CD4+ T cell immunity is dependent on an intrinsic stem-like program.

CD4+ T cells are central to various immune responses, but the molecular programs that drive and maintain CD4+ T cell immunity are not entirely clear. Here we identify a stem-like program that governs the CD4+ T cell response in transplantation models. Single-cell-transcriptomic analysis revealed that naive alloantigen-specific CD4+ T cells develop into TCF1hi effector precursor (TEP) cells and TCF1-CXCR6+ effectors in transplant recipients. The TCF1-CXCR6+CD4+ effectors lose proliferation capacity and do not reject allografts upon adoptive transfer into secondary hosts. By contrast, the TCF1hiCD4+ TEP cells have dual features of self-renewal and effector differentiation potential, and allograft rejection depends on continuous replenishment of TCF1-CXCR6+ effectors from TCF1hiCD4+ TEP cells. Mechanistically, TCF1 sustains the CD4+ TEP cell population, whereas the transcription factor IRF4 and the glycolytic enzyme LDHA govern the effector differentiation potential of CD4+ TEP cells. Deletion of IRF4 or LDHA in T cells induces transplant acceptance. These findings unravel a stem-like program that controls the self-renewal capacity and effector differentiation potential of CD4+ TEP cells and have implications for T cell-related immunotherapies.

Ablation of Transcription Factor IRF4 Promotes Transplant Acceptance by Driving Allogenic CD4+ T Cell Dysfunction.

CD4+ T cells orchestrate immune responses and destruction of allogeneic organ transplants, but how this process is regulated on a transcriptional level remains unclear. Here, we demonstrated that interferon regulatory factor 4 (IRF4) was a key transcriptional determinant controlling T cell responses during transplantation. IRF4 deletion in mice resulted in progressive establishment of CD4+ T cell dysfunction and long-term allograft survival. Mechanistically, IRF4 repressed PD-1, Helios, and other molecules associated with T cell dysfunction. In the absence of IRF4, chromatin accessibility and binding of Helios at PD-1 cis-regulatory elements were increased, resulting in enhanced PD-1 expression and CD4+ T cell dysfunction. The dysfunctional state of Irf4-deficient T cells was initially reversible by PD-1 ligand blockade, but it progressively developed into an irreversible state. Hence, IRF4 controls a core regulatory circuit of CD4+ T cell dysfunction, and targeting IRF4 represents a potential therapeutic strategy for achieving transplant acceptance.

Innate immune cell dysfunction and systemic inflammation in children with chronic liver diseases undergoing transplantation.

Advanced liver diseases (ALD) can affect immune function and compromise host defense against infections. In this study, we examined the phenotypic and functional alterations in circulating monocyte and dendritic cells (DCs) in children with ALD undergoing liver transplantation (LT). Children were stratified into 2 clusters, C1 (mild) and C2 (severe), on the basis of laboratory parameters of ALD and compared with healthy pediatric controls. Children in C2 had a significant reduction in frequencies of nonclassical monocytes and myeloid DCs. Children in C2 displayed monocyte and DC dysfunction, characterized by lower human leucocyte antigen DR expression and reduced interleukin 12 production, and had an increased incidence of infections before and after LT. Children in C2 demonstrated immune dysregulation with elevations of pro- and anti-inflammatory cytokines in plasma. Alterations of innate immune cells correlated with multiple laboratory parameters of ALD, including plasma bile acids. In vitro, monocytes cultured with specific bile acids demonstrated a dose-dependent reduction in interleukin 12 production, similar to alterations in children with ALD. In conclusion, a cohort of children with ALD undergoing LT exhibited innate immune dysfunction, which may be related to the chronic elevation of serum bile acids. Identifying at-risk patients may permit personalized management pre- and post-transplant, thereby reducing the incidence of infection-related complications.

Genetically targeting the BATF family transcription factors BATF and BATF3 in the mouse abrogates effector T cell activities and enables long-term heart allograft survival.

T cells must be activated and become effectors first before executing allograft rejection, a process that is regulated by diverse signals and transcription factors. In this study, we studied the basic leucine zipper ATF-like transcription factor (BATF) family members in regulating T cell activities in a heart transplant model and found that mice deficient for both BATF and BATF3 (Batf-/- Batf3-/- mice) spontaneously accept the heart allografts long-term without tolerizing therapies. Similarly, adoptive transfer of wild type T cells into Rag1-/- hosts induced prompt rejection of heart and skin allografts, whereas the Batf-/- Batf3-/- T cells failed to do so. Analyses of graft-infiltrating cells showed that Batf-/- Batf3-/- T cells infiltrate the graft but fail to acquire an effector phenotype (CD44high KLRG1+ ). Co-transfer experiments in a T cell receptor transgenic TEa model revealed that the Batf-/- Batf3-/- T cells fail to expand in vivo, retain a quiescent phenotype (CD62L+ CD127+ ), and unable to produce effector cytokines to alloantigen stimulation, which contrasted sharply to that of wild type T cells. Together, our data demonstrate that the BATF and BATF3 are critical regulators of T effector functions, thus making them attractive targets for therapeutic interventions in transplant settings.

