Lymphocyte Activation Gene-3 Regulates Dendritic Cell Metabolic Programing and T Cell Priming Function.

Deficiency of lymphocyte activation gene-3 (LAG3) is significantly associated with increased cardiovascular disease risk with in vitro results demonstrating increased TNF-α and decreased IL-10 secretion from LAG3-deficient human B lymphoblasts. The hypothesis tested in this study was that Lag3 deficiency in dendritic cells (DCs) would significantly affect cytokine expression, alter cellular metabolism, and prime naive T cells to greater effector differentiation. Experimental approaches used included differentiation of murine bone marrow-derived DCs (BMDCs) to measure secreted cytokines, cellular metabolism, RNA sequencing, whole cell proteomics, adoptive OT-II CD4+Lag3 +/+ donor cells into wild-type (WT) C57BL/6 and Lag3 -/- recipient mice, and ex vivo measurements of IFN-γ from cultured splenocytes. Results showed that Lag3 -/- BMDCs secreted more TNF-α, were more glycolytic, used fewer fatty acids for mitochondrial respiration, and glycolysis was significantly reduced by exogenous IL-10 treatment. Under basal conditions, RNA sequencing revealed increased expression of CD40 and CD86 and other cytokine-signaling targets as compared with WT. Whole cell proteomics identified a significant number of proteins up- and downregulated in Lag3 -/- BMDCs, with significant differences noted in exogenous IL-10 responsiveness compared with WT cells. Ex vivo, IFN-γ expression was significantly higher in Lag3 -/- mice as compared with WT. With in vivo adoptive T cell and in vitro BMDC:T coculture experiments, Lag3 -/- BMDCs showed greater T cell effector differentiation and proliferation, respectively, compared with WT BMDCs. In conclusion, Lag3 deficiency in DCs is associated with an inflammatory phenotype that provides a plausible mechanism for increased cardiovascular disease risk in humans with LAG3 deficiency.

Cancer Immunology and Immunotherapy: From Defining Basic Immunology to Leading the Fight Against Cancer.

The past decade has seen the advent and widespread use of several immunotherapeutic modalities that have markedly improved treatment outcomes for patients with various cancers. Nevertheless, the study of cancer immunology traces its roots back to the inception of modern immunology, and played a critical role in the of discovery of central immunological concepts and development of key technologies and methodologies and that have propelled advances in all areas of immunology.

An Immunotherapeutic CD137 Agonist Releases Eomesodermin from ThPOK Repression in CD4 T Cells.

Agonists to the TNF/TNFR costimulatory receptors CD134 (OX40) and CD137 (4-1BB) elicit antitumor immunity. Dual costimulation with anti-CD134 plus anti-CD137 is particularly potent because it programs cytotoxic potential in CD8+ and CD4+ T cells. Cytotoxicity in dual-costimulated CD4 T cells depends on the T-box transcription factor eomesodermin (Eomes), which we report is induced via a mechanism that does not rely on IL-2, in contrast to CD8+ CTL, but rather depends on the CD8 T cell lineage commitment transcription factor Runx3, which supports Eomes expression in mature CD8+ CTLs. Further, Eomes and Runx3 were indispensable for dual-costimulated CD4 T cells to mediate antitumor activity in an aggressive melanoma model. Runx3 is also known to be expressed in standard CD4 Th1 cells where it fosters IFN-γ expression; however, the CD4 T cell lineage commitment factor ThPOK represses transcription of Eomes and other CD8 lineage genes, such as Cd8a Hence, CD4 T cells can differentiate into Eomes+ cytotoxic CD4+CD8+ double-positive T cells by terminating ThPOK expression. In contrast, dual-costimulated CD4 T cells express Eomes, despite the continued expression of ThPOK and the absence of CD8α, indicating that Eomes is selectively released from ThPOK repression. Finally, although Eomes was induced by CD137 agonist, but not CD134 agonist, administered individually, CD137 agonist failed to induce CD134-/- CD4 T cells to express Eomes or Runx3, indicating that both costimulatory pathways are required for cytotoxic Th1 programming, even when only CD137 is intentionally engaged with a therapeutic agonist.

