The CD4 Versus CD8 T Cell Fate Decision: A Multiomics-Informed Perspective.

The choice of developing thymocytes to become CD8+ cytotoxic or CD4+ helper T cells has been intensely studied, but many of the underlying mechanisms remain to be elucidated. Recent multiomics approaches have provided much higher resolution analysis of gene expression in developing thymocytes than was previously achievable, thereby offering a fresh perspective on this question. Focusing on our recent studies using CITE-seq (cellular indexing of transcriptomes and epitopes) analyses of mouse thymocytes, we present a detailed timeline of RNA and protein expression changes during CD8 versus CD4 T cell differentiation. We also revisit our current understanding of the links between T cell receptor signaling and expression of the lineage-defining transcription factors ThPOK and RUNX3. Finally, we propose a sequential selection model to explain the tight linkage between MHC-I versus MHC-II recognition and T cell lineage choice. This model incorporates key aspects of previously proposed kinetic signaling, instructive, and stochastic/selection models.

Beyond the Barrier: Unraveling the Mechanisms of Immunotherapy Resistance.

Immune checkpoint blockade (ICB) induces a remarkable and durable response in a subset of cancer patients. However, most patients exhibit either primary or acquired resistance to ICB. This resistance arises from a complex interplay of diverse dynamic mechanisms within the tumor microenvironment (TME). These mechanisms include genetic, epigenetic, and metabolic alterations that prevent T cell trafficking to the tumor site, induce immune cell dysfunction, interfere with antigen presentation, drive heightened expression of coinhibitory molecules, and promote tumor survival after immune attack. The TME worsens ICB resistance through the formation of immunosuppressive networks via immune inhibition, regulatory metabolites, and abnormal resource consumption. Finally, patient lifestyle factors, including obesity and microbiome composition, influence ICB resistance. Understanding the heterogeneity of cellular, molecular, and environmental factors contributing to ICB resistance is crucial to develop targeted therapeutic interventions that enhance the clinical response. This comprehensive overview highlights key mechanisms of ICB resistance that may be clinically translatable.

Osr2 functions as a biomechanical checkpoint to aggravate CD8+ T cell exhaustion in tumor.

Alterations in extracellular matrix (ECM) architecture and stiffness represent hallmarks of cancer. Whether the biomechanical property of ECM impacts the functionality of tumor-reactive CD8+ T cells remains largely unknown. Here, we reveal that the transcription factor (TF) Osr2 integrates biomechanical signaling and facilitates the terminal exhaustion of tumor-reactive CD8+ T cells. Osr2 expression is selectively induced in the terminally exhausted tumor-specific CD8+ T cell subset by coupled T cell receptor (TCR) signaling and biomechanical stress mediated by the Piezo1/calcium/CREB axis. Consistently, depletion of Osr2 alleviates the exhaustion of tumor-specific CD8+ T cells or CAR-T cells, whereas forced Osr2 expression aggravates their exhaustion in solid tumor models. Mechanistically, Osr2 recruits HDAC3 to rewire the epigenetic program for suppressing cytotoxic gene expression and promoting CD8+ T cell exhaustion. Thus, our results unravel Osr2 functions as a biomechanical checkpoint to exacerbate CD8+ T cell exhaustion and could be targeted to potentiate cancer immunotherapy.

Blockade of LAG-3 and PD-1 leads to co-expression of cytotoxic and exhaustion gene modules in CD8+ T cells to promote antitumor immunity.

Relatlimab (rela; anti-LAG-3) plus nivolumab (nivo; anti-PD-1) is safe and effective for treatment of advanced melanoma. We designed a trial (NCT03743766) where advanced melanoma patients received rela, nivo, or rela+nivo to interrogate the immunologic mechanisms of rela+nivo. Analysis of biospecimens from this ongoing trial demonstrated that rela+nivo led to enhanced capacity for CD8+ T cell receptor signaling and altered CD8+ T cell differentiation, leading to heightened cytotoxicity despite the retention of an exhaustion profile. Co-expression of cytotoxic and exhaustion signatures was driven by PRDM1, BATF, ETV7, and TOX. Effector function was upregulated in clonally expanded CD8+ T cells that emerged after rela+nivo. A rela+nivo intratumoral CD8+ T cell signature was associated with a favorable prognosis. This intratumoral rela+nivo signature was validated in peripheral blood as an elevated frequency of CD38+TIM3+CD8+ T cells. Overall, we demonstrated that cytotoxicity can be enhanced despite the retention of exhaustion signatures, which will inform future therapeutic strategies.

