PSGL-1 attenuates early TCR signaling to suppress CD8+ T cell progenitor differentiation and elicit terminal CD8+ T cell exhaustion.

PSGL-1 (P-selectin glycoprotein-1) is a T cell-intrinsic checkpoint regulator of exhaustion with an unknown mechanism of action. Here, we show that PSGL-1 acts upstream of PD-1 and requires co-ligation with the T cell receptor (TCR) to attenuate activation of mouse and human CD8+ T cells and drive terminal T cell exhaustion. PSGL-1 directly restrains TCR signaling via Zap70 and maintains expression of the Zap70 inhibitor Sts-1. PSGL-1 deficiency empowers CD8+ T cells to respond to low-affinity TCR ligands and inhibit growth of PD-1-blockade-resistant melanoma by enabling tumor-infiltrating T cells to sustain an elevated metabolic gene signature supportive of increased glycolysis and glucose uptake to promote effector function. This outcome is coupled to an increased abundance of CD8+ T cell stem cell-like progenitors that maintain effector functions. Additionally, pharmacologic blockade of PSGL-1 curtails T cell exhaustion, indicating that PSGL-1 represents an immunotherapeutic target for PD-1-blockade-resistant tumors.

Ubiquitin Ligases Siah1a/2 Control Alveolar Macrophage Functions to Limit Carcinogen-Induced Lung Adenocarcinoma.

Cellular components of the tumor microenvironment, including myeloid cells, play important roles in the progression of lung adenocarcinoma (LUAD) and its response to therapy. Here, we characterize the function of the ubiquitin ligases Siah1a/2 in regulating the differentiation and activity of alveolar macrophages (AM) and assess the implication of Siah1a/2 control of AMs for carcinogen-induced LUAD. Macrophage-specific genetic ablation of Siah1a/2 promoted accumulation of AMs with an immature phenotype and increased expression of protumorigenic and pro-inflammatory Stat3 and ß-catenin gene signatures. Administration of urethane to wild-type mice promoted enrichment of immature-like AMs and lung tumor development, which was enhanced by macrophage-specific Siah1a/2 ablation. The profibrotic gene signature seen in Siah1a/2-ablated immature-like macrophages was associated with increased tumor infiltration of CD14+ myeloid cells and poorer survival of patients with LUAD. Single-cell RNA-seq confirmed the presence of a cluster of immature-like AMs expressing a profibrotic signature in lungs of patients with LUAD, a signature enhanced in smokers. These findings identify Siah1a/2 in AMs as gatekeepers of lung cancer development. SIGNIFICANCE: The ubiquitin ligases Siah1a/2 control proinflammatory signaling, differentiation, and profibrotic phenotypes of alveolar macrophages to suppress lung carcinogenesis.

Induction of the activating transcription factor-4 in the intratumoral CD8+ T cells sustains their viability and anti-tumor activities.

Immune suppressive factors of the tumor microenvironment (TME) undermine viability and exhaust the activities of the intratumoral cytotoxic CD8 + T lymphocytes (CTL) thereby evading anti-tumor immunity and decreasing the benefits of immune therapies. To counteract this suppression and improve the efficacy of therapeutic regimens, it is important to identify and understand the critical regulators within CD8 + T cells that respond to TME stress and tumor-derived factors. Here we investigated the regulation and importance of activating transcription factor-4 (ATF4) in CTL using a novel Atf4ΔCD8 mouse model lacking ATF4 specifically in CD8 + cells. Induction of ATF4 in CD8 + T cells occurred in response to antigenic stimulation and was further increased by exposure to tumor-derived factors and TME conditions. Under these conditions, ATF4 played a critical role in the maintenance of survival and activities of CD8 + T cells. Conversely, selective ablation of ATF4 in CD8 + T cells in mice rendered these Atf4ΔCD8 hosts prone to accelerated growth of implanted tumors. Intratumoral ATF4-deficient CD8 + T cells were under-represented compared to wild-type counterparts and exhibited impaired activation and increased apoptosis. These findings identify ATF4 as an important regulator of viability and activity of CD8 + T cells in the TME and argue for caution in using agents that could undermine these functions of ATF4 for anti-cancer therapies.

PSGL-1 Is a T Cell Intrinsic Inhibitor That Regulates Effector and Memory Differentiation and Responses During Viral Infection.

