic variants of Nfkbid contribute to the efficiency of intrathymic deletion of diabetogenic CD8(+) T cells. However, although enhancing thymic deletion of pathogenic CD8(+) T cells, ablating Nfkbid expression surprisingly accelerated T1D onset that was associated with numeric decreases in both regulatory T and B lymphocytes in NOD mice.