CXCL16-dependent scavenging of oxidized lipids by islet macrophages promotes differentiation of pathogenic CD8+ T cells in diabetic autoimmunity.

The pancreatic islet microenvironment is highly oxidative, rendering ß cells vulnerable to autoinflammatory insults. Here, we examined the role of islet resident macrophages in the autoimmune attack that initiates type 1 diabetes. Islet macrophages highly expressed CXCL16, a chemokine and scavenger receptor for oxidized low-density lipoproteins (OxLDLs), regardless of autoimmune predisposition. Deletion of Cxcl16 in nonobese diabetic (NOD) mice suppressed the development of autoimmune diabetes. Mechanistically, Cxcl16 deficiency impaired clearance of OxLDL by islet macrophages, leading to OxLDL accumulation in pancreatic islets and a substantial reduction in intra-islet transitory (Texint) CD8+ T cells displaying proliferative and effector signatures. Texint cells were vulnerable to oxidative stress and diminished by ferroptosis; PD-1 blockade rescued this population and reversed diabetes resistance in NOD.Cxcl16-/- mice. Thus, OxLDL scavenging in pancreatic islets inadvertently promotes differentiation of pathogenic CD8+ T cells, presenting a paradigm wherein tissue homeostasis processes can facilitate autoimmune pathogenesis in predisposed individuals.

Human epigenetic and transcriptional T cell differentiation atlas for identifying functional T cell-specific enhancers.

The clinical benefit of T cell immunotherapies remains limited by incomplete understanding of T cell differentiation and dysfunction. We generated an epigenetic and transcriptional atlas of T cell differentiation from healthy humans that included exhausted CD8 T cells and applied this resource in three ways. First, we identified modules of gene expression and chromatin accessibility, revealing molecular coordination of differentiation after activation and between central memory and effector memory. Second, we applied this healthy molecular framework to three settings-a neoadjuvant anti-PD1 melanoma trial, a basal cell carcinoma scATAC-seq dataset, and autoimmune disease-associated SNPs-yielding insights into disease-specific biology. Third, we predicted genome-wide cis-regulatory elements and validated this approach for key effector genes using CRISPR interference, providing functional annotation and demonstrating the ability to identify targets for non-coding cellular engineering. These studies define epigenetic and transcriptional regulation of human T cells and illustrate the utility of interrogating disease in the context of a healthy T cell atlas.

Polycomb Repressive Complex 2-Mediated Chromatin Repression Guides Effector CD8+ T Cell Terminal Differentiation and Loss of Multipotency.

Understanding immunological memory formation depends on elucidating how multipotent memory precursor (MP) cells maintain developmental plasticity and longevity to provide long-term immunity while other effector cells develop into terminally differentiated effector (TE) cells with limited survival. Profiling active (H3K27ac) and repressed (H3K27me3) chromatin in naive, MP, and TE CD8+ T cells during viral infection revealed increased H3K27me3 deposition at numerous pro-memory and pro-survival genes in TE relative to MP cells, indicative of fate restriction, but permissive chromatin at both pro-memory and pro-effector genes in MP cells, indicative of multipotency. Polycomb repressive complex 2 deficiency impaired clonal expansion and TE cell differentiation, but minimally impacted CD8+ memory T cell maturation. Abundant H3K27me3 deposition at pro-memory genes occurred late during TE cell development, probably from diminished transcription factor FOXO1 expression. These results outline a temporal model for loss of memory cell potential through selective epigenetic silencing of pro-memory genes in effector T cells.

Hobit and Blimp-1 regulate TRM abundance after LCMV infection by suppressing tissue exit pathways of TRM precursors.

Tissue-resident memory T cells (Trm) are retained in peripheral tissues after infection for enhanced protection against secondary encounter with the same pathogen. We have previously shown that the transcription factor Hobit and its homolog Blimp-1 drive Trm development after viral infection, but how and when these transcription factors mediate Trm formation remains poorly understood. In particular, the major impact of Blimp-1 in regulating several aspects of effector T-cell differentiation impairs study of its specific role in Trm development. Here, we used the restricted expression of Hobit in the Trm lineage to develop mice with a conditional deletion of Blimp-1 in Trm, allowing us to specifically investigate the role of both transcription factors in Trm differentiation. We found that Hobit and Blimp-1 were required for the upregulation of CD69 and suppression of CCR7 and S1PR1 on virus-specific Trm precursors after LCMV infection, underlining a role in their retention within tissues. The early impact of Hobit and Blimp-1 favored Trm formation and prevented the development of circulating memory T cells. Thus, our findings highlight a role of Hobit and Blimp-1 at the branching point of circulating and resident memory lineages by suppressing tissue egress of Trm precursors early during infection.