Effective T cell differentiation during acute virus infections leads to the generation of effector T cells that mediate viral clearance, as well as memory T cells that confer protection against subsequent reinfection. While inhibitory immune checkpoints have been shown to promote T cell dysfunction during chronic virus infections and in tumors, their roles in fine tuning the differentiation and responses of effector and memory T cells are only just beginning to be appreciated. We previously identified PSGL-1 as a fundamental regulator of T cell exhaustion that sustains expression of several inhibitory receptors, including PD-1. We now show that PSGL-1 can restrict the magnitude of effector T cell responses and memory T cell development to acute LCMV virus infection by limiting survival, sustaining PD-1 expression, and reducing effector responses. After infection, PSGL-1-deficient effector T cells accumulated to a greater extent than wild type T cells, and preferentially generated memory precursor cells that displayed enhanced accumulation and functional capacity in response to TCR stimulation as persisting memory cells. Although, PSGL-1-deficient memory cells did not exhibit inherent greater sensitivity to cell death, they failed to respond to a homologous virus challenge after adoptive transfer into naïve hosts indicating an impaired capacity to generate memory effector T cell responses in the context of viral infection. These studies underscore the function of PSGL-1 as a key negative regulator of effector and memory T cell differentiation and suggest that PSGL-1 may limit excessive stimulation of memory T cells during acute viral infection.

FBXO44 promotes DNA replication-coupled repetitive element silencing in cancer cells.

Repetitive elements (REs) compose ∼50% of the human genome and are normally transcriptionally silenced, although the mechanism has remained elusive. Through an RNAi screen, we identified FBXO44 as an essential repressor of REs in cancer cells. FBXO44 bound H3K9me3-modified nucleosomes at the replication fork and recruited SUV39H1, CRL4, and Mi-2/NuRD to transcriptionally silence REs post-DNA replication. FBXO44/SUV39H1 inhibition reactivated REs, leading to DNA replication stress and stimulation of MAVS/STING antiviral pathways and interferon (IFN) signaling in cancer cells to promote decreased tumorigenicity, increased immunogenicity, and enhanced immunotherapy response. FBXO44 expression inversely correlated with replication stress, antiviral pathways, IFN signaling, and cytotoxic T cell infiltration in human cancers, while a FBXO44-immune gene signature correlated with improved immunotherapy response in cancer patients. FBXO44/SUV39H1 were dispensable in normal cells. Collectively, FBXO44/SUV39H1 are crucial repressors of RE transcription, and their inhibition selectively induces DNA replication stress and viral mimicry in cancer cells.

Tumor necrosis factor overcomes immune evasion in p53-mutant medulloblastoma.

Many immunotherapies act by enhancing the ability of cytotoxic T cells to kill tumor cells. Killing depends on T cell recognition of antigens presented by class I major histocompatibility complex (MHC-I) proteins on tumor cells. In this study, we showed that medulloblastomas lacking the p53 tumor suppressor do not express surface MHC-I and are therefore resistant to immune rejection. Mechanistically, this is because p53 regulates expression of the peptide transporter Tap1 and the aminopeptidase Erap1, which are required for MHC-I trafficking to the cell surface. In vitro, tumor necrosis factor (TNF) or lymphotoxin-ß receptor agonist can rescue expression of Erap1, Tap1 and MHC-I on p53-mutant tumor cells. In vivo, low doses of TNF prolong survival and synergize with immune checkpoint inhibitors to promote tumor rejection. These studies identified p53 as a key regulator of immune evasion and suggest that TNF could be used to enhance sensitivity of tumors to immunotherapy.

Prebiotic-Induced Anti-tumor Immunity Attenuates Tumor Growth.

Growing evidence supports the importance of gut microbiota in the control of tumor growth and response to therapy. Here, we select prebiotics that can enrich bacterial taxa that promote anti-tumor immunity. Addition of the prebiotics inulin or mucin to the diet of C57BL/6 mice induces anti-tumor immune responses and inhibition of BRAF mutant melanoma growth in a subcutaneously implanted syngeneic mouse model. Mucin fails to inhibit tumor growth in germ-free mice, indicating that the gut microbiota is required for the activation of the anti-tumor immune response. Inulin and mucin drive distinct changes in the microbiota, as inulin, but not mucin, limits tumor growth in syngeneic mouse models of colon cancer and NRAS mutant melanoma and enhances the efficacy of a MEK inhibitor against melanoma while delaying the emergence of drug resistance. We highlight the importance of gut microbiota in anti-tumor immunity and the potential therapeutic role for prebiotics in this process.

Siah2 control of T-regulatory cells limits anti-tumor immunity.

Understanding the mechanisms underlying anti-tumor immunity is pivotal for improving immune-based cancer therapies. Here, we report that growth of BRAF-mutant melanoma cells is inhibited, up to complete rejection, in Siah2-/- mice. Growth-inhibited tumors exhibit increased numbers of intra-tumoral activated T cells and decreased expression of Ccl17, Ccl22, and Foxp3. Marked reduction in Treg proliferation and tumor infiltration coincide with G1 arrest in tumor infiltrated Siah2-/- Tregs in vivo or following T cell stimulation in culture, attributed to elevated expression of the cyclin-dependent kinase inhibitor p27, a Siah2 substrate. Growth of anti-PD-1 therapy resistant melanoma is effectively inhibited in Siah2-/- mice subjected to PD-1 blockade, indicating synergy between PD-1 blockade and Siah2 loss. Low SIAH2 and FOXP3 expression is identified in immune responsive human melanoma tumors. Overall, Siah2 regulation of Treg recruitment and cell cycle progression effectively controls melanoma development and Siah2 loss in the host sensitizes melanoma to anti-PD-1 therapy.

The gut microbiome: an unexpected player in cancer immunity.

Numerous independent studies link gut microbiota composition and disease and imply a causal role of select commensal microbes in disease etiology. In the gut, commensal microbiota or pathobionts secrete metabolites that underlie pathological conditions, often impacting proximal tissues and gaining access to the bloodstream. Here we focus on extrinsic and intrinsic factors affecting composition of gut microbiota and their impact on the immune system, as key drivers of anti-tumor immunity. In discussing exciting advances relevant to microbiome-tumor interaction, we note existing knowledge gaps that need to be filled to advance basic and clinical research initiatives.

Striking a Balance-Cellular and Molecular Drivers of Memory T Cell Development and Responses to Chronic Stimulation.

Effective adaptive immune responses are characterized by stages of development and maturation of T and B cell populations that respond to disturbances in the host homeostasis in cases of both infections and cancer. For the T cell compartment, this begins with recognition of specific peptides by naïve, antigen-inexperienced T cells that results in their activation, proliferation, and differentiation, which generates an effector population that clears the antigen. Loss of stimulation eventually returns the host to a homeostatic state, with a heterogeneous memory T cell population that persists in the absence of antigen and is primed for rapid responses to a repeat antigen exposure. However, in chronic infections and cancers, continued antigen persistence impedes a successful adaptive immune response and the formation of a stereotypical memory population of T cells is compromised. With repeated antigen stimulation, responding T cells proceed down an altered path of differentiation that allows for antigen persistence, but much less is known regarding the heterogeneity of these cells and the extent to which they can become "memory-like," with a capacity for self-renewal and recall responses that are characteristic of bona fide memory cells. This review focuses on the differentiation of CD4+ and CD8+ T cells in the context of chronic antigen stimulation, highlighting the central observations in both human and mouse studies regarding the differentiation of memory or "memory-like" T cells. The importance of both the cellular and molecular drivers of memory T cell development are emphasized to better understand the consequences of persisting antigen on T cell fates. Integrating what is known and is common across model systems and patients can instruct future studies aimed at further understanding T cell differentiation and development, with the goal of developing novel methods to direct T cells toward the generation of effective memory populations.

Gut microbiota dependent anti-tumor immunity restricts melanoma growth in Rnf5-/- mice.

Accumulating evidence points to an important role for the gut microbiome in anti-tumor immunity. Here, we show that altered intestinal microbiota contributes to anti-tumor immunity, limiting tumor expansion. Mice lacking the ubiquitin ligase RNF5 exhibit attenuated activation of the unfolded protein response (UPR) components, which coincides with increased expression of inflammasome components, recruitment and activation of dendritic cells and reduced expression of antimicrobial peptides in intestinal epithelial cells. Reduced UPR expression is also seen in murine and human melanoma tumor specimens that responded to immune checkpoint therapy. Co-housing of Rnf5-/- and WT mice abolishes the anti-tumor immunity and tumor inhibition phenotype, whereas transfer of 11 bacterial strains, including B. rodentium, enriched in Rnf5-/- mice, establishes anti-tumor immunity and restricts melanoma growth in germ-free WT mice. Altered UPR signaling, exemplified in Rnf5-/- mice, coincides with altered gut microbiota composition and anti-tumor immunity to control melanoma growth.

Microenvironment-Dependent Gradient of CTL Exhaustion in the AE17sOVA Murine Mesothelioma Tumor Model.

The immune system, and in particular, cytotoxic CD8+ T cells (CTLs), plays a vital part in the prevention and elimination of tumors. In many patients, however, CTL-mediated tumor killing ultimately fails in the clearance of cancer cells resulting in disease progression, in large part due to the progression of effector CTL into exhausted CTL. While there have been major breakthroughs in the development of CTL-mediated "reinvigoration"-driven immunotherapies such as checkpoint blockade therapy, there remains a need to better understand the drivers behind the development of T cell exhaustion. Our study highlights the unique differences in T cell exhaustion development in tumor-specific CTL which arises over time in a mouse model of mesothelioma. Importantly, we also show that peripheral tumor-specific T cells have a unique expression profile compared to exhausted tumor-infiltrating CTL at a late-stage of tumor progression in mice. Together, these data suggest that greater emphasis should be placed on understanding contributions of individual microenvironments in the development of T cell exhaustion.

Regulation of S100A8 Stability by RNF5 in Intestinal Epithelial Cells Determines Intestinal Inflammation and Severity of Colitis.

Inflammatory bowel disease (IBD) is prevalent, but the mechanisms underlying disease development remain elusive. We identify a role for the E3 ubiquitin ligase RNF5 in IBD. Intestinal epithelial cells (IECs) express a high level of RNF5, while the colon of Rnf5-/- mice exhibits activated dendritic cells and intrinsic inflammation. Rnf5-/- mice exhibit severe acute colitis following dextran sodium sulfate (DSS) treatment. S100A8 is identified as an RNF5 substrate, resulting in S100A8 ubiquitination and proteasomal-dependent degradation that is attenuated upon inflammatory stimuli. Loss of RNF5 from IECs leads to enhanced S100A8 secretion, which induces mucosal CD4+ T cells, resulting in Th1 pro-inflammatory responses. Administration of S100A8-neutralizing antibodies to DSS-treated Rnf5-/- mice attenuates acute colitis development and increases survival. An inverse correlation between RNF5 and S100A8 protein expression in IECs of IBD patients coincides with disease severity. Collectively, RNF5-mediated regulation of S100A8 stability in IECs is required for the maintenance of intestinal homeostasis.

Fucosyltransferase Induction during Influenza Virus Infection Is Required for the Generation of Functional Memory CD4+ T Cells.

T cells mediating influenza viral control are instructed in lymphoid and nonlymphoid tissues to differentiate into memory T cells that confer protective immunity. The mechanisms by which influenza virus-specific memory CD4+ T cells arise have been attributed to changes in transcription factors, cytokines and cytokine receptors, and metabolic programming. The molecules involved in these biosynthetic pathways, including proteins and lipids, are modified to varying degrees of glycosylation, fucosylation, sialation, and sulfation, which can alter their function. It is currently unknown how the glycome enzymatic machinery regulates CD4+ T cell effector and memory differentiation. In a murine model of influenza virus infection, we found that fucosyltransferase enzymatic activity was induced in effector and memory CD4+ T cells. Using CD4+ T cells deficient in the Fut4/7 enzymes that are expressed only in hematopoietic cells, we found decreased frequencies of effector cells with reduced expression of T-bet and NKG2A/C/E in the lungs during primary infection. Furthermore, Fut4/7-/- effector CD4+ T cells had reduced survival with no difference in proliferation or capacity for effector function. Although Fut4/7-/- CD4+ T cells seeded the memory pool after primary infection, they failed to form tissue-resident cells, were dysfunctional, and were unable to re-expand after secondary infection. Our findings highlight an important regulatory axis mediated by cell-intrinsic fucosyltransferase activity in CD4+ T cell effectors that ensure the development of functional memory CD4+ T cells.

PSGL-1 Is an Immune Checkpoint Regulator that Promotes T Cell Exhaustion.

Chronic viruses and cancers thwart immune responses in humans by inducing T cell dysfunction. Using a murine chronic virus that models human infections, we investigated the function of the adhesion molecule, P-selectin glycoprotein ligand-1 (PSGL-1), that is upregulated on responding T cells. PSGL-1-deficient mice cleared the virus due to increased intrinsic survival of multifunctional effector T cells that had downregulated PD-1 as well as other inhibitory receptors. Notably, this response resulted in CD4(+)-T-cell-dependent immunopathology. Mechanistically, PSGL-1 ligation on exhausted CD8(+) T cells inhibited T cell receptor (TCR) and interleukin-2 (IL-2) signaling and upregulated PD-1, leading to diminished survival with TCR stimulation. In models of melanoma cancer in which T cell dysfunction occurs, PSGL-1 deficiency led to PD-1 downregulation, improved T cell responses, and tumor control. Thus, PSGL-1 plays a fundamental role in balancing viral control and immunopathology and also functions to regulate T cell responses in the tumor microenvironment.