EphA2 phosphorylates NLRP3 and inhibits inflammasomes in airway epithelial cells.

Inflammasomes are intracellular complexes that form in the cytosol of inflammatory cells. NLRP3 is one of the sensor proteins in the complex that can recognize a wide variety of stimuli ranging from microbial components to environmental particulates. Here, we report that in mouse airway epithelial cells (AECs), inflammasome activation is inhibited by EphA2, a member of the transmembrane tyrosine kinase receptor family, via tyrosine phosphorylation of NLRP3 in a model of reovirus infection. We find that EphA2 depletion markedly enhances interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) production in response to the virus. EphA2-/- mice show stronger inflammatory infiltration and enhanced inflammasome activation upon viral infection, and aggravated asthma symptoms upon ovalbumin (ova) induction. Mechanistically, EphA2 binds to NLRP3 and induces its phosphorylation at Tyr132, thereby interfering with ASC speck formation and blocking the activation of the NLRP3-inflammasome. These data demonstrate that reovirus employs EphA2 to suppress inflammasome activation in AECs and that EphA2 deficiency causes a pathological exacerbation of asthma in an ova-induced asthma model.

Pre-transplant T-cell Clonality: An Observational Study of a Biomarker for Prediction of Sepsis in Liver Transplant Recipients.

OBJECTIVE: This study investigated the ability of pre-transplant T-cell clonality to predict sepsis after liver transplant (LT). SUMMARY BACKGROUND DATA: Sepsis is a leading cause of death in LT recipients. Currently, no biomarkers predict sepsis before clinical symptom manifestation. METHODS: Between December 2013 and March 2018, our institution performed 478 LTs. After exclusions (eg, patients with marginal donor livers, autoimmune disorders, nonabdominal multi-organ, and liver retransplantations), 180 consecutive LT were enrolled. T-cell characterization was assessed within 48 hours before LT (immunoSEQ Assay, Adaptive Biotechnologies, Seattle, WA). Sepsis-2 and Sepsis-3 cases, defined by presence of acute infection plus ≥2 SIRS criteria, or clinical documentation of sepsis, were identified by chart review. Receiver-operating characteristic analyses determined optimal T-cell repertoire clonality for predicting post-LT sepsis. Kaplan-Meier and Cox proportional hazard modeling assessed outcome-associated prognostic variables. RESULTS: Patients with baseline T-cell repertoire clonality ≥0.072 were 3.82 (1.25, 11.40; P = 0.02), and 2.40 (1.00, 5.75; P = 0.049) times more likely to develop sepsis 3 and 12 months post-LT, respectively, when compared to recipients with lower (<0.072) clonality. T-cell repertoire clonality was the only predictor of sepsis 3 months post-LT in multivariate analysis (C-Statistic, 0.75). Adequate treatment resulted in equivalent survival rates between both groups: (93.4% vs 96.2%, respectively, P = 0.41) at 12 months post-LT. CONCLUSIONS: T-cell repertoire clonality is a novel biomarker predictor of sepsis before development of clinical symptoms. Early sepsis monitoring and management may reduce post-LT mortality. These findings have implications for developing sepsis-prevention protocols in transplantation and potentially other populations.

Identification of the E3 Ligase TRIM29 as a Critical Checkpoint Regulator of NK Cell Functions.

NK cells play an important role in immune surveillance and protective immunity, mainly through rapid cytokine release and cytolytic activities. But how such responses are negatively regulated remains poorly defined. In this study, we demonstrated that the E3 ubiquitin ligase TRIM29 is a crucial regulator of NK cell functions. We found that TRIM29 was not expressed in resting NK cells, but was readily upregulated following activation, especially after IL-12 plus IL-18 stimulation. The levels of TRIM29 expression were inversely correlated with IFN-γ production by NK cells, suggesting that TRIM29 inhibits NK cell functions. Indeed, deficiency of TRIM29, specifically in NK cells, resulted in an enhanced IFN-γ production and consequently protected mice from murine CMV infection. Mechanistically, we showed that once induced in NK cells, TRIM29 ubiquitinates and degrades the TGF-ß-activated kinase 1 binding protein 2 (TAB2), a key adaptor protein in IFN-γ production by NK cells. These results identify TRIM29 as a negative regulator of NK cell functions and may have important clinical implications.

The Pursuit of Regulatory T Cells in the Induction of Transplant Tolerance.

Organ transplantation is a preferred treatment option for patients with end-stage organ failure. However, transplant induces a robust rejection response that necessitates life-long immunosuppression, which often leads to a plethora of comorbidities. Thus, the goal of transplantation is to achieve a state of tolerance wherein the host permanently accepts the transplanted organ while maintaining normal immune responses to other antigens. Regulatory T cells (Tregs) play an important role in realizing this goal and are being explored in both animal models and human clinical trials. In this chapter, we discuss the key principles of transplant rejection and Treg biology, as well as the status of human clinical trials utilizing Tregs as cellular therapy. We discuss how the current immunosuppressive drugs are utilized in transplantation in favoring an increased Treg to T effector cell ratio, different approaches in generation of therapeutic Tregs, and various facets in Treg trial designs in the clinic. Such clinical trials provided many opportunities to leverage our understanding of Tregs in transplantation. They also demonstrated Tregs as a safe cellular therapy for human use, but the efficacy of this treatment has yet to be fully realized.

T cell exhaustion is associated with antigen abundance and promotes transplant acceptance.

Exhaustion of T cells limits their ability to clear chronic infections or eradicate tumors. Here, in the context of transplant, we investigated whether T cell exhaustion occurs and has a role in determining transplant outcome. A peptide/MHC tetramer-based approach was used to track exhausted CD8+ T cells in a male-to-female skin transplant model. Transplant of large whole-tail skins, but not small tail skins (0.8 cm × 0.8 cm), led to exhaustion of anti-male tetramer+ CD8+ T cells and subsequently the acceptance of skin grafts. To study CD4+ T cell exhaustion, we used the TCR-transgenic B6 TEa cells that recognize a major transplant antigen I-Eα from Balb/c mice. TEa cells were adoptively transferred either into B6 recipients that received Balb/c donor skins or into CB6F1 mice that contained an excessive amount of I-Eα antigen. Adoptively transferred TEa cells in skin-graft recipients were not exhausted. By contrast, virtually all adoptively transferred TEa cells were exhausted in CB6F1 mice. Those exhausted TEa cells lost ability to reject Balb/c skins upon further transfer into lymphopenic B6.Rag1-/- mice. Hence, T cell exhaustion develops in the presence of abundant antigen and promotes transplant acceptance. These findings are essential for better understanding the nature of transplant tolerance.

Epigenetically modifying the Foxp3 locus for generation of stable antigen-specific Tregs as cellular therapeutics.

Foxp3+ regulatory T cells (Tregs) are potent immunoregulatory cells, prompting strong interests in manipulating them for therapeutic purposes. However, significant challenges remain, including their heterogeneity and functional instability. Here we focused on the inducible Tregs (iTregs) and studied whether the Foxp3 locus can be epigenetically edited ex vivo to produce stable therapeutic iTregs. Under iTreg-inducing condition where activated CD4+ T effector cells were converted to Foxp3+ Tregs, we tested approximately 30 compounds and identified 3 chromatin-modifying chemical compounds (3C) consisting of sodium butyrate (a broad histone deacetylase inhibitor), UNC0646 (a histone methyltransferase inhibitor), and vitamin C (a TET dioxygenase co-activator), that together produced complete demethylation at the conserved noncoding sequence 2 (CNS2) region of Foxp3 locus. We found that iTregs induced in the presence of 3C (3C-iTregs) are stable, even after exposure to inflammatory cytokines. They expressed high levels of Foxp3 and exhibited potent suppressive activities both in vitro and in vivo. We showed that in models of autoimmunity and transplant rejection, adoptive transfer of antigen-specific 3C-iTregs prevented the induction of experimental autoimmune encephalitis and enabled long-term skin allograft survival. Our data demonstrate that the Foxp3 locus can be epigenetically edited ex vivo to generate stable therapeutic iTregs.

The many shades of macrophages in regulating transplant outcome.

