CD8+ T cell metabolic flexibility elicited by CD28-ARS2 axis-driven alternative splicing of PKM supports antitumor immunity.

Metabolic flexibility has emerged as a critical determinant of CD8+ T-cell antitumor activity, yet the mechanisms driving the metabolic flexibility of T cells have not been determined. In this study, we investigated the influence of the nuclear cap-binding complex (CBC) adaptor protein ARS2 on mature T cells. In doing so, we discovered a novel signaling axis that endows activated CD8+ T cells with flexibility of glucose catabolism. ARS2 upregulation driven by CD28 signaling reinforced splicing factor recruitment to pre-mRNAs and affected approximately one-third of T-cell activation-induced alternative splicing events. Among these effects, the CD28-ARS2 axis suppressed the expression of the M1 isoform of pyruvate kinase in favor of PKM2, a key determinant of CD8+ T-cell glucose utilization, interferon gamma production, and antitumor effector function. Importantly, PKM alternative splicing occurred independently of CD28-driven PI3K pathway activation, revealing a novel means by which costimulation reprograms glucose metabolism in CD8+ T cells.

Neutrophil heterogeneity and emergence of a distinct population of CD11b/CD18-activated low-density neutrophils after trauma.

INTRODUCTION: Multiple large clinical trauma trials have documented an increased susceptibility to infection after injury. Although neutrophils (polymorphonuclear leukocytes [PMNs]) were historically considered a homogeneous cell type, we hypothesized that injury could alter neutrophil heterogeneity and predispose to dysfunction. To explore whether trauma modifies PMN heterogeneity, we performed an observational mass-spectrometry-based cytometry study on total leukocytes and low-density PMNs found in the peripheral blood mononuclear cell fraction of leukocytes from healthy controls and trauma patients. METHODS: A total of 74 samples from 12 trauma patients, each sampled at 1 or more time points, and matched controls were fractionated and profiled by mass-spectrometry-based cytometry using a panel of 44 distinct markers. After deconvolution and conservative gating on neutrophils, data were analyzed using Seurat, followed by clustering of principal components. RESULTS: Eleven distinct neutrophil populations were resolved in control and trauma neutrophils based on differential protein surface marker expression. Trauma markedly altered the basal heterogeneity of neutrophil subgroups seen in the control samples, with loss of a dominant population of resting neutrophils marked by high expression of C3AR and low levels of CD63, CD64, and CD177 (cluster 1), and expansion of two alternative neutrophil populations, one of which is marked by high expression of CD177 with suppression of CD10, CD16, C3AR, CD63, and CD64 (cluster 6). Remarkably, following trauma, a substantially larger percentage of neutrophils sediment in the monocyte fraction. These low-density neutrophils bear markers of functional exhaustion and form a unique trauma-induced population (cluster 9) with markedly upregulated expression of active surface adhesion molecules (activated CD11b/CD18), with suppression of nearly all other surface markers, including receptors for formyl peptides, leukotrienes, chemokines, and complement. CONCLUSION: Circulating neutrophils demonstrate considerable evidence of functional heterogeneity that is markedly altered by trauma. Trauma induces evolution of a novel, exhausted, low-density neutrophil population with immunosuppressive features.

Neutrophil interactions with T cells, platelets, endothelial cells, and of course tumor cells.

Neutrophils sense microbes and host inflammatory mediators, and traffic to sites of infection where they direct a broad armamentarium of antimicrobial products against pathogens. Neutrophils are also activated by damage-associated molecular patterns (DAMPs), which are products of cellular injury that stimulate the innate immune system through pathways that are similar to those activated by microbes. Neutrophils and platelets become activated by injury, and cluster and cross-signal to each other with the cumulative effect of driving antimicrobial defense and hemostasis. In addition, neutrophil extracellular traps are extracellular chromatin and granular constituents that are generated in response to microbial and damage motifs and are pro-thrombotic and injurious. Although neutrophils can worsen tissue injury, neutrophils may also have a role in facilitating wound repair following injury. A central theme of this review relates to how critical functions of neutrophils that evolved to respond to infection and damage modulate the tumor microenvironment (TME) in ways that can promote or limit tumor progression. Neutrophils are reprogrammed by the TME, and, in turn, can cross-signal to tumor cells and reshape the immune landscape of tumors. Importantly, promising new therapeutic strategies have been developed to target neutrophil recruitment and function to make cancer immunotherapy more effective.