Islet antigen-specific Th17 cells can induce TNF-α-dependent autoimmune diabetes.

Type 1 diabetes (T1D) results from autoimmune destruction of pancreatic ß-cells. Although Th1 cells are key orchestrators of T1D, the function(s) of the more recently identified Th17 subset are unclear due to inherent plasticity. In this study, we analyzed Th17 cells for stability and diabetogenicity in NOD mice. We found that like Th1 cells, Th17 are a distinct population throughout the prediabetic phase. At diabetes onset, there were marked increases in IL-17-producing Th17 cells and IFN-γ-producing Th1 cells in the pancreas as well as in the serum levels of these cytokines, indicating that these proinflammatory mediators serve as biomarkers of advanced autoimmunity. Although naturally occurring Th17 cells in diabetic mice did not contribute to diabetes development in transfer models, islet-specific Th17 cells were diabetogenic independently of IL-17 and displayed inflammation-induced Th17-to-Th1 reprogramming that could be elicited by Th1 cells. However, an inability to generate Th1 cells because of Stat4, Ifngr, and Ifng deficiencies did not prevent diabetes. Instead, TNF-α could mediate diabetes in response to either Th17 cells or Th1 cells. The results identify a previously unknown mechanism by which Th17 cells can contribute to T1D. Our studies also suggest that when developing interventions for T1D, it will be potentially advantageous to focus on mechanisms common to effector T cells rather than on the signature cytokines of various subsets.

Memory CD4+ T-cell-mediated protection depends on secondary effectors that are distinct from and superior to primary effectors.

Whether differences between naive cell-derived primary (1°) and memory cell-derived secondary (2°) CD4(+) T-cell effectors contribute to protective recall responses is unclear. Here, we compare these effectors directly after influenza A virus infection. Both develop with similar kinetics, but 2° effectors accumulate in greater number in the infected lung and are the critical component of memory CD4(+) T-cell-mediated protection against influenza A virus, independent of earlier-acting memory-cell helper functions. Phenotypic, functional, and transcriptome analyses indicate that 2° effectors share organ-specific expression patterns with 1° effectors but are more multifunctional, with more multicytokine (IFN-γ(+)/IL-2(+)/TNF(+))-producing cells and contain follicular helper T-cell populations not only in the spleen and draining lymph nodes but also in the lung. In addition, they express more CD127 and NKG2A but less ICOS and Lag-3 than 1° effectors and express higher levels of several genes associated with survival and migration. Targeting two differentially expressed molecules, NKG2A and Lag-3, reveals differential regulation of 1° and 2° effector functions during pathogen challenge.

Matrix metalloprotease 9 mediates neutrophil migration into the airways in response to influenza virus-induced toll-like receptor signaling.

The early inflammatory response to influenza virus infection contributes to severe lung disease and continues to pose a serious threat to human health. The mechanisms by which neutrophils gain entry to the respiratory tract and their role during pathogenesis remain unclear. Here, we report that neutrophils significantly contributed to morbidity in a pathological mouse model of influenza virus infection. Using extensive immunohistochemistry, bone marrow transfers, and depletion studies, we identified neutrophils as the predominant pulmonary cellular source of the gelatinase matrix metalloprotease (MMP) 9, which is capable of digesting the extracellular matrix. Furthermore, infection of MMP9-deficient mice showed that MMP9 was functionally required for neutrophil migration and control of viral replication in the respiratory tract. Although MMP9 release was toll-like receptor (TLR) signaling-dependent, MyD88-mediated signals in non-hematopoietic cells, rather than neutrophil TLRs themselves, were important for neutrophil migration. These results were extended using multiplex analyses of inflammatory mediators to show that neutrophil chemotactic factor, CCL3, and TNFα were reduced in the Myd88⁻/⁻ airways. Furthermore, TNFα induced MMP9 secretion by neutrophils and blocking TNFα in vivo reduced neutrophil recruitment after infection. Innate recognition of influenza virus therefore provides the mechanisms to induce recruitment of neutrophils through chemokines and to enable their motility within the tissue via MMP9-mediated cleavage of the basement membrane. Our results demonstrate a previously unknown contribution of MMP9 to influenza virus pathogenesis by mediating excessive neutrophil migration into the respiratory tract in response to viral replication that could be exploited for therapeutic purposes.