Organized immune cell interactions within tumors sustain a productive T-cell response.

Tumor-infiltrating CD8 T cells are associated with improved patient survival and response to immunotherapy in various cancers. Persistent antigen leads to CD8 T-cell exhaustion, where proliferation/self-renewal and killing are divided within distinct subsets of CD8 T cells in the tumor. CD8 T-cell responses in chronic antigen settings must be maintained for long periods of time, suggesting that mechanisms that regulate chronic CD8 T-cell responses may differ from those in acute settings. Currently, factors that regulate the maintenance of stem-like CD8 T cells in the tumor or their differentiation into terminally differentiated cells are unknown. In this review, we discuss the role of dendritic cells in the activation and differentiation of CD8 T-cell subsets within secondary lymphoid tissue and tumors. In addition, we examine changes in CD4 T-cell differentiation in response to chronic antigens and consider how subset-specific mechanisms could assist the stem-like and terminally differentiated CD8 T-cell subsets. Finally, we highlight how tumor-infiltrating CD4 T cells and dendritic cells interact with CD8 T cells within organized lymphoid-like areas in the tumor and propose a CD8 T-cell differentiation model that requires the collaboration of CD4 T cells and dendritic cells. These organized interactions coordinate the anti-tumor response and control disease progression by mechanisms that regulate CD8 T-cell differentiation, which permit the maintenance of an effective balance of stem-like and terminally differentiated CD8 T cells.

In vivo RNAi screens: concepts and applications.

Functional genomics approaches that leverage the RNAi pathway have been applied in vivo to examine the roles of hundreds or thousands of genes; mainly in the context of cancer. Here, we discuss principles guiding the design of RNAi screens, parameters that determine success and recent developments that have improved accuracy and expanded the applicability of these approaches to other in vivo settings, including the immune system. We review recent studies that have applied in vivo RNAi screens in T cells to examine genes that regulate T cell differentiation during viral infection, and that control their accumulation in tumors in a model of adoptive T cell therapy. In this context, we put forward an argument as to why RNAi approaches in vivo are likely to provide particularly salient insight into immunology.

Triclocarban disrupts the activation and differentiation of human CD8+ T cells by suppressing the vitamin D receptor signaling.

Triclocarban (TCC) is a widely applied environmental endocrine-disrupting chemical (EDC). Similar to most of EDCs, TCC potentially damages the immunity of various species. However, whether and how TCC impacts the adaptive immunity in mammals has yet to be determined. Herein, we discovered that TCC disrupts the activation and differentiation of CD8+ T cells in primary human peripheral blood samples, purified CD8+ T cells, and in mice in vivo. Mechanistically, TCC might block the activation of the vitamin D receptor (VDR) and reduce the synthesis of cholesterol, a precursor of vitamin D, resulting in inhibition of VDR signaling due to the suppression of both its ligand and the receptor itself by TCC. Our findings elucidate the hazard and potential mechanisms of TCC in mammalian adaptive immunity and highlighted VDR as a potential therapeutic target for the immunodeficiency caused by TCC.

Cell-intrinsic PD-L1 ablation sustains effector CD8+ T cell responses and promotes antitumor T cell therapy.

Adoptive cell therapies are emerging forms of immunotherapy that reprogram T cells for enhanced antitumor responses. Although surface programmed cell death-ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) engagement inhibits antitumor immunity, the role of cell-intrinsic PD-L1 in adoptive T cell therapy remains unknown. Here, we found that intracellular PD-L1 was enriched in tumor-infiltrating CD8+ T cells of cancer patients. PD-L1 ablation promoted antitumor immune responses and the maintenance of an effector-like state of therapeutic CD8+ T cells, while blockade of surface PD-L1 was unable to impact on their expansion and function. Moreover, cell-intrinsic PD-L1 impeded CD8+ T cell activity, which partially relied on mTORC1 signaling. Furthermore, endogenous tumor-reactive CD8+ T cells were motivated by BATF3-driven dendritic cells after adoptive transfer of PD-L1-deficient therapeutic CD8+ T cells. This role of cell-intrinsic PD-L1 in therapeutic CD8+ T cell dysfunction highlights that disrupting cell-intrinsic PD-L1 in CD8+ T cells represents a viable approach to improving T cell-based cancer immunotherapy.