Carboplatin enhances lymphocyte-endothelial interactions to promote CD8+ T cell trafficking into the ovarian tumor microenvironment.

OBJECTIVES: Standard chemotherapy agents, including carboplatin, have known immunogenic properties. We sought to determine how carboplatin may influence lymphocyte trafficking to tumor sites. METHODS: Murine models of ovarian cancer were utilized to examine lymphocyte trafficking with common clinically used agents including carboplatin, anti-PD-1 antibody, or anti-VEGFR-2 antibody. Adhesion interactions of lymphocytes with tumor vasculature were measured using intravital microscopy, lymphocyte homing with immunohistochemistry, and treatment groups followed for overall survival. RESULTS: Carboplatin chemotherapy profoundly alters the tumor microenvironment to promote lymphocyte adhesive interactions with tumor vasculature and resultant improvement in lymphocyte trafficking. The measured results seen with carboplatin in the tumor microenvironment were superior to anti-PD-1 treatment or anti-VEGFR-2 which may have contributed to increased overall survival in carboplatin treated groups. CONCLUSIONS: These novel findings suggest a role for chemotherapeutic agents to broadly influence anti-tumor immune responses beyond the induction of immunogenic tumor cell death.

Immunogenic cell stress and injury versus immunogenic cell death: implications for improving cancer treatment with immune checkpoint blockade.

Inducing immunogenic tumor cell death to stimulate the response to immune checkpoint blockade has not yet been effectively translated into clinical practice. We recently discovered that stressed/injured but still viable tumor cells are critical for T-cell priming and substantially improve responses to systemic anti-PD1/CTLA4. Therapeutic tumor cell injury, rather than complete killing, in the tumor microenvironment may enhance efficacy of immunotherapy in various cancers.

VSSP abrogates murine ovarian tumor-associated myeloid cell-driven immune suppression and induces M1 polarization in tumor-associated macrophages from ovarian cancer patients.

The ovarian tumor microenvironment (TME) is characterized by the accumulation of immunosuppressive tumor-associated macrophages (TAMs) and granulocytic cells. Very small size particles (VSSP), comprised of the ganglioside NAcGM3 and Neisseria meningitidis derived outer membrane vesicles, is being developed as a nanoparticulated modulator of innate immunity. Prior studies have shown that VSSP enhanced antigen-specific cytotoxic T cell responses and reduced the suppressive phenotype of splenic granulocytic cells in tumor-bearing mice. Here, we hypothesized that intraperitoneal VSSP would modify myeloid cell accumulation and phenotypes in the ovarian TME and abrogate suppressor function of TAMs and tumor-associated granulocytic cells. In the ID8 syngeneic model of epithelial ovarian cancer, VSSP reduced peritoneal TAMs and induced M1-like polarization in TAMs. In addition, VSSP stimulated peritoneal inflammation characterized by increased granulocytes and monocytes, including inflammatory monocytic cells. VSSP treatment resulted in peritoneal TAMs and granulocytic cells being less suppressive of ex vivo stimulated CD8+ T cell responses. VSSP alone and combined with anti-PD-1 modestly but significantly prolonged survival in tumor-bearing mice. In addition, ex vivo treatment with VSSP induced M1-like polarization in TAMs from patients with metastatic ovarian cancer and variably abrogated their suppressor phenotype. VSSP treatment also partially abrogated the induction of suppressor function in healthy donor neutrophils exposed to ascites supernatants from patients with ovarian cancer. Together, these results point to VSSP reprogramming myeloid responses resulting in abrogation of suppressive pathways and raise the potential for administration of VSSP into the TME to enhance anti-tumor immunity.

Mechanisms Driving Neutrophil-Induced T-cell Immunoparalysis in Ovarian Cancer.

T-cell activation and expansion in the tumor microenvironment (TME) are critical for antitumor immunity. Neutrophils in the TME acquire a complement-dependent T-cell suppressor phenotype that is characterized by inhibition of T-cell proliferation and activation through mechanisms distinct from those of myeloid-derived suppressor cells. In this study, we used ascites fluid supernatants (ASC) from patients with ovarian cancer as an authentic component of the TME to evaluate the effects of ASC on neutrophil function and mechanisms for neutrophil-driven immune suppression. ASC prolonged neutrophil life span, decreased neutrophil density, and induced nuclear hypersegmentation. Mass cytometry analysis showed that ASC induced 15 distinct neutrophil clusters. ASC stimulated complement deposition and signaling in neutrophils, resulting in surface mobilization of granule constituents, including NADPH oxidase. NADPH oxidase activation and phosphatidylserine signaling were required for neutrophil suppressor function, although we did not observe a direct role of extracellular reactive oxygen species in inhibiting T-cell proliferation. Postoperative surgical drainage fluid also induced a complement-dependent neutrophil suppressor phenotype, pointing to this effect as a general response to injury. Like circulating lymphocytes, ASC-activated neutrophils caused complement-dependent suppression of tumor-associated lymphocytes. ASC-activated neutrophils adhered to T cells and caused trogocytosis of T-cell membranes. These injury and signaling cues resulted in T-cell immunoparalysis characterized by impaired NFAT translocation, IL2 production, glucose uptake, mitochondrial function, and mTOR activation. Our results demonstrate that complement-dependent priming of neutrophil effector functions in the TME induces a T-cell nonresponsiveness distinct from established checkpoint pathways and identify targets for immunotherapy.See related Spotlight by Cassatella, p. 725.

RNA editing enzyme APOBEC3A promotes pro-inflammatory M1 macrophage polarization.

Pro-inflammatory M1 macrophage polarization is associated with microbicidal and antitumor responses. We recently described APOBEC3A-mediated cytosine-to-uracil (C > U) RNA editing during M1 polarization. However, the functional significance of this editing is unknown. Here we find that APOBEC3A-mediated cellular RNA editing can also be induced by influenza or Maraba virus infections in normal human macrophages, and by interferons in tumor-associated macrophages. Gene knockdown and RNA_Seq analyses show that APOBEC3A mediates C>U RNA editing of 209 exonic/UTR sites in 203 genes during M1 polarization. The highest level of nonsynonymous RNA editing alters a highly-conserved amino acid in THOC5, which encodes a nuclear mRNA export protein implicated in M-CSF-driven macrophage differentiation. Knockdown of APOBEC3A reduces IL6, IL23A and IL12B gene expression, CD86 surface protein expression, and TNF-α, IL-1ß and IL-6 cytokine secretion, and increases glycolysis. These results show a key role of APOBEC3A cytidine deaminase in transcriptomic and functional polarization of M1 macrophages.

Quantification of Early-Stage Myeloid-Derived Suppressor Cells in Cancer Requires Excluding Basophils.

Myeloid derived suppressor cells (MDSC) are a heterogeneous group of immature cells that accumulate in the peripheral blood and tumor microenvironment and are barriers to cancer therapy. MDSCs serve as prognostic biomarkers and are targets for therapy. On the basis of surface markers, three subsets of MDSCs have been defined in humans: granulocytic, monocytic, and early stage (e-MDSC). The markers attributed to e-MDSCs overlap with those of basophils, which are rare circulating myeloid cells with unrecognized roles in cancer. Thus, we asked whether e-MDSCs in circulation and the tumor microenvironment include basophils. On average, 58% of cells with e-MDSC surface markers in blood and 36% in ascites from patients with ovarian cancer were basophils based on CD123high expression and cytology, whereas cells with immature features were rare. Circulating and ascites basophils did not suppress proliferation of stimulated T cells, a key feature of MDSCs. Increased accumulation of basophils and basogranulin, a marker of basophil degranulation, were observed in ascites compared to serum in patients with newly diagnosed ovarian cancer. Basophils recruited to the tumor microenvironment may exacerbate fluid accumulation by their release of proinflammatory granular constituents that promote vascular leakage. No significant correlation was observed between peripheral basophil counts and survival in patients with ovarian cancer. Our results suggest that studies in which e-MDSCs were defined solely by surface markers should be reevaluated to exclude basophils. Both immaturity and suppression are criteria to define e-MDSCs in future studies.

Mature neutrophils suppress T cell immunity in ovarian cancer microenvironment.

Epithelial ovarian cancer (EOC) often presents with metastases and ascites. Granulocytic myeloid-derived suppressor cells are an immature population that impairs antitumor immunity. Since suppressive granulocytes in the ascites of patients with newly diagnosed EOC were morphologically mature, we hypothesized that PMN were rendered suppressive in the tumor microenvironment (TME). Circulating PMN from patients were not suppressive but acquired a suppressor phenotype (defined as ≥1 log10 reduction of anti-CD3/CD28-stimulated T cell proliferation) after ascites supernatant exposure. Ascites supernatants (20 of 31 supernatants) recapitulated the suppressor phenotype in PMN from healthy donors. T cell proliferation was restored with ascites removal and restimulation. PMN suppressors also inhibited T cell activation and cytokine production. PMN suppressors completely suppressed proliferation in naive, central memory, and effector memory T cells and in engineered tumor antigen-specific cytotoxic T lymphocytes, while antigen-specific cell lysis was unaffected. Inhibition of complement C3 activation and PMN effector functions, including CR3 signaling, protein synthesis, and vesicular trafficking, abrogated the PMN suppressor phenotype. Moreover, malignant effusions from patients with various metastatic cancers also induced the C3-dependent PMN suppressor phenotype. These results point to PMN impairing T cell expansion and activation in the TME and the potential for complement inhibition to abrogate this barrier to antitumor immunity.

Mitochondrial DNA in the tumour microenvironment activates neutrophils and is associated with worse outcomes in patients with advanced epithelial ovarian cancer.

BACKGROUND: Advanced cancer causes necrosis and releases damage-associated molecular patterns (DAMPs). Mitochondrial DAMPs activate neutrophils, including generation of neutrophil extracellular traps (NETs), which are injurious, thrombogenic, and implicated in metastasis. We hypothesised that extracellular mitochondrial DNA (mtDNA) in ascites from patients with epithelial ovarian cancer (EOC) would correlate with worse outcomes. METHODS: Banked ascites supernatants from patients with newly diagnosed advanced EOC were analysed for mtDNA, neutrophil elastase, and activation of healthy donor neutrophils and platelets. TCGA was mined for expression of SELP and ELANE. RESULTS: The highest quartile of ascites mtDNA correlated with reduced progression-free survival (PFS) and a higher likelihood of disease progression within 12-months following primary surgery (n = 68, log-rank, p = 0.0178). NETs were detected in resected tumours. Ascites supernatants chemoattracted neutrophils, induced NETs, and activated platelets. Ascites exposure rendered neutrophils suppressive, based on abrogation of ex vivo stimulated T cell proliferation. Increased SELP mRNA expression correlated with worse overall survival (n = 302, Cox model, p = 0.02). CONCLUSION: In this single-centre retrospective analysis, ascites mtDNA correlated with worse PFS in advanced EOC. Mitochondrial and other DAMPs in ascites may activate neutrophil and platelet responses that facilitate metastasis and obstruct anti-tumour immunity. These pathways are potential prognostic markers and therapeutic targets.

Computational detection and quantification of human and mouse neutrophil extracellular traps in flow cytometry and confocal microscopy.

Neutrophil extracellular traps (NETs) are extracellular defense mechanisms used by neutrophils, where chromatin is expelled together with histones and granular/cytoplasmic proteins. They have become an immunology hotspot, implicated in infections, but also in a diverse array of diseases such as systemic lupus erythematosus, diabetes, and cancer. However, the precise assessment of in vivo relevance in different disease settings has been hampered by limited tools to quantify occurrence of extracellular traps in experimental models and human samples. To expedite progress towards improved quantitative tools, we have developed computational pipelines to identify extracellular traps from an in vitro human samples visualized using the ImageStream® platform (Millipore Sigma, Darmstadt, Germany), and confocal images of an in vivo mouse disease model of aspergillus fumigatus pneumonia. Our two in vitro methods, tested on n = 363/n =145 images respectively, achieved holdout sensitivity/specificity 0.98/0.93 and 1/0.92. Our unsupervised method for thin lung tissue sections in murine fungal pneumonia achieved sensitivity/specificity 0.99/0.98 in n = 14 images. Our supervised method for thin lung tissue classified NETs with sensitivity/specificity 0.86/0.90. We expect that our approach will be of value for researchers, and have application in infectious and inflammatory diseases.

Impact of ascites volume on clinical outcomes in ovarian cancer: A cohort study.

OBJECTIVES: To investigate the impact of ascites volume on ovarian cancer outcomes. METHODS: Clinicopathologic features of a cohort of patients with ovarian cancer were obtained from a curated database at a single institution. Progression free survival (PFS) and overall survival (OS) were recorded. Ascites volume at primary surgery was dichotomized at 2000mL and comparisons for high and low volume ascites were made. Additionally, to elucidate interactions between ascites and ovarian tumor progression, we evaluated the effect of intraperitoneal administrations of murine cell-free ascites versus saline in a syngeneic mouse model of epithelial ovarian cancer. RESULTS: Out of 685 patients identified, 58% had ascites present at the time of initial surgery. Considering the volume of ascites continuously, each liter of ascites was associated with shorter PFS (HR=1.12, 95% CI: 1.07-1.17) and OS (HR=1.12, 95%CI: 1.07-1.17). Patients with ascites greater than the median of 2000mL had significantly shorter PFS (14.5months vs. 22.7months; p<0.001) and OS (27.7months vs. 42.9months; p<0.001). After adjusting for stage, presence of ascites was inversely associated with ability to achieve optimal cytoreductive surgery. Consistent with these correlative results in patients, intraperitoneal administrations of murine cell-free ascites accelerated ovarian cancer progression in mice. CONCLUSIONS: The volume of ascites at initial diagnosis of ovarian cancer correlated with worse PFS and OS. The effect of large volume on prognosis is likely to be in part related to reduced likelihood for complete resection of tumor (R0). If these findings are confirmed in independent studies, consideration should be made to add the presence of large volume ascites at diagnosis to the staging criteria for ovarian cancer.

In vitro exposure to the herbicide atrazine inhibits T cell activation, proliferation, and cytokine production and significantly increases the frequency of Foxp3+ regulatory T cells.

The herbicide atrazine (2-chloro-4-[ethylamino]-6-[isopropylamino]-s-triazine) is the most common water contaminant in the United States. Atrazine is a phosphodiesterase inhibitor and is classified as an estrogen disrupting compound because it elevates estrogen levels via induction of the enzyme aromatase. Previous studies have shown that atrazine exposure alters the function of innate immune cells such as NK cells, DC, mast cells, and macrophages. In this study we have examined the impact of in vitro atrazine exposure on the activation, proliferation, and effector cytokine production by primary murine CD4(+) T lymphocytes. We found that atrazine exposure significantly inhibited CD4(+) T cell proliferation and accumulation as well as the expression of the activation markers CD25 and CD69 in a dose-dependent manner. Interestingly, the effects were more pronounced in cells from male animals. These effects were partially mimicked by pharmacological reagents that elevate intracellular cAMP levels and addition of exogenous rmIL-2 further inhibited proliferation and CD25 expression. Consistent with these findings, atrazine exposure during T cell activation resulted in a 2- to 5-fold increase in the frequency of Foxp3(+) CD4(+) T cells.