Autoregulated splicing of TRA2ß programs T cell fate in response to antigen-receptor stimulation.

T cell receptor (TCR) sensitivity to peptide-major histocompatibility complex (MHC) dictates T cell fate. Canonical models of TCR sensitivity cannot be fully explained by transcriptional regulation. In this work, we identify a posttranscriptional regulatory mechanism of TCR sensitivity that guides alternative splicing of TCR signaling transcripts through an evolutionarily ultraconserved poison exon (PE) in the RNA-binding protein (RBP) TRA2ß in mouse and human. TRA2ß-PE splicing, seen during cancer and infection, was required for TCR-induced effector T cell expansion and function. Tra2ß-PE skipping enhanced T cell response to antigen by increasing TCR sensitivity. As antigen levels decreased, Tra2ß-PE reinclusion allowed T cell survival. Finally, we found that TRA2ß-PE was first included in the genome of jawed vertebrates that were capable of TCR gene rearrangements. We propose that TRA2ß-PE splicing acts as a gatekeeper of TCR sensitivity to shape T cell fate.

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.

GRK2 enforces androgen receptor dependence in the prostate and prostate tumors.

Metastatic tumors that have become resistant to androgen deprivation therapy represent the major challenge in treating prostate cancer. Although these recurrent tumors typically remain dependent on the androgen receptor (AR), non-AR-driven tumors that also emerge are particularly deadly and becoming more prevalent. Here, we present a new genetically engineered mouse model for non-AR-driven prostate cancer that centers on a negative regulator of G protein-coupled receptors that is downregulated in aggressive human prostate tumors. Thus, prostate-specific expression of a dominant-negative G protein-coupled receptor kinase 2 (GRK2-DN) transgene diminishes AR and AR target gene expression in the prostate, and confers resistance to castration-induced involution. Further, the GRK2-DN transgene dramatically accelerates oncogene-initiated prostate tumorigenesis by increasing primary tumor size, potentiating visceral organ metastasis, suppressing AR, and inducing neuroendocrine marker mRNAs. In summary, GRK2 enforces AR-dependence in the prostate, and the loss of GRK2 function in prostate tumors accelerates disease progression toward the deadliest stage.

IL-17 inhibits CXCL9/10-mediated recruitment of CD8+ cytotoxic T cells and regulatory T cells to colorectal tumors.

BACKGROUND: The IL-17 family cytokines are potent drivers of colorectal cancer (CRC) development. We and others have shown that IL-17 mainly signals to tumor cells to promote CRC, but the underlying mechanism remains unclear. IL-17 also dampens Th1-armed anti-tumor immunity, in part by attracting myeloid cells to tumor. Whether IL-17 controls the activity of adaptive immune cells in a more direct manner, however, is unknown. METHODS: Using mouse models of sporadic or inducible colorectal cancers, we ablated IL-17RA in the whole body or specifically in colorectal tumor cells. We also performed adoptive bone marrow reconstitution to knockout CXCR3 in hematopoietic cells. Histological and immunological experimental methods were used to reveal the link among IL-17, chemokine production, and CRC development. RESULTS: Loss of IL-17 signaling in mouse CRC resulted in marked increase in the recruitment of CD8+ cytotoxic T lymphocytes (CTLs) and regulatory T cells (Tregs), starting from early stage CRC lesions. This is accompanied by the increased expression of anti-inflammatory cytokines IL-10 and TGF-ß. IL-17 signaling also inhibits the production of T cell attracting chemokines CXCL9 and CXCL10 by tumor cells. Conversely, the inability of hematopoietic cells to respond to CXCL9/10 resulted in decreased tumor infiltration by CTLs and Tregs, decreased levels of IL-10 and TGF-ß, and increased numbers of tumor lesions. Blockade of IL-17 signaling resulted in increased expression of immune checkpoint markers. On the other hand, treatment of mouse CRC with anti-CTLA-4 antibody led to increased expression of pro-tumor IL-17. CONCLUSION: IL-17 signals to colorectal tumor cells and inhibits their production of CXCL9/10 chemokines. By doing so, IL-17 inhibits the infiltration of CD8+ CTLs and Tregs to CRC, thus promoting CRC development. Cancer immunotherapy may be benefited by the use of anti-IL-17 agents as adjuvant therapies, which serve to block both IL-17-mediated tumor promotion and T cell exclusion.