BATF-mediated regulation of exhausted CD8+ T-cell responses and potential implications for chimeric antigen receptor-T therapy.

Chimeric antigen receptor (CAR) T-cell therapy for malignant tumors has reached a crucial stage, with recent studies underscoring the role of T-cell exhaustion in determining the efficacy of CAR-T therapy. This trailblazing discovery has opened new avenues to augment the potency of CAR-T therapy. Basic leucine zipper ATF-like transcription factor (BATF) is indispensable in alleviating T-cell exhaustion and is pivotal in the early stages of CD8+ T-cell differentiation. In cooperation with other transcription factors, it plays a key role in the differentiation and maturation processes of exhausted T cells. A deeper comprehension of BATF's mechanisms in T-cell biology may yield novel insights into amplifying the efficacy of CAR-T therapy.

Chimeric antigen receptor (CAR) T-cell therapy, a treatment that boosts the body's immune system to fight cancer, has made significant progress. Recent research has shown that T-cell exhaustion, which is when the body's immune cells become less effective, affects how well this therapy works. This finding has opened new possibilities to make CAR-T therapy more effective. There is a specific protein called BATF that plays an important role in reducing T-cell exhaustion and influencing the early development of certain immune cells. This review describes how BATF interacts with exhausted T cells, to improve CAR-T therapy. By understanding how BATF works in the immune system, new ways to enhance CAR-T therapy and its ability to fight cancer may be found.

Themis suppresses the effector function of CD8+ T cells in acute viral infection.

CD8+ T cells play a central role in antiviral immune responses. Upon infection, naive CD8+ T cells differentiate into effector cells to eliminate virus-infected cells, and some of these effector cells further differentiate into memory cells to provide long-term protection after infection is resolved. Although extensively investigated, the underlying mechanisms of CD8+ T-cell differentiation remain incompletely understood. Themis is a T-cell-specific protein that plays critical roles in T-cell development. Recent studies using Themis T-cell conditional knockout mice also demonstrated that Themis is required to promote mature CD8+ T-cell homeostasis, cytokine responsiveness, and antibacterial responses. In this study, we used LCMV Armstrong infection as a probe to explore the role of Themis in viral infection. We found that preexisting CD8+ T-cell homeostasis defects and cytokine hyporesponsiveness do not impair viral clearance in Themis T-cell conditional knockout mice. Further analyses showed that in the primary immune response, Themis deficiency promoted the differentiation of CD8+ effector cells and increased their TNF and IFNγ production. Moreover, Themis deficiency impaired memory precursor cell (MPEC) differentiation but promoted short-lived effector cell (SLEC) differentiation. Themis deficiency also enhanced effector cytokine production in memory CD8+ T cells while impairing central memory CD8+ T-cell formation. Mechanistically, we found that Themis mediates PD-1 expression and its signaling in effector CD8+ T cells, which explains the elevated cytokine production in these cells when Themis is disrupted.

The diverse effects of transforming growth factor-ß and SMAD signaling pathways during the CTL response.

Cytotoxic T lymphocytes (CTLs) play an important role in defense against infections with intracellular pathogens and anti-tumor immunity. Efficient migration is required to locate and destroy infected cells in different regions of the body. CTLs accomplish this task by differentiating into specialized subsets of effector and memory CD8 T cells that traffic to different tissues. Transforming growth factor-beta (TGFß) belongs to a large family of growth factors that elicit diverse cellular responses via canonical and non-canonical signaling pathways. Canonical SMAD-dependent signaling pathways are required to coordinate changes in homing receptor expression as CTLs traffic between different tissues. In this review, we discuss the various ways that TGFß and SMAD-dependent signaling pathways shape the cellular immune response and transcriptional programming of newly activated CTLs. As protective immunity requires access to the circulation, emphasis is placed on cellular processes that are required for cell-migration through the vasculature.