Identification of a Sjögren's syndrome susceptibility locus at OAS1 that influences isoform switching, protein expression, and responsiveness to type I interferons.

Sjögren's syndrome (SS) is a common, autoimmune exocrinopathy distinguished by keratoconjunctivitis sicca and xerostomia. Patients frequently develop serious complications including lymphoma, pulmonary dysfunction, neuropathy, vasculitis, and debilitating fatigue. Dysregulation of type I interferon (IFN) pathway is a prominent feature of SS and is correlated with increased autoantibody titers and disease severity. To identify genetic determinants of IFN pathway dysregulation in SS, we performed cis-expression quantitative trait locus (eQTL) analyses focusing on differentially expressed type I IFN-inducible transcripts identified through a transcriptome profiling study. Multiple cis-eQTLs were associated with transcript levels of 2'-5'-oligoadenylate synthetase 1 (OAS1) peaking at rs10774671 (PeQTL = 6.05 × 10-14). Association of rs10774671 with SS susceptibility was identified and confirmed through meta-analysis of two independent cohorts (Pmeta = 2.59 × 10-9; odds ratio = 0.75; 95% confidence interval = 0.66-0.86). The risk allele of rs10774671 shifts splicing of OAS1 from production of the p46 isoform to multiple alternative transcripts, including p42, p48, and p44. We found that the isoforms were differentially expressed within each genotype in controls and patients with and without autoantibodies. Furthermore, our results showed that the three alternatively spliced isoforms lacked translational response to type I IFN stimulation. The p48 and p44 isoforms also had impaired protein expression governed by the 3' end of the transcripts. The SS risk allele of rs10774671 has been shown by others to be associated with reduced OAS1 enzymatic activity and ability to clear viral infections, as well as reduced responsiveness to IFN treatment. Our results establish OAS1 as a risk locus for SS and support a potential role for defective viral clearance due to altered IFN response as a genetic pathophysiological basis of this complex autoimmune disease.

A novel biologic platform elicits profound T cell costimulatory activity and antitumor immunity in mice.

Combination immunotherapies utilizing complementary modalities that target distinct tumor attributes or immunosuppressive mechanisms, or engage different arms of the antitumor immune response, can elicit greater therapeutic efficacy than the component monotherapies. Increasing the number of agents included in a therapeutic cocktail can further increase efficacy, however, this approach poses numerous challenges for clinical translation. Here, a novel platform to simplify combination immunotherapy by covalently linking immunotherapeutic agonists to the costimulatory receptors CD134 and CD137 into a single heterodimeric drug, "OrthomAb", is shown. This reagent not only retains costimulatory T cell activity, but also elicits unique T cell functions that are not programmed by either individual agonist, and preferentially expands effector T cells over Tregs. Finally, in an aggressive melanoma model OrthomAb elicits better therapeutic efficacy compared to the unlinked agonists. This demonstration that two drugs can be combined into one provides a framework for distilling complex combination drug cocktails into simpler delivery platforms.

Costimulation Endows Immunotherapeutic CD8 T Cells with IL-36 Responsiveness during Aerobic Glycolysis.

CD134- and CD137-primed CD8 T cells mount powerful effector responses upon recall, but even without recall these dual-costimulated T cells respond to signal 3 cytokines such as IL-12. We searched for alternative signal 3 receptor pathways and found the IL-1 family member IL-36R. Although IL-36 alone did not stimulate effector CD8 T cells, in combination with IL-12, or more surprisingly IL-2, it induced striking and rapid TCR-independent IFN-γ synthesis. To understand how signal 3 responses functioned in dual-costimulated T cells we showed that IL-2 induced IL-36R gene expression in a JAK/STAT-dependent manner. These data help delineate a sequential stimulation process where IL-2 conditioning must precede IL-36 for IFN-γ synthesis. Importantly, this responsive state was transient and functioned only in effector T cells capable of aerobic glycolysis. Specifically, as the effector T cells metabolized glucose and consumed O2, they also retained potential to respond through IL-36R. This suggests that T cells use innate receptor pathways such as the IL-36R/axis when programmed for aerobic glycolysis. To explore a function for IL-36R in vivo, we showed that dual costimulation therapy reduced B16 melanoma tumor growth while increasing IL-36R gene expression. In summary, cytokine therapy to eliminate tumors may target effector T cells, even outside of TCR specificity, as long as the effectors are in the correct metabolic state.

