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Filamentous fungi (moulds) are ubiquitous soil inhabitants whose conidia are inhaled into the respiratory tract, where they may cause life-threatening infections. Among these infections is invasive aspergillosis, which is a major cause of morbidity and mortality in the severely immunocompromised. Risk factors for invasive aspergillosis include prolonged and severe neutropenia, hematopoietic stem cell and solid organ transplantation, advanced AIDS, and chronic granulomatous disease. Invasive aspergillosis most commonly involves the sinopulmonary tract reflecting inhalation as the principal portal of entry. Chest computed tomography scans and new non-culture-based assays such as antigen detection and polymerase chain reaction may facilitate the early diagnosis of invasive aspergillosis, but have limitations. Reflecting an important unmet need, there has been a significant expansion in the antifungal armamentarium. The second-generation triazole, voriconazole, was superior to conventional amphotericin B as primary therapy for invasive aspergillosis, and is the new standard of care for this infection. There is significant interest in combination antifungal therapy pairing an echinocandin with either an azole or amphotericin B formulation as therapy for invasive aspergillosis. In addition, there has been an increased understanding of the immunology of Aspergillus infection, paving the way to novel immune augmentation strategies in animal models that merit evaluation in phase I clinic trials. [ABSTRACT FROM AUTHOR]

Evidence has shown that T-cell receptors (TCRs) that recognize the same epitopes may not be the exact TCR clonotypes but have slightly different TCR sequences. However, the changes in the genomic and transcriptomic signatures of these highly homologous T cells during immunotherapy remain unknown. Here, we examined the evolutionary features in circulating TCR clonotypes observed in tumors (tumor-infiltrating lymphocyte (TIL)-TCRs) by combining single-cell RNA/TCR sequencing of longitudinal blood samples and TCR sequencing of tumor tissue from a patient treated with anti-cytotoxic T-lymphocyte-associated protein 4/programmed cell death protein-1 therapy. We found frequent circulating CD8 + TIL-TCRs with identical complementarity determining region 3 (CDR3)α amino acid sequences but quasi-identical CDR3β and TCR α/β (TRA/TRB) sequences. Despite their sequence similarities, these highly homologous TIL-TCRs responded differently to immunotherapy, and exhibited distinct transcriptional signatures that were uniquely distinguished by the expression of GZMK Overall, the expression of IFNG in CD8 + T-cell subsets including highly homologous TIL-TCRs increased when the patient achieved a response, but gradually decreased as the patient developed acquired resistance. Our findings provide insight into the cross-talk between T cells in the tumor microenvironment and those in the blood, and highlight that CD8 + T cells with highly homologous TCR sequences might display divergent transcriptional states and kinetics in response to immunotherapy., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2025. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ Group.)

Recurrent ovarian cancer patients, especially those resistant to platinum, lack effective curative treatments. To address this, we conducted a phase 2 clinical trial (NCT02853318) combining pembrolizumab with bevacizumab, to increase T cell infiltration into the tumor, and oral cyclophosphamide, to reduce the number of regulatory T cells. The trial accrued 40 heavily pretreated recurrent ovarian cancer patients. The primary endpoint, progression free survival, was extended to a median of 10.2 months. The secondary endpoints demonstrated an objective response rate of 47.5%, and disease control in 30% of patients for over a year while maintaining a good quality of life. We performed comprehensive molecular, immune, microbiome, and metabolic profiling on samples of trial patients. Here, we show increased T and B cell clusters and distinct microbial patterns with amino acid and lipid metabolism are linked to exceptional clinical responses. This study suggests the immune milieu and host-microbiome can be leveraged to improve antitumor response in future immunotherapy trials., Competing Interests: Competing interests: The authors declare no competing interests. This study was approved by the Roswell Park Institutional Review Board (IRB), and all patients provided written informed consent before initiating any study procedures. Participants did not receive financial compensation. This study followed the Consolidated Standards of Reporting Trials ( CONSORT ) reporting guideline. While all work was completed at the Roswell Park Comprehensive Cancer Center by author Shanmuga Chilakapata, this author has relocated to Northeastern University., (© 2024. The Author(s).)

Background: Actinomyces are mucous membrane commensals that infrequently cause invasive disease. Our goal was to define Actinomyces species prevalence, the predominant disease site and risk factors for actinomycosis., Methods: We retrospectively reviewed patients with growth of Actinomyces species from cultures in a single-cancer center from July 2007 to June 2020. Proven invasive actinomycosis was defined as the presence of compatible clinical syndrome and radiographic findings with histopathological confirmation or culture from a normally sterile site. Probable invasive actinomycosis was defined based on the same criteria but without histologic confirmation. Contaminants were defined as culture growth in the absence of clinical or radiological findings consistent with disease. Speciation of Actinomyces was performed by the bioMerieux VITEK 2 anaerobic and coryneform identification card., Results: Of 235 patients, 179 (76.2%) had malignancy. Among 90 (38.3%) patients with invasive actinomycosis, A odontolyticus was isolated in 32 (35.6%), followed by A meyeri in 20 (22.2%), and A naeslundii in 17 (18.9%). Among 145 (61.7%) colonized patients, A odontolyticus was isolated in 67 (46.2%), followed by A naeslundii in 27 (18.6%). Abdominopelvic infection was the most common site for invasive actinomycosis documented in 54 patients (60.0%) followed by orocervicofacial in 14 (15.6%) and thoracic in 10 (11.1%)., Conclusions: A odontolyticus, A meyeri, and A naeslundi were the most frequently isolated species causing invasive actinomycosis, and A odontolyticus and A nauslendii among colonizers. Abdominopelvic represented the most frