Autonomous IL-36R signaling in neutrophils activates potent antitumor effector functions.

While the rapid advancement of immunotherapies has revolutionized cancer treatment, only a small fraction of patients derive clinical benefit. Eradication of large, established tumors appears to depend on engaging and activating both innate and adaptive immune system components to mount a rigorous and comprehensive immune response. Identifying such agents is a high unmet medical need, because they are sparse in the therapeutic landscape of cancer treatment. Here, we report that IL-36 cytokine can engage both innate and adaptive immunity to remodel an immune-suppressive tumor microenvironment (TME) and mediate potent antitumor immune responses via signaling in host hematopoietic cells. Mechanistically, IL-36 signaling modulates neutrophils in a cell-intrinsic manner to greatly enhance not only their ability to directly kill tumor cells but also promote T and NK cell responses. Thus, while poor prognostic outcomes are typically associated with neutrophil enrichment in the TME, our results highlight the pleiotropic effects of IL-36 and its therapeutic potential to modify tumor-infiltrating neutrophils into potent effector cells and engage both the innate and adaptive immune system to achieve durable antitumor responses in solid tumors.

The adaptor protein TRAF3 is an immune checkpoint that inhibits myeloid-derived suppressor cell expansion.

Myeloid-derived suppressor cells (MDSCs) are aberrantly expanded in cancer patients and under other pathological conditions. These cells orchestrate the immunosuppressive and inflammatory network to facilitate cancer metastasis and mediate patient resistance to therapies, and thus are recognized as a prime therapeutic target of human cancers. Here we report the identification of the adaptor protein TRAF3 as a novel immune checkpoint that critically restrains MDSC expansion. We found that myeloid cell-specific Traf3-deficient (M-Traf3 -/-) mice exhibited MDSC hyperexpansion during chronic inflammation. Interestingly, MDSC hyperexpansion in M-Traf3 -/- mice led to accelerated growth and metastasis of transplanted tumors associated with an altered phenotype of T cells and NK cells. Using mixed bone marrow chimeras, we demonstrated that TRAF3 inhibited MDSC expansion via both cell-intrinsic and cell-extrinsic mechanisms. Furthermore, we elucidated a GM-CSF-STAT3-TRAF3-PTP1B signaling axis in MDSCs and a novel TLR4-TRAF3-CCL22-CCR4-G-CSF axis acting in inflammatory macrophages and monocytes that coordinately control MDSC expansion during chronic inflammation. Taken together, our findings provide novel insights into the complex regulatory mechanisms of MDSC expansion and open up unique perspectives for the design of new therapeutic strategies that aim to target MDSCs in cancer patients.

Neoantigen Identification and Response to Adoptive Cell Transfer in Anti-PD-1 Naïve and Experienced Patients with Metastatic Melanoma.

PURPOSE: Immune checkpoint blockade (ICB) agents and adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL) are prominent immunotherapies used for the treatment of advanced melanoma. Both therapies rely on activation of lymphocytes that target shared tumor antigens or neoantigens. Recent analysis of patients with metastatic melanoma who underwent treatment with TIL ACT at the NCI demonstrated decreased responses in patients previously treated with anti-PD-1 agents. We aimed to find a basis for the difference in response rates between anti-PD-1 naïve and experienced patients. PATIENTS AND METHODS: We examined the tumor mutational burden (TMB) of resected tumors and the repertoire of neoantigens targeted by autologous TIL in a cohort of 112 anti-PD-1 naïve and 69 anti-PD-1 experienced patients. RESULTS: Anti-PD-1 naïve patients were found to possess tumors with higher TMBs (352.0 vs. 213.5, P = 0.005) and received TIL reactive with more neoantigens (2 vs. 1, P = 0.003) compared with anti-PD-1 experienced patients. Among patients treated with TIL ACT, TMB and number of neoantigens identified were higher in ACT responders than ACT nonresponders in both anti-PD-1 naïve and experienced patients. Among patients with comparable TMBs and predicted neoantigen loads, treatment products administered to anti-PD-1 naïve patients were more likely to contain T cells reactive against neoantigens than treatment products for anti-PD-1 experienced patients (2.5 vs. 1, P = 0.02). CONCLUSIONS: These results indicate that decreases in TMB and targeted neoantigens partially account for the difference in response to ACT and that additional factors likely influence responses in these patients. See related commentary by Blass and Ott, p. 2980.

A machine learning model for ranking candidate HLA class I neoantigens based on known neoepitopes from multiple human tumor types.

Tumor neoepitopes presented by major histocompatibility complex (MHC) class I are recognized by tumor-infiltrating lymphocytes (TIL) and are targeted by adoptive T-cell therapies. Identifying which mutant neoepitopes from tumor cells are capable of recognition by T cells can assist in the development of tumor-specific, cell-based therapies and can shed light on antitumor responses. Here, we generate a ranking algorithm for class I candidate neoepitopes by using next-generation sequencing data and a dataset of 185 neoepitopes that are recognized by HLA class I-restricted TIL from individuals with metastatic cancer. Random forest model analysis showed that the inclusion of multiple factors impacting epitope presentation and recognition increased output sensitivity and specificity compared to the use of predicted HLA binding alone. The ranking score output provides a set of class I candidate neoantigens that may serve as therapeutic targets and provides a tool to facilitate in vitro and in vivo studies aimed at the development of more effective immunotherapies.

Engineered IL-21 Cytokine Muteins Fused to Anti-PD-1 Antibodies Can Improve CD8+ T Cell Function and Anti-tumor Immunity.

Inhibitors that block the programmed cell death-1 (PD-1) pathway can potentiate endogenous antitumor immunity and have markedly improved cancer survival rates across a broad range of indications. However, these treatments work for only a minority of patients. The efficacy of anti-PD-1 inhibitors may be extended by cytokines, however, the incorporation of cytokines into therapeutic regimens has significant challenges. In their natural form when administered as recombinant proteins, cytokine treatments are often associated with low response rates. Most cytokines have a short half-life which limits their exposure and efficacy. In addition, cytokines can activate counterregulatory pathways, in the case of immune-potentiating cytokines this can lead to immune suppression and thereby diminish their potential efficacy. Improving the drug-like properties of natural cytokines using protein engineering can yield synthetic cytokines with improved bioavailability and tissue targeting, allowing for enhanced efficacy and reduced off-target effects. Using structure guided engineering we have designed a novel class of antibody-cytokine fusion proteins consisting of a PD-1 targeting antibody fused together with an interleukin-21 (IL-21) cytokine mutein. Our bifunctional fusion proteins can block PD-1/programmed death-ligand 1 (PD-L1) interaction whilst simultaneously delivering IL-21 cytokine to PD-1 expressing T cells. Targeted delivery of IL-21 can improve T cell function in a manner that is superior to anti-PD-1 monotherapy. Fusion of engineered IL-21 variants to anti-PD1 antibodies can improve the drug-like properties of IL-21 cytokine leading to improved cytokine serum half-life allowing for less frequent dosing. In addition, we show that targeted delivery of IL-21 can minimize any potential detrimental effect on local antigen-presenting cells. A highly attenuated IL-21 mutein variant (R9E:R76A) fused to a PD-1 antibody provides protection in a humanized mouse model of cancer that is refractory to anti-PD-1 monotherapy. Collectively, our preclinical data demonstrate that this approach may improve upon and extend the utility of anti-PD-1 therapeutics currently in the clinic.

Unique Neoantigens Arise from Somatic Mutations in Patients with Gastrointestinal Cancers.

Immunotherapies can mediate regression of human tumors with high mutation rates, but responses are rarely observed in patients with common epithelial cancers. This raises the question of whether patients with these common cancers harbor T lymphocytes that recognize mutant proteins expressed by autologous tumors that may represent ideal targets for immunotherapy. Using high-throughput immunologic screening of mutant gene products identified via whole-exome sequencing, we identified neoantigen-reactive tumor-infiltrating lymphocytes (TIL) from 62 of 75 (83%) patients with common gastrointestinal cancers. In total, 124 neoantigen-reactive TIL populations were identified, and all but one of the neoantigenic determinants were unique. The results of in vitro T-cell recognition assays demonstrated that 1.6% of the gene products encoded by somatic nonsynonymous mutations were immunogenic. These findings demonstrate that the majority of common epithelial cancers elicit immune recognition and open possibilities for cell-based immunotherapies for patients bearing these cancers. SIGNIFICANCE: TILs cultured from 62 of 75 (83%) patients with gastrointestinal cancers recognized neoantigens encoded by 1.6% of somatic mutations expressed by autologous tumor cells, and 99% of the neoantigenic determinants appeared to be unique and not shared between patients.This article is highlighted in the In This Issue feature, p. 983.

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay.

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

TRAF molecules in inflammation and inflammatory diseases.

PURPOSE OF REVIEW: This review presents an overview of the current knowledge of TRAF molecules in inflammation with an emphasis on available human evidence and direct in vivo evidence of mouse models that demonstrate the contribution of TRAF molecules in the pathogenesis of inflammatory diseases. RECENT FINDINGS: The tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) family of cytoplasmic proteins was initially identified as signaling adaptors that bind directly to the intracellular domains of receptors of the TNF-R superfamily. It is now appreciated that TRAF molecules are widely employed in signaling by a variety of adaptive and innate immune receptors as well as cytokine receptors. TRAF-dependent signaling pathways typically lead to the activation of nuclear factor-κBs (NF-κBs), mitogen-activated protein kinases (MAPKs), or interferon-regulatory factors (IRFs). Most of these signaling pathways have been linked to inflammation, and therefore TRAF molecules were expected to regulate inflammation and inflammatory responses since their discovery in 1990s. However, direct in vivo evidence of TRAFs in inflammation and especially in inflammatory diseases had been lacking for many years, partly due to the difficulty imposed by early lethality of TRAF2-/-, TRAF3-/-, and TRAF6-/- mice. With the creation of conditional knockout and lineage-specific transgenic mice of different TRAF molecules, our understanding about TRAFs in inflammation and inflammatory responses has rapidly advanced during the past decade. SUMMARY: Increasing evidence indicates that TRAF molecules are versatile and indispensable regulators of inflammation and inflammatory responses and that aberrant expression or function of TRAFs contributes to the pathogenesis of inflammatory diseases.

Regulation of Physical Microglia-Neuron Interactions by Fractalkine Signaling after Status Epilepticus.

Microglia, the resident immune cells of the brain, perform elaborate surveillance in which they physically interact with neuronal elements. A novel form of microglia-neuron interaction named microglial process convergence (MPC) toward neuronal axons and dendrites has recently been described. However, the molecular regulators and pathological relevance of MPC have not been explored. Here, using high-resolution two-photon imaging in vivo and ex vivo, we observed a dramatic increase in MPCs after kainic acid- or pilocarpine-induced experimental seizures that was reconstituted after glutamate treatment in slices from mice. Interestingly, a deficiency of the fractalkine receptor (CX3CR1) decreased MPCs, whereas fractalkine (CX3CL1) treatment increased MPCs, suggesting that fractalkine signaling is a critical regulator of these microglia-neuron interactions. Furthermore, we found that interleukin-1ß was necessary and sufficient to trigger CX3CR1-dependent MPCs. Finally, we show that a deficiency in fractalkine signaling corresponds with increased seizure phenotypes. Together, our results identify the neuroglial CX3CL1-CX3CR1 communication axis as a modulator of potentially neuroprotective microglia-neuron physical interactions during conditions of neuronal hyperactivity.

TRAF3: a novel tumor suppressor gene in macrophages.

Tumor necrosis factor receptor-associated factor 3 (TRAF3), a member of the TRAF family of cytoplasmic adaptor proteins with E3 ligase activity, is ubiquitously expressed in various cell types of the immune system. It is shared for signaling by a variety of adaptive and innate immune receptors as well as cytokine receptors. Previous studies examining conditional TRAF3-deficient mouse models that have the Traf3 gene specifically deleted in B lymphocytes or T lymphocytes have revealed the diverse and critical in vivo functions of TRAF3 in adaptive immunity. Although in vitro evidence points to a pivotal and indispensable role for TRAF3 in type I interferon production induced by pattern recognition receptors in macrophages and dendritic cells, the in vivo functions of TRAF3 in the innate immune system had long remained unclear. Three laboratories have recently addressed this gap in knowledge by investigating myeloid cell-specific TRAF3-deficient (genotype: TRAF3flox/floxLysM+/Cre) mice. The new evidence together demonstrates that specific ablation of TRAF3 in myeloid cells leads to inflammatory diseases, altered progression of diabetes, and spontaneous development of different types of tumors and infections in mice. These new findings indicate that TRAF3 acts as an anti-inflammatory factor and is required for optimal innate immunity in myeloid cells. Strikingly, the new evidence also identifies TRAF3 as a novel tumor suppressor gene in macrophages and other myeloid cells. In this review, we discuss and summarize the new findings and current knowledge about the multi-faceted regulatory roles and complex signaling mechanisms of myeloid cell TRAF3 in inflammation, innate immunity, and tumor development.

Myeloid cell TRAF3 regulates immune responses and inhibits inflammation and tumor development in mice.

Myeloid cells, including granulocytes, monocytes, macrophages, and dendritic cells, are crucial players in innate immunity and inflammation. These cells constitutively or inducibly express a number of receptors of the TNFR and TLR families, whose signals are transduced by TNFR-associated factor (TRAF) molecules. In vitro studies showed that TRAF3 is required for TLR-induced type I IFN production, but the in vivo function of TRAF3 in myeloid cells remains unknown. In this article, we report the generation and characterization of myeloid cell-specific TRAF3-deficient (M-TRAF3(-/-)) mice, which allowed us to gain insights into the in vivo functions of TRAF3 in myeloid cells. We found that TRAF3 ablation did not affect the maturation or homeostasis of myeloid cells in young adult mice, even though TRAF3-deficient macrophages and neutrophils exhibited constitutive NF-κB2 activation. However, in response to injections with LPS (a bacterial mimic) or polyinosinic-polycytidylic acid (a viral mimic), M-TRAF3(-/-) mice exhibited an altered profile of cytokine production. M-TRAF3(-/-) mice immunized with T cell-independent and -dependent Ags displayed elevated T cell-independent IgG3 and T cell-dependent IgG2b responses. Interestingly, 15- to 22-mo-old M-TRAF3(-/-) mice spontaneously developed chronic inflammation or tumors, often affecting multiple organs. Taken together, our findings indicate that TRAF3 expressed in myeloid cells regulates immune responses in myeloid cells and acts to inhibit inflammation and tumor development in mice.