Two functional lupus-associated BLK promoter variants control cell-type- and developmental-stage-specific transcription.

Efforts to identify lupus-associated causal variants in the FAM167A/BLK locus on 8p21 are hampered by highly associated noncausal variants. In this report, we used a trans-population mapping and sequencing strategy to identify a common variant (rs922483) in the proximal BLK promoter and a tri-allelic variant (rs1382568) in the upstream alternative BLK promoter as putative causal variants for association with systemic lupus erythematosus. The risk allele (T) at rs922483 reduced proximal promoter activity and modulated alternative promoter usage. Allelic differences at rs1382568 resulted in altered promoter activity in B progenitor cell lines. Thus, our results demonstrated that both lupus-associated functional variants contribute to the autoimmune disease association by modulating transcription of BLK in B cells and thus potentially altering immune responses.

Tumor-Unrelated CD4 T Cell Help Augments CD134 plus CD137 Dual Costimulation Tumor Therapy.

The ability of immune-based cancer therapies to elicit beneficial CD8(+) CTLs is limited by tolerance pathways that inactivate tumor-specific CD4 Th cells. A strategy to bypass this problem is to engage tumor-unrelated CD4 Th cells. Thus, CD4 T cells, regardless of their specificity per se, can boost CD8(+) CTL priming as long as the cognate epitopes are linked via presentation on the same dendritic cell. In this study, we assessed the therapeutic impact of engaging tumor-unrelated CD4 T cells during dual costimulation with CD134 plus CD137 that provide help via the above-mentioned classical linked pathway, as well as provide nonlinked help that facilitates CTL function in T cells not directly responding to cognate Ag. We found that engagement of tumor-unrelated CD4 Th cells dramatically boosted the ability of dual costimulation to control the growth of established B16 melanomas. Surprisingly, this effect depended upon a CD134-dependent component that was extrinsic to the tumor-unrelated CD4 T cells, suggesting that the dual costimulated helper cells are themselves helped by a CD134(+) cell(s). Nevertheless, the delivery of therapeutic help tracked with an increased frequency of tumor-infiltrating granzyme B(+) effector CD8 T cells and a reciprocal decrease in Foxp3(+)CD4(+) cell frequency. Notably, the tumor-unrelated CD4 Th cells also infiltrated the tumors, and their deletion several days following initial T cell priming negated their therapeutic impact. Taken together, dual costimulation programs tumor-unrelated CD4 T cells to deliver therapeutic help during both the priming and effector stages of the antitumor response.

Preferential association of a functional variant in complement receptor 2 with antibodies to double-stranded DNA.

OBJECTIVES: Systemic lupus erythematosus (SLE; OMIM 152700) is characterised by the production of antibodies to nuclear antigens. We previously identified variants in complement receptor 2 (CR2/CD21) that were associated with decreased risk of SLE. This study aimed to identify the causal variant for this association. METHODS: Genotyped and imputed genetic variants spanning CR2 were assessed for association with SLE in 15 750 case-control subjects from four ancestral groups. Allele-specific functional effects of associated variants were determined using quantitative real-time PCR, quantitative flow cytometry, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-PCR. RESULTS: The strongest association signal was detected at rs1876453 in intron 1 of CR2 (pmeta=4.2×10(-4), OR 0.85), specifically when subjects were stratified based on the presence of dsDNA autoantibodies (case-control pmeta=7.6×10(-7), OR 0.71; case-only pmeta=1.9×10(-4), OR 0.75). Although allele-specific effects on B cell CR2 mRNA or protein levels were not identified, levels of complement receptor 1 (CR1/CD35) mRNA and protein were significantly higher on B cells of subjects harbouring the minor allele (p=0.0248 and p=0.0006, respectively). The minor allele altered the formation of several DNA protein complexes by EMSA, including one containing CCCTC-binding factor (CTCF), an effect that was confirmed by ChIP-PCR. CONCLUSIONS: These data suggest that rs1876453 in CR2 has long-range effects on gene regulation that decrease susceptibility to lupus. Since the minor allele at rs1876453 is preferentially associated with reduced risk of the highly specific dsDNA autoantibodies that are present in preclinical, active and severe lupus, understanding its mechanisms will have important therapeutic implications.