Bispecific dendritic-T cell engager potentiates anti-tumor immunity.

Immune checkpoint inhibition treatment using aPD-1 monoclonal antibodies is a promising cancer immunotherapy approach. However, its effect on tumor immunity is narrow, as most patients do not respond to the treatment or suffer from recurrence. We show that the crosstalk between conventional type I dendritic cells (cDC1) and T cells is essential for an effective aPD-1-mediated anti-tumor response. Accordingly, we developed a bispecific DC-T cell engager (BiCE), a reagent that facilitates physical interactions between PD-1+ T cells and cDC1. BiCE treatment promotes the formation of active dendritic/T cell crosstalk in the tumor and tumor-draining lymph nodes. In vivo, single-cell and physical interacting cell analysis demonstrates the distinct and superior immune reprogramming of the tumors and tumor-draining lymph nodes treated with BiCE as compared to conventional aPD-1 treatment. By bridging immune cells, BiCE potentiates cell circuits and communication pathways needed for effective anti-tumor immunity.

A versatile CRISPR-Cas13d platform for multiplexed transcriptomic regulation and metabolic engineering in primary human T cells.

CRISPR technologies have begun to revolutionize T cell therapies; however, conventional CRISPR-Cas9 genome-editing tools are limited in their safety, efficacy, and scope. To address these challenges, we developed multiplexed effector guide arrays (MEGA), a platform for programmable and scalable regulation of the T cell transcriptome using the RNA-guided, RNA-targeting activity of CRISPR-Cas13d. MEGA enables quantitative, reversible, and massively multiplexed gene knockdown in primary human T cells without targeting or cutting genomic DNA. Applying MEGA to a model of CAR T cell exhaustion, we robustly suppressed inhibitory receptor upregulation and uncovered paired regulators of T cell function through combinatorial CRISPR screening. We additionally implemented druggable regulation of MEGA to control CAR activation in a receptor-independent manner. Lastly, MEGA enabled multiplexed disruption of immunoregulatory metabolic pathways to enhance CAR T cell fitness and anti-tumor activity in vitro and in vivo. MEGA offers a versatile synthetic toolkit for applications in cancer immunotherapy and beyond.

Cancer SLC6A6-mediated taurine uptake transactivates immune checkpoint genes and induces exhaustion in CD8+ T cells.

Taurine is used to bolster immunity, but its effects on antitumor immunity are unclear. Here, we report that cancer-related taurine consumption causes T cell exhaustion and tumor progression. The taurine transporter SLC6A6 is correlated with aggressiveness and poor outcomes in multiple cancers. SLC6A6-mediated taurine uptake promotes the malignant behaviors of tumor cells but also increases the survival and effector function of CD8+ T cells. Tumor cells outcompete CD8+ T cells for taurine by overexpressing SLC6A6, which induces T cell death and malfunction, thereby fueling tumor progression. Mechanistically, taurine deficiency in CD8+ T cells increases ER stress, promoting ATF4 transcription in a PERK-JAK1-STAT3 signaling-dependent manner. Increased ATF4 transactivates multiple immune checkpoint genes and induces T cell exhaustion. In gastric cancer, we identify a chemotherapy-induced SP1-SLC6A6 regulatory axis. Our findings suggest that tumoral-SLC6A6-mediated taurine deficiency promotes immune evasion and that taurine supplementation reinvigorates exhausted CD8+ T cells and increases the efficacy of cancer therapies.

Neoadjuvant PARPi or chemotherapy in ovarian cancer informs targeting effector Treg cells for homologous-recombination-deficient tumors.

Homologous recombination deficiency (HRD) is prevalent in cancer, sensitizing tumor cells to poly (ADP-ribose) polymerase (PARP) inhibition. However, the impact of HRD and related therapies on the tumor microenvironment (TME) remains elusive. Our study generates single-cell gene expression and T cell receptor profiles, along with validatory multimodal datasets from >100 high-grade serous ovarian cancer (HGSOC) samples, primarily from a phase II clinical trial (NCT04507841). Neoadjuvant monotherapy with the PARP inhibitor (PARPi) niraparib achieves impressive 62.5% and 73.6% response rates per RECIST v.1.1 and GCIG CA125, respectively. We identify effector regulatory T cells (eTregs) as key responders to HRD and neoadjuvant therapies, co-occurring with other tumor-reactive T cells, particularly terminally exhausted CD8+ T cells (Tex). TME-wide interferon signaling correlates with cancer cells upregulating MHC class II and co-inhibitory ligands, potentially driving Treg and Tex fates. Depleting eTregs in HRD mouse models, with or without PARP inhibition, significantly suppresses tumor growth without observable toxicities, underscoring the potential of eTreg-focused therapeutics for HGSOC and other HRD-related tumors.