Loss of PBAF promotes expansion and effector differentiation of CD8+ T cells during chronic viral infection and cancer.

During chronic viral infection and cancer, it has been established that a subset of progenitor CD8+ T cells continuously gives rise to terminally exhausted cells and cytotoxic effector cells. Although multiple transcriptional programs governing the bifurcated differentiation trajectories have been previously studied, little is known about the chromatin structure changes regulating CD8+ T cell-fate decision. In this study, we demonstrate that the chromatin remodeling complex PBAF restrains expansion and promotes exhaustion of CD8+ T cells during chronic viral infection and cancer. Mechanistically, transcriptomic and epigenomic analyses reveal the role of PBAF in maintaining chromatin accessibility of multiple genetic pathways and transcriptional programs to restrain proliferation and promote T cell exhaustion. Harnessing this knowledge, we demonstrate that perturbation of PBAF complex constrained exhaustion and promoted expansion of tumor-specific CD8+ T cells resulting in antitumor immunity in a preclinical melanoma model, implicating PBAF as an attractive target for cancer immunotherapeutic.

Asymmetric cell division safeguards memory CD8 T cell development.

The strength of T cell receptor (TCR) stimulation and asymmetric distribution of fate determinants are both implied to affect T cell differentiation. Here, we uncover asymmetric cell division (ACD) as a safeguard mechanism for memory CD8 T cell generation specifically upon strong TCR stimulation. Using live imaging approaches, we find that strong TCR stimulation induces elevated ACD rates, and subsequent single-cell-derived colonies comprise both effector and memory precursor cells. The abundance of memory precursor cells emerging from a single activated T cell positively correlates with first mitosis ACD. Accordingly, preventing ACD by inhibition of protein kinase Cζ (PKCζ) during the first mitosis upon strong TCR stimulation markedly curtails the formation of memory precursor cells. Conversely, no effect of ACD on fate commitment is observed upon weak TCR stimulation. Our data provide relevant mechanistic insights into the role of ACD for CD8 T cell fate regulation upon different activation conditions.

Exhaustion of CD8+ central memory responder T cell differentiation provokes non-melanoma skin cancer in elderly kidney transplant recipients.

Introduction: Immunosuppressive therapy prevents graft rejection but increases the risk of non-melanoma skin cancer (NMSC), especially in elderly kidney transplant recipients (KTR). Methods: In this study, we separately investigated the differentiation of CD8+ regulatory T cells (Tregs) and responder T cells (Tresps) between healthy KTR without NMSC, KTR developing de-novo NMSC within two years after the enrolment, and KTR with NMSC at the time of enrolment. Antigen-unexperienced CCR7+CD45RA+CD31+ recent thymic emigrant (RTE) cells differentiate via CD45RA-CD31+ memory (CD31+ memory) cells, via resting mature naïve (MN) cells or via direct proliferation into CD45RA-CD31- memory (CD31- memory) cells, consisting of both CCR7+CD45RA- central memory (CM) and CCR7-CD45RA- effector memory (EM) cells. Results: We found that both RTE Treg and Tresp differentiation via CD31+ memory Tregs/Tresps was age-independently increased in KTR, who developed de novo NMSC during the follow-up period, causing abundant CM Treg/Tresp production, which may be crucial for cancer immunity. These changes favored a strongly increased CD8+ Treg/Tresp ratio, suggesting this ratio as a reliable marker for de-novo NMSC development in KTR. However, with age, this differentiation was replaced by increased conversion of resting MN Tregs/Tresps into CM Tregs/Tresps, which exhausted for Tresps but not for Tregs. In KTR with already existing NMSC at enrolment, differentiation was maintained via conversion and proliferation of resting MN Tregs/Tresps, which however increasingly exhausted with age, especially for Tresps. This resulted in a strong accumulation of terminally differentiated effector memory (TEMRA) Tresps in elderly individuals. Patients with NMSC recurrence showed increased proliferation of resting MN Tregs/Tresps into EM Tregs/Tresps, which tended to exhaust more rapidly, particularly for Tresps, than in patients without NMSC recurrence. Discussion: In conclusion, we provide evidence that immunosuppressive therapy inhibits differentiation of CD8+ Tregs more than that of CD8+ Tresps, resulting in an exhausted Tresp profile, thus providing a possible therapeutic approach to improve poor cancer immunity in elderly KTR.