Identification of IRF8, TMEM39A, and IKZF3-ZPBP2 as susceptibility loci for systemic lupus erythematosus in a large-scale multiracial replication study.

Systemic lupus erythematosus (SLE) is a chronic heterogeneous autoimmune disorder characterized by the loss of tolerance to self-antigens and dysregulated interferon responses. The etiology of SLE is complex, involving both heritable and environmental factors. Candidate-gene studies and genome-wide association (GWA) scans have been successful in identifying new loci that contribute to disease susceptibility; however, much of the heritable risk has yet to be identified. In this study, we sought to replicate 1,580 variants showing suggestive association with SLE in a previously published GWA scan of European Americans; we tested a multiethnic population consisting of 7,998 SLE cases and 7,492 controls of European, African American, Asian, Hispanic, Gullah, and Amerindian ancestry to find association with the disease. Several genes relevant to immunological pathways showed association with SLE. Three loci exceeded the genome-wide significance threshold: interferon regulatory factor 8 (IRF8; rs11644034; p(meta-Euro) = 2.08 × 10(-10)), transmembrane protein 39A (TMEM39A; rs1132200; p(meta-all) = 8.62 × 10(-9)), and 17q21 (rs1453560; p(meta-all) = 3.48 × 10(-10)) between IKAROS family of zinc finger 3 (AIOLOS; IKZF3) and zona pellucida binding protein 2 (ZPBP2). Fine mapping, resequencing, imputation, and haplotype analysis of IRF8 indicated that three independent effects tagged by rs8046526, rs450443, and rs4843869, respectively, were required for risk in individuals of European ancestry. Eleven additional replicated effects (5 × 10(-8) < p(meta-Euro) < 9.99 × 10(-5)) were observed with CFHR1, CADM2, LOC730109/IL12A, LPP, LOC63920, SLU7, ADAMTSL1, C10orf64, OR8D4, FAM19A2, and STXBP6. The results of this study increase the number of confirmed SLE risk loci and identify others warranting further investigation.

Presensitizing with a Toll-like receptor 3 ligand impairs CD8 T-cell effector differentiation and IL-33 responsiveness.

The synthetic double-stranded RNA poly(I:C) is commonly used as an adjuvant to boost CD8 T-cell function; however, polyinosinic:polycytidylic acid [poly(I:C)] can also suppress autoimmune disease. The mechanism by which a single adjuvant achieves two distinct immunoregulatory roles is unknown. Although it is clear that coadministration of poly(I:C) with antigen elicits strong adjuvant effects in mice, we found that poly(I:C) injection before antigen substantially reduced antigen-dependent CD8 T-cell responses. Notably, CD8 T cells sensitized in poly(I:C)-pretreated mice failed to fully up-regulate IL-33R (ST2), which led to impaired T-cell receptor-independent responses to IL-33. In contrast, nonsensitized effector CD8 T cells responded robustly to IL-33 using a two-signal cytokine mechanism. During an acute lung response to Staphylococcus aureus enterotoxin, peripheral injection of poly(I:C) manifested a suppressive process by inhibiting the differentiation of both antigen- and IL-33-responsive CD8 effectors systemically. These findings highlight that early exposure to double-stranded RNA reverses its role as an adjuvant and, importantly, prevents IL-33R up-regulation on CD8 effector T cells to dampen inflammation.

Lymphatic endothelial cells induce tolerance via PD-L1 and lack of costimulation leading to high-level PD-1 expression on CD8 T cells.

Lymphatic endothelial cells (LECs) induce peripheral tolerance by direct presentation to CD8 T cells (T(CD8)). We demonstrate that LECs mediate deletion only via programmed cell death-1 (PD-1) ligand 1, despite expressing ligands for the CD160, B- and T-lymphocyte attenuator, and lymphocyte activation gene-3 inhibitory pathways. LECs induce activation and proliferation of T(CD8), but lack of costimulation through 4-1BB leads to rapid high-level expression of PD-1, which in turn inhibits up-regulation of the high-affinity IL-2 receptor that is necessary for T(CD8) survival. Rescue of tyrosinase-specific T(CD8) by interference with PD-1 or provision of costimulation results in autoimmune vitiligo, demonstrating that LECs are significant, albeit suboptimal, antigen-presenting cells. Because LECs express numerous peripheral tissue antigens, lack of costimulation coupled to rapid high-level up-regulation of inhibitory receptors may be generally important in systemic peripheral tolerance.

CD134/CD137 dual costimulation-elicited IFN-γ maximizes effector T-cell function but limits Treg expansion.

T-cell tolerance to tumor antigens represents a major hurdle in generating tumor immunity. Combined administration of agonistic monoclonal antibodies (mAbs) to the costimulatory receptors CD134 plus CD137 can program T-cells responding to tolerogenic antigen to undergo expansion, and effector T-cell differentiation, and also elicits tumor immunity. Nevertheless, CD134 and CD137 agonists can also engage inhibitory immune components. To understand how immune stimulatory versus inhibitory components are regulated during CD134 plus CD137 dual costimulation (DCo), the current study utilized a model where DCo programs T-cells encountering a highly tolerogenic self-antigen to undergo effector differentiation. IFN-γ was found to have a pivotal role in maximizing the function of effector T-cells, while simultaneously limiting the expansion of CD4(+)CD25(+)Foxp3(+) Tregs. In antigen-responding effector T-cells, IFN-γ operates via a direct cell-intrinsic mechanism to cooperate with IL-2 to program maximal expression of granzyme B. Simultaneously, IFN-γ limits expression of the IL-2 receptor alpha chain (CD25) and IL-2 signaling through a mechanism that does not involve T-bet-mediated repression of IL-2. IFN-γ also limited CD25 and Foxp3 expression on bystanding CD4(+)Foxp3(+) Tregs, and limited the potential of these Tregs to expand. These effects could not be explained by the ability of IFN-γ to limit IL-2 availability. Taken together, during DCo IFN-γ interacts with IL-2 through distinct mechanisms to program maximal expression of effector molecules in antigen-responding T-cells, while simultaneously limiting Treg expansion.

Association of two independent functional risk haplotypes in TNIP1 with systemic lupus erythematosus.

OBJECTIVE: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibody production and altered type I interferon expression. Genetic surveys and genome-wide association studies have identified >30 SLE susceptibility genes. One of these genes, TNIP1, encodes the ABIN1 protein. ABIN1 functions in the immune system by restricting NF-κB signaling. The present study was undertaken to investigate the genetic factors that influence association with SLE in genes that regulate the NF-κB pathway. METHODS: We analyzed a dense set of genetic markers spanning TNIP1 and TAX1BP1, as well as the TNIP1 homolog TNIP2, in case-control populations of diverse ethnic origins. TNIP1, TNIP2, and TAX1BP1 were fine-mapped in a total of 8,372 SLE cases and 7,492 healthy controls from European-ancestry, African American, Hispanic, East Asian, and African American Gullah populations. Levels of TNIP1 messenger RNA (mRNA) and ABIN1 protein in Epstein-Barr virus-transformed human B cell lines were analyzed by quantitative reverse transcription-polymerase chain reaction and Western blotting, respectively. RESULTS: We found significant associations between SLE and genetic variants within TNIP1, but not in TNIP2 or TAX1BP1. After resequencing and imputation, we identified 2 independent risk haplotypes within TNIP1 in individuals of European ancestry that were also present in African American and Hispanic populations. Levels of TNIP1 mRNA and ABIN1 protein were reduced among subjects with these haplotypes, suggesting that they harbor hypomorphic functional variants that influence susceptibility to SLE by restricting ABIN1 expression. CONCLUSION: Our results confirm the association signals between SLE and TNIP1 variants in multiple populations and provide new insight into the mechanism by which TNIP1 variants may contribute to SLE pathogenesis.