The shift of emphasis from short-term to long-term graft outcomes has led to renewed interests in how the innate immune cells regulate transplant survival, an area that is traditionally dominated by T cells in the adaptive system. This shift is driven largely by the limited efficacy of current immunosuppression protocols which primarily target T cells in preventing chronic graft loss, as well as by the rapid advance of basic sciences in the realm of innate immunity. In fact, the innate immune cells have emerged as key players in the allograft response in various models, contributing to both graft rejection and graft acceptance. Here, we focus on the macrophages, highlighting their diversity, plasticity and emerging features in transplant models, as well as recent developments in our studies of diverse subsets of macrophages. We also discuss challenges, unsolved questions, and emerging approaches in therapeutically modulating macrophages in further improvement of transplant outcomes.

Role of the NF-κB Family Member RelB in Regulation of Foxp3+ Regulatory T Cells In Vivo.

The NF-κB family member RelB is an important transcription factor that is capable of regulating diverse immune and inflammatory responses. However, its role in the regulation of Foxp3+ regulatory T cells (Tregs) in vivo is poorly defined. In this study, we demonstrated that germline deletion of Relb resulted in systemic autoimmunity, which is associated with significant accumulation of Foxp3+ Tregs in lymphoid and nonlymphoid organs. Foxp3+ Tregs from RelB-deficient mice were functional and capable of suppressing T effector cells in vitro and in vivo, but Foxp3- T effector cells from RelB-deficient mice showed features of hyperactivation and spontaneously produced high levels of IL-2. Surprisingly, mice with conditional deletion of Relb in T cells (Cd4CreRelbf/f mice) or specifically in Foxp3+ Tregs (Foxp3CreRelbf/f mice) did not show signs of autoimmunity and had similar frequencies of Foxp3+ Tregs in the periphery as wild-type C57BL/6 controls. Both strains of conditional knockout mice also had a normal conventional T cell compartment. However, reconstituting Rag-1-/-Relb-/- hosts with wild-type C57BL/6 bone marrow cells led to hyperactivation of T effector cells, as well as marked expansion of Foxp3+ T cells. These data suggest that the autoimmune phenotype in germline RelB-deficient mice is most likely caused by T cell-extrinsic mechanisms, and further studies are warranted to uncover such mechanisms.

Ablation of interferon regulatory factor 4 in T cells induces "memory" of transplant tolerance that is irreversible by immune checkpoint blockade.

Achieving transplant tolerance remains the ultimate goal in the field of organ transplantation. We demonstrated previously that ablation of the transcription factor interferon regulatory factor 4 (IRF4) in T cells induced heart transplant acceptance by driving allogeneic CD4+ T cell dysfunction. Herein, we showed that heart-transplanted mice with T cell-specific IRF4 deletion were tolerant to donor-specific antigens and accepted the subsequently transplanted donor-type but not third-party skin allografts. Moreover, despite the rejection of the primary heart grafts in T cell-specific Irf4 knockout mice under immune checkpoint blockade, the establishment of donor-specific tolerance in these mice was unhindered. By tracking alloantigen-specific CD4+ T cells in vivo, we revealed that checkpoint blockade restored the expression levels of the majority of wild-type T cell-expressed genes in Irf4-deficient T cells on day 6 post-heart grafting, indicating the initial reinvigoration of Irf4-deficient T cells. Nevertheless, checkpoint blockade did not restore cell frequency, effector memory cell generation, and IFN-γ/TNF-α production of Irf4-/- alloreactive T cells at day 30 post-heart grafting. Hence, targeting IRF4 represents a potential therapeutic strategy for driving intrinsic T cell dysfunction and achieving alloantigen-specific transplant tolerance.

Adaptive features of innate immune cells and their relevance to graft rejection.

PURPOSE OF REVIEW: Allograft rejection involves both innate and adaptive immune cells, and the adaptive immune cells have dominated transplant studies for decades. Recent studies have identified surprising new features for the innate immune cells, including memory recall responses, which may have significant implications in further improvement of transplant outcomes. RECENT FINDINGS: Transplant survival is excellent in the short-term, but the long-term graft outcomes are not so, and most grafts are continuously lost to chronic rejection in the clinic. In both animal models and clinical settings, graft loss to chronic rejection is often dominated by innate immune cells, especially macrophages and natural killer (NK) cells in the grafts. Recent studies suggest that innate immune cells can acquire features of adaptive cells in that they either directly sense allogeneic nonself or become 'trained' in the allogeneic milieu, where they show features of memory recall responses. In certain models, targeting the adaptive features of such innate immune cells can promote long-term allograft survival. These findings may open new therapeutic opportunities in promoting transplant survival in the clinic. SUMMARY: The discovery of donor specificity and memory recall responses of certain innate immune cells, which are prominently featured in chronic allograft rejection, may open novel therapeutic opportunities in transplantation, as well as in treatment of cancers and autoimmune diseases.