Differentiation of Memory CD8 T Cells Unravel Gene Expression Pattern Common to Effector and Memory Precursors.

Long-term immunological protection relies on the differentiation and maintenance of memory lymphocytes. Since the knowledge of memory generation has been centered on in vivo models of infection, there are obstacles to deep molecular analysis of differentiating subsets. Here we defined a novel in vitro CD8 T cell activation and culture regimen using low TCR engagement and cytokines to generate differentiated cells consistent with central memory-like cells, as shown by surface phenotype, gene expression profile and lack of cytotoxic function after challenge. Our results showed an effector signature expressed by in vitro memory precursors and their plasticity under specific conditions. Moreover, memory CD8 T cells conferred long-term protection against bacterial infection and slowed in vivo tumor growth more efficiently than effector cells. This model may allow further understanding of CD8 T cell memory molecular differentiation subsets and be suited for generating cells to be used for immunotherapy.

Autocrine and paracrine IL-2 signals collaborate to regulate distinct phases of CD8 T cell memory.

Differential interleukin-2 (IL-2) signaling and production are associated with disparate effector and memory fates. Whether the IL-2 signals perceived by CD8 T cells come from autocrine or paracrine sources, the timing of IL-2 signaling and their differential impact on CD8 T cell responses remain unclear. Using distinct models of germline and conditional IL-2 ablation in post-thymic CD8 T cells, this study shows that paracrine IL-2 is sufficient to drive optimal primary expansion, effector and memory differentiation, and metabolic function. In contrast, autocrine IL-2 is uniquely required during primary expansion to program robust secondary expansion potential in memory-fated cells. This study further shows that IL-2 production by antigen-specific CD8 T cells is largely independent of CD4 licensing of dendritic cells (DCs) in inflammatory infections with robust DC activation. These findings bear implications for immunizations and adoptive T cell immunotherapies, where effector and memory functions may be commandeered through IL-2 programming.

SMAD4 and TGFß are architects of inverse genetic programs during fate determination of antiviral CTLs.

Transforming growth factor ß (TGFß) is an important differentiation factor for cytotoxic T lymphocytes (CTLs) and alters the expression levels of several of homing receptors during infection. SMAD4 is part of the canonical signaling network used by members of the transforming growth factor family. For this study, genetically modified mice were used to determine how SMAD4 and TGFß receptor II (TGFßRII) participate in transcriptional programming of pathogen-specific CTLs. We show that these molecules are essential components of opposing signaling mechanisms, and cooperatively regulate a collection of genes that determine whether specialized populations of pathogen-specific CTLs circulate around the body, or settle in peripheral tissues. TGFß uses a canonical SMAD-dependent signaling pathway to downregulate Eomesodermin (EOMES), KLRG1, and CD62L, while CD103 is induced. Conversely, in vivo and in vitro data show that EOMES, KLRG1, CX3CR1, and CD62L are positively regulated via SMAD4, while CD103 and Hobit are downregulated. Intravascular staining also shows that signaling via SMAD4 promotes formation of long-lived terminally differentiated CTLs that localize in the vasculature. Our data show that inflammatory molecules play a key role in lineage determination of pathogen-specific CTLs, and use SMAD-dependent signaling to alter the expression levels of multiple homing receptors and transcription factors with known functions during memory formation.

Chronic Kidney Failure Provokes the Enrichment of Terminally Differentiated CD8+ T Cells, Impairing Cytotoxic Mechanisms After Kidney Transplantation.

Chronic kidney failure (KF) provokes the development of immune senescent CD8+ cytotoxic T cells, affecting the occurrence of graft rejection, viral infections, and malignancies after kidney transplantation. In this study, we analyzed the impact of KF, subsequent dialysis treatment, and kidney transplantation on the differentiation of CD8+CD31+CD45RA+CCR7+ recent thymic emigrant (CCR7+ RTE) Tregs/Tresps into CD8+CD31-CD45RA- memory (CD31- memory) Tregs/Tresps and its effect on the release of cytokines, Fas receptor, Fas ligand as well as cytotoxic mediators by naïve, central memory (CM), effector memory (EM), and terminally differentiated effector memory (TEMRA) Tresps. We found that normal age-dependent differentiation of CD8+ Tregs/Tresps generally differs in the way that TEMRA cells only arise in Tresps. Compared to healthy controls, KF patients revealed an age-independently decreased frequency of CCR7+ RTE Tregs/Tresps, but increased frequencies of CCR7+ MN Tregs/Tresps and CD31- memory Tregs/Tresps, suggesting an increased differentiation via CD31+CD45RA- memory (CD31+ memory) Tregs/Tresps into CD31- memory Tregs/Tresps. Intensified differentiation via CD31+ memory Tresps increased the emergence of apoptosis-resistant CM Tresps with strong Fas ligand-mediated cytotoxicity. CCR7+ RTE Tresp proliferation generated TEMRA Tresps, secreting high levels of cytotoxic mediators. In dialysis and transplant patients, CD31+ TEMRA Tregs/Tresps accumulated, proposing an impaired CCR7+ RTE Treg/Tresp differentiation via CD31+ memory Tregs/Tresps into CD31- memory Tregs/Tresps. Increased percentages of CD31- TEMRA Tresps, but not of CD31- TEMRA Tregs, were observed in all patient groups, indicating impaired proliferation of CCR7+ RTE Tresps, but not of CCR7+ RTE Tregs, into CD31- memory Tregs/Tresps. In transplant patients, CCR7+ RTE Tregs accumulated, while frequencies of CCR7+ RTE Tresps were decreased, suggesting that the immunosuppressive therapy only prevented excessive CCR7+ RTE Treg differentiation but not that of CCR7+ RTE Tresps. Presumably, this caused the accumulation of TEMRA Tresps with decreased release of cytotoxic mediators, such as perforin. In conclusion, we propose that chronic KF affects both the differentiation of CD8+ Tregs and CD8+ Tresps. However, the immunosuppressive therapy after transplantation may successfully prevent excessive Treg differentiation, but not as suffciently that of Tresps. Therefore, the risk for graft rejection may be reduced, while the susceptibility for infections and malignancies may be increased in these patients.

CXCR3 regulates stem and proliferative CD8+ T cells during chronic infection by promoting interactions with DCs in splenic bridging channels.

Production of effector CD8+ T cells during persistent infection requires a stable pool of stem-like cells that can give rise to effector cells via a proliferative intermediate population. In infection models marked by T cell exhaustion, this process can be transiently induced by checkpoint blockade but occurs spontaneously in mice chronically infected with the protozoan intracellular parasite Toxoplasma gondii. We observe distinct locations for parasite-specific T cell subsets, implying a link between differentiation and anatomical niches in the spleen. Loss of the chemokine receptor CXCR3 on T cells does not prevent white pulp-to-red pulp migration but reduces interactions with CXCR3 ligand-producing dendritic cells (DCs) and impairs memory-to-intermediate transition, leading to a buildup of memory T cells in the red pulp. Thus, CXCR3 increases T cell exposure to differentiation-inducing signals during red pulp migration, providing a dynamic mechanism for modulating effector differentiation in response to environmental signals.

Single-cell analysis of CD8 T lymphocyte diversity during adaptive immunity.

An effective adaptive immune response to microbial infection relies on the generation of heterogeneous T lymphocyte fates and functions. CD8 T lymphocytes play a pivotal role in mediating immediate and long-term protective immune responses to intracellular pathogen infection. Systems-based analysis of the immune response to infection has begun to identify cell fate determinants and the molecular mechanisms underpinning CD8 T lymphocyte diversity at single-cell resolution. Resolving CD8 T lymphocyte heterogeneity during adaptive immunity highlights the advantages of single-cell technologies and computational approaches to better understand the ontogeny of CD8 T cellular diversity following infection. Future directions of integrating single-cell multiplex approaches capitalize on the importance of systems biology in the understanding of immune CD8 T cell differentiation and functional diversity. This article is categorized under: Physiology > Mammalian Physiology in Health and Disease Biological Mechanisms > Cell Fates.