CD134 plus CD137 dual costimulation induces Eomesodermin in CD4 T cells to program cytotoxic Th1 differentiation.

Cytotoxic CD4 Th1 cells are emerging as a therapeutically useful T cell lineage that can effectively target tumors, but until now the pathways that govern their differentiation have been poorly understood. We demonstrate that CD134 (OX40) costimulation programs naive self- and virus-reactive CD4 T cells to undergo in vivo differentiation into cytotoxic Th1 effectors. CD137 (4-1BB) costimulation maximized clonal expansion, and IL-2 was necessary for cytotoxic Th1 differentiation. Importantly, the T-box transcription factor Eomesodermin was critical for inducing the cytotoxic marker granzyme B. CD134 plus CD137 dual costimulation also imprinted a cytotoxic phenotype on bystanding CD4 T cells. Thus, to our knowledge, the current study identifies for the first time a specific costimulatory pathway and an intracellular mechanism relying on Eomesodermin that induces both Ag-specific and bystander cytotoxic CD4 Th1 cells. This mechanism might be therapeutically useful because CD134 plus CD137 dual costimulation induced CD4 T cell-dependent tumoricidal function in a mouse melanoma model.

Androgen ablation mitigates tolerance to a prostate/prostate cancer-restricted antigen.

To understand the T cell response to prostate cancer, we created transgenic mice that express a model antigen in a prostate-restricted pattern and crossed these animals to TRAMP mice that develop spontaneous prostate cancer. Adoptive transfer of prostate-specific CD4 T cells shows that, in the absence of prostate cancer, the prostate gland is mostly ignored. Tumorigenesis allows T cell recognition of the prostate gland--but this recognition is tolerogenic, resulting in abortive proliferation and ultimately in hyporesponsiveness at the systemic level. Androgen ablation (the most common treatment for metastatic prostate cancer) was able to mitigate this tolerance--allowing prostate-specific T cells to expand and develop effector function after vaccination. These results suggest that immunotherapy for prostate cancer may be most efficacious when administered after androgen ablation.

High glucose promotes regulatory T cell differentiation.

The consumption of processed foods and sugary sodas in Western diets correlates with an increased incidence of obesity, metabolic syndromes such as type 2 diabetes, cardiovascular diseases, and autoimmune diseases including inflammatory bowel disease and rheumatoid arthritis. All these diseases have an inflammatory component, of which T lymphocytes can play a critical role in driving. Much has been learned regarding the importance of sugar, particularly glucose, in fueling effector versus regulatory T cells that can promote or dampen inflammation, respectively. In particular, glucose and its metabolic breakdown products via glycolysis are essential for effector T cell differentiation and function, while fatty acid-fueled oxidative phosphorylation supports homeostasis and function of regulatory T cells. Nevertheless, a critical knowledge gap, given the prevalence of diabetes in Western societies, is the impact of elevated glucose concentrations on the balance between effector versus regulatory T cells. To begin addressing this, we cultured naïve CD4+ T cells with different concentrations of glucose, and examined their differentiation into effector versus regulatory lineages. Surprisingly, high glucose promoted regulatory T cell differentiation and inhibited Th1 effector differentiation. This skewing towards the regulatory lineage occurred via an indirect mechanism that depends on lactate produced by activated glycolytic T cells. Addition of lactate to the T cell differentiation process promotes the differentiation of Treg cells, and activates Akt/mTOR signaling cascade. Hence, our findings suggest the existence of a novel feedback mechanism in which lactate produced by activated, differentiating T cells skews their lineage commitment towards the regulatory fate.