A mathematical model of combined CD8 T cell costimulation by 4-1BB (CD137) and OX40 (CD134) receptors.

Combined agonist stimulation of the TNFR costimulatory receptors 4-1BB (CD137) and OX40(CD134) has been shown to generate supereffector CD8 T cells that clonally expand to greater levels, survive longer, and produce a greater quantity of cytokines compared to T cells stimulated with an agonist of either costimulatory receptor individually. In order to understand the mechanisms for this effect, we have created a mathematical model for the activation of the CD8 T cell intracellular signaling network by mono- or dual-costimulation. We show that supereffector status is generated via downstream interacting pathways that are activated upon engagement of both receptors, and in silico simulations of the model are supported by published experimental results. The model can thus be used to identify critical molecular targets of T cell dual-costimulation in the context of cancer immunotherapy.

Costimulation Induces CD4 T Cell Antitumor Immunity via an Innate-like Mechanism.

Chronic exposure to tumor-associated antigens inactivates cognate T cells, restricting the repertoire of tumor-specific effector T cells. This problem was studied here by transferring TCR transgenic CD4 T cells into recipient mice that constitutively express a cognate self-antigen linked to MHC II on CD11c-bearing cells. Immunotherapeutic agonists to CD134 plus CD137, "dual costimulation," induces specific CD4 T cell expansion and expression of the receptor for the Th2-associated IL-1 family cytokine IL-33. Rather than producing IL-4, however, they express the tumoricidal Th1 cytokine IFNγ when stimulated with IL-33 or IL-36 (a related IL-1 family member) plus IL-12 or IL-2. IL-36, which is induced within B16-F10 melanomas by dual costimulation, reduces tumor growth when injected intratumorally as a monotherapy and boosts the efficacy of tumor-nonspecific dual costimulated CD4 T cells. Dual costimulation thus enables chronic antigen-exposed CD4 T cells, regardless of tumor specificity, to elaborate tumoricidal function in response to tumor-associated cytokines.

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.

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.

Macrophage polarization and meta-inflammation.

Chronic overnutrition and obesity induces low-grade inflammation throughout the body. Termed "meta-inflammation," this chronic state of inflammation is mediated by macrophages located within the colon, liver, muscle, and adipose tissue. A sentinel orchestrator of immune activity and homeostasis, macrophages adopt variable states of activation as a function of time and environmental cues. Meta-inflammation phenotypically skews these polarization states and has been linked to numerous metabolic disorders. The past decade has revealed several key regulators of macrophage polarization, including the signal transducer and activator of transcription family, the peroxisome proliferator-activated receptor gamma, the CCAAT-enhancer-binding proteins (C/EBP) family, and the interferon regulatory factors. Recent studies have also suggested that microRNAs and long noncoding RNA influence macrophage polarization. The pathogenic alteration of macrophage polarization in meta-inflammation is regulated by both extracellular and intracellular cues, resulting in distinct secretome profiles. Meta-inflammation-altered macrophage polarization has been linked to insulin insensitivity, atherosclerosis, inflammatory bowel disease, cancer, and autoimmunity. Thus, further mechanistic exploration into the skewing of macrophage polarization promises to have profound impacts on improving global health.

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.

Addressing current challenges in cancer immunotherapy with mathematical and computational modelling.

The goal of cancer immunotherapy is to boost a patient's immune response to a tumour. Yet, the design of an effective immunotherapy is complicated by various factors, including a potentially immunosuppressive tumour microenvironment, immune-modulating effects of conventional treatments and therapy-related toxicities. These complexities can be incorporated into mathematical and computational models of cancer immunotherapy that can then be used to aid in rational therapy design. In this review, we survey modelling approaches under the umbrella of the major challenges facing immunotherapy development, which encompass tumour classification, optimal treatment scheduling and combination therapy design. Although overlapping, each challenge has presented unique opportunities for modellers to make contributions using analytical and numerical analysis of model outcomes, as well as optimization algorithms. We discuss several examples of models that have grown in complexity as more biological information has become available, showcasing how model development is a dynamic process interlinked with the rapid advances in tumour-immune biology. We conclude the review with recommendations for modellers both with respect to methodology and biological direction that might help keep modellers at the forefront of cancer immunotherapy development.

Cytokines and metabolic factors regulate tumoricidal T-cell function during cancer immunotherapy.

Recent advances in cancer biology and genetics have fostered precision therapies targeting tumor-specific attributes. Immune-based therapies that elicit cytolytic T cells (CTL) specific for tumor antigens can provide therapeutic benefit to cancer patients, however, cure rates are typically low. This largely results from immunosuppressive mechanisms operating within the tumor microenvironment, many of which inflict metabolic stresses upon CTL. Conversely, immunotherapies can mitigate specific metabolic stressors. For instance, dual costimulation immunotherapy with CD134 (OX40) plus CD137 (4-1BB) agonists appears to mediate tumor control in part by engaging cytokine networks that enable infiltrating CTL to compete for limiting supplies of glucose. Future efforts combining modalities that endow CTL with complimentary metabolic advantages should improve therapeutic efficacies.

Enhancing the safety of antibody-based immunomodulatory cancer therapy without compromising therapeutic benefit: Can we have our cake and eat it too?

INTRODUCTION: Monoclonal antibodies (mAbs) targeting checkpoint inhibitors have demonstrated clinical benefit in treating patients with cancer and have paved the way for additional immune-modulating mAbs such as those targeting costimulatory receptors. The full clinical utility of these agents, however, is hampered by immune-related adverse events (irAEs) that can occur during therapy. AREAS COVERED: We first provide a general overview of tumor immunity, followed by a review of the two major classes of immunomodulatory mAbs being developed as cancer therapeutics: checkpoint inhibitors and costimulatory receptor agonists. We then discuss therapy-associated adverse events. Finally, we describe in detail the mechanisms driving their therapeutic activity, with an emphasis on interactions between antibody fragment crystallizable (Fc) domains and Fc receptors (FcR). EXPERT OPINION: Given that Fc-FcR interactions appear critical in facilitating the ability of immunomodulatory mAbs to elicit both therapeutically useful as well as adverse effects, the engineering of mAbs that can effectively engage their targets while limiting interaction with FcRs might represent a promising future avenue for developing the next generation of immune-enhancing tumoricidal agents with increased safety and retention of efficacy.

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.

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.

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.

Betting on improved cancer immunotherapy by doubling down on CD134 and CD137 co-stimulation.

The ability of T cells to recognize a vast array of antigens enables them to destroy tumor cells while inflicting minimal collateral damage. Nevertheless, tumor antigens often are a form of self-antigen, and thus tumor immunity can be dampened by tolerance mechanisms that evolved to prevent autoimmunity. Since tolerance can be induced by steady-state antigen-presenting cells that provide insufficient co-stimulation, the exogenous administration of co-stimulatory agonists can favor the expansion and tumoricidal functions of tumor-specific T cells. Agonists of the co-stimulatory tumor necrosis factor receptor (TNFR) family members CD134 and CD137 exert antitumor activity in mice, and as monotherapies have exhibited encouraging results in clinical trials. This review focuses on how the dual administration of CD134 and CD137 agonists synergistically boosts T-cell priming and elaborates a multi-pronged antitumor immune response, as well as how such dual co-stimulation might be translated into effective anticancer therapies.

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.

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.