LAG-3 sustains TOX expression and regulates the CD94/NKG2-Qa-1b axis to govern exhausted CD8 T cell NK receptor expression and cytotoxicity.

Exhausted CD8 T (Tex) cells in chronic viral infection and cancer have sustained co-expression of inhibitory receptors (IRs). Tex cells can be reinvigorated by blocking IRs, such as PD-1, but synergistic reinvigoration and enhanced disease control can be achieved by co-targeting multiple IRs including PD-1 and LAG-3. To dissect the molecular changes intrinsic when these IR pathways are disrupted, we investigated the impact of loss of PD-1 and/or LAG-3 on Tex cells during chronic infection. These analyses revealed distinct roles of PD-1 and LAG-3 in regulating Tex cell proliferation and effector functions, respectively. Moreover, these studies identified an essential role for LAG-3 in sustaining TOX and Tex cell durability as well as a LAG-3-dependent circuit that generated a CD94/NKG2+ subset of Tex cells with enhanced cytotoxicity mediated by recognition of the stress ligand Qa-1b, with similar observations in humans. These analyses disentangle the non-redundant mechanisms of PD-1 and LAG-3 and their synergy in regulating Tex cells.

Progestogen-driven B7-H4 contributes to onco-fetal immune tolerance.

Immune tolerance mechanisms are shared in cancer and pregnancy. Through cross-analyzing single-cell RNA-sequencing data from multiple human cancer types and the maternal-fetal interface, we found B7-H4 (VTCN1) is an onco-fetal immune tolerance checkpoint. We showed that genetic deficiency of B7-H4 resulted in immune activation and fetal resorption in allogeneic pregnancy models. Analogously, B7-H4 contributed to MPA/DMBA-induced breast cancer progression, accompanied by CD8+ T cell exhaustion. Female hormone screening revealed that progesterone stimulated B7-H4 expression in placental and breast cancer cells. Mechanistically, progesterone receptor (PR) bound to a newly identified -58 kb enhancer, thereby mediating B7-H4 transcription via the PR-P300-BRD4 axis. PR antagonist or BRD4 degrader potentiated immunotherapy in a murine B7-H4+ breast cancer model. Thus, our work unravels a mechanistic and biological connection of a female sex hormone (progesterone) to onco-fetal immune tolerance via B7-H4 and suggests that the PR-P300-BRD4 axis is targetable for treating B7-H4+ cancer.

Therapeutic potential of co-signaling receptor modulation in hepatitis B.

Reversing CD8+ T cell dysfunction is crucial in treating chronic hepatitis B virus (HBV) infection, yet specific molecular targets remain unclear. Our study analyzed co-signaling receptors during hepatocellular priming and traced the trajectory and fate of dysfunctional HBV-specific CD8+ T cells. Early on, these cells upregulate PD-1, CTLA-4, LAG-3, OX40, 4-1BB, and ICOS. While blocking co-inhibitory receptors had minimal effect, activating 4-1BB and OX40 converted them into antiviral effectors. Prolonged stimulation led to a self-renewing, long-lived, heterogeneous population with a unique transcriptional profile. This includes dysfunctional progenitor/stem-like (TSL) cells and two distinct dysfunctional tissue-resident memory (TRM) populations. While 4-1BB expression is ubiquitously maintained, OX40 expression is limited to TSL. In chronic settings, only 4-1BB stimulation conferred antiviral activity. In HBeAg+ chronic patients, 4-1BB activation showed the highest potential to rejuvenate dysfunctional CD8+ T cells. Targeting all dysfunctional T cells, rather than only stem-like precursors, holds promise for treating chronic HBV infection.

Heterologous survey of 130 DNA transposons in human cells highlights their functional divergence and expands the genome engineering toolbox.

Experimental studies on DNA transposable elements (TEs) have been limited in scale, leading to a lack of understanding of the factors influencing transposition activity, evolutionary dynamics, and application potential as genome engineering tools. We predicted 130 active DNA TEs from 102 metazoan genomes and evaluated their activity in human cells. We identified 40 active (integration-competent) TEs, surpassing the cumulative number (20) of TEs found previously. With this unified comparative data, we found that the Tc1/mariner superfamily exhibits elevated activity, potentially explaining their pervasive horizontal transfers. Further functional characterization of TEs revealed additional divergence in features such as insertion bias. Remarkably, in CAR-T therapy for hematological and solid tumors, Mariner2_AG (MAG), the most active DNA TE identified, largely outperformed two widely used vectors, the lentiviral vector and the TE-based vector SB100X. Overall, this study highlights the varied transposition features and evolutionary dynamics of DNA TEs and increases the TE toolbox diversity.

LAG-3 and PD-1 synergize on CD8+ T cells to drive T cell exhaustion and hinder autocrine IFN-γ-dependent anti-tumor immunity.

Overcoming immune-mediated resistance to PD-1 blockade remains a major clinical challenge. Enhanced efficacy has been demonstrated in melanoma patients with combined nivolumab (anti-PD-1) and relatlimab (anti-LAG-3) treatment, the first in its class to be FDA approved. However, how these two inhibitory receptors synergize to hinder anti-tumor immunity remains unknown. Here, we show that CD8+ T cells deficient in both PD-1 and LAG-3, in contrast to CD8+ T cells lacking either receptor, mediate enhanced tumor clearance and long-term survival in mouse models of melanoma. PD-1- and LAG-3-deficient CD8+ T cells were transcriptionally distinct, with broad TCR clonality and enrichment of effector-like and interferon-responsive genes, resulting in enhanced IFN-γ release indicative of functionality. LAG-3 and PD-1 combined to drive T cell exhaustion, playing a dominant role in modulating TOX expression. Mechanistically, autocrine, cell-intrinsic IFN-γ signaling was required for PD-1- and LAG-3-deficient CD8+ T cells to enhance anti-tumor immunity, providing insight into how combinatorial targeting of LAG-3 and PD-1 enhances efficacy.

The CD8+ T cell tolerance checkpoint triggers a distinct differentiation state defined by protein translation defects.

Peripheral CD8+ T cell tolerance is a checkpoint in both autoimmune disease and anti-cancer immunity. Despite its importance, the relationship between tolerance-induced states and other CD8+ T cell differentiation states remains unclear. Using flow cytometric phenotyping, single-cell RNA sequencing (scRNA-seq), and chromatin accessibility profiling, we demonstrated that in vivo peripheral tolerance to a self-antigen triggered a fundamentally distinct differentiation state separate from exhaustion, memory, and functional effector cells but analogous to cells defectively primed against tumors. Tolerant cells diverged early and progressively from effector cells, adopting a transcriptionally and epigenetically distinct state within 60 h of antigen encounter. Breaching tolerance required the synergistic actions of strong T cell receptor (TCR) signaling and inflammation, which cooperatively induced gene modules that enhanced protein translation. Weak TCR signaling during bystander infection failed to breach tolerance due to the uncoupling of effector gene expression from protein translation. Thus, tolerance engages a distinct differentiation trajectory enforced by protein translation defects.

The cytokine Meteorin-like inhibits anti-tumor CD8+ T cell responses by disrupting mitochondrial function.

Tumor-infiltrating lymphocyte (TIL) hypofunction contributes to the progression of advanced cancers and is a frequent target of immunotherapy. Emerging evidence indicates that metabolic insufficiency drives T cell hypofunction during tonic stimulation, but the signals that initiate metabolic reprogramming in this context are largely unknown. Here, we found that Meteorin-like (METRNL), a metabolically active cytokine secreted by immune cells in the tumor microenvironment (TME), induced bioenergetic failure of CD8+ T cells. METRNL was secreted by CD8+ T cells during repeated stimulation and acted via both autocrine and paracrine signaling. Mechanistically, METRNL increased E2F-peroxisome proliferator-activated receptor delta (PPARδ) activity, causing mitochondrial depolarization and decreased oxidative phosphorylation, which triggered a compensatory bioenergetic shift to glycolysis. Metrnl ablation or downregulation improved the metabolic fitness of CD8+ T cells and enhanced tumor control in several tumor models, demonstrating the translational potential of targeting the METRNL-E2F-PPARδ pathway to support bioenergetic fitness of CD8+ TILs.

Signaling via a CD27-TRAF2-SHP-1 axis during naive T cell activation promotes memory-associated gene regulatory networks.

The interaction of the tumor necrosis factor receptor (TNFR) family member CD27 on naive CD8+ T (Tn) cells with homotrimeric CD70 on antigen-presenting cells (APCs) is necessary for T cell memory fate determination. Here, we examined CD27 signaling during Tn cell activation and differentiation. In conjunction with T cell receptor (TCR) stimulation, ligation of CD27 by a synthetic trimeric CD70 ligand triggered CD27 internalization and degradation, suggesting active regulation of this signaling axis. Internalized CD27 recruited the signaling adaptor TRAF2 and the phosphatase SHP-1, thereby modulating TCR and CD28 signals. CD27-mediated modulation of TCR signals promoted transcription factor circuits that induced memory rather than effector associated gene programs, which are induced by CD28 costimulation. CD27-costimulated chimeric antigen receptor (CAR)-engineered T cells exhibited improved tumor control compared with CD28-costimulated CAR-T cells. Thus, CD27 signaling during Tn cell activation promotes memory properties with relevance to T cell immunotherapy.

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.

Bile acids modified by the intestinal microbiota promote colorectal cancer growth by suppressing CD8+ T cell effector functions.

Concentrations of the secondary bile acid, deoxycholic acid (DCA), are aberrantly elevated in colorectal cancer (CRC) patients, but the consequences remain poorly understood. Here, we screened a library of gut microbiota-derived metabolites and identified DCA as a negative regulator for CD8+ T cell effector function. Mechanistically, DCA suppressed CD8+ T cell responses by targeting plasma membrane Ca2+ ATPase (PMCA) to inhibit Ca2+-nuclear factor of activated T cells (NFAT)2 signaling. In CRC patients, CD8+ T cell effector function negatively correlated with both DCA concentration and expression of a bacterial DCA biosynthetic gene. Bacteria harboring DCA biosynthetic genes suppressed CD8+ T cells effector function and promoted tumor growth in mice. This effect was abolished by disrupting bile acid metabolism via bile acid chelation, genetic ablation of bacterial DCA biosynthetic pathway, or specific bacteriophage. Our study demonstrated causation between microbial DCA metabolism and anti-tumor CD8+ T cell response in CRC, suggesting potential directions for anti-tumor therapy.

The tissue-resident regulatory T cell pool is shaped by transient multi-tissue migration and a conserved residency program.

The tissues are the site of many important immunological reactions, yet how the immune system is controlled at these sites remains opaque. Recent studies have identified Foxp3+ regulatory T (Treg) cells in non-lymphoid tissues with unique characteristics compared with lymphoid Treg cells. However, tissue Treg cells have not been considered holistically across tissues. Here, we performed a systematic analysis of the Treg cell population residing in non-lymphoid organs throughout the body, revealing shared phenotypes, transient residency, and common molecular dependencies. Tissue Treg cells from different non-lymphoid organs shared T cell receptor (TCR) sequences, with functional capacity to drive multi-tissue Treg cell entry and were tissue-agnostic on tissue homing. Together, these results demonstrate that the tissue-resident Treg cell pool in most non-lymphoid organs, other than the gut, is largely constituted by broadly self-reactive Treg cells, characterized by transient multi-tissue migration. This work suggests common regulatory mechanisms may allow pan-tissue Treg cells to safeguard homeostasis across the body.

The amalgam of naive CD4+ T cell transcriptional states is reconfigured by helminth infection to dampen the amplitude of the immune response.

Naive CD4+ T cells in specific pathogen-free (SPF) mice are characterized by transcriptional heterogeneity and subpopulations distinguished by the expression of quiescence, the extracellular matrix (ECM) and cytoskeleton, type I interferon (IFN-I) response, memory-like, and T cell receptor (TCR) activation genes. We demonstrate that this constitutive heterogeneity, including the presence of the IFN-I response cluster, is commensal independent insofar as being identical in germ-free and SPF mice. By contrast, Nippostrongylus brasiliensis infection altered this constitutive heterogeneity. Naive T cell-intrinsic transcriptional changes acquired during helminth infection correlated with and accounted for decreased immunization response to an unrelated antigen. These compositional and functional changes were dependent variables of helminth infection, as they disappeared at the established time point of its clearance in mice. Collectively, our results indicate that the naive T cell pool is subject to dynamic transcriptional changes in response to certain environmental cues, which in turn permutes the magnitude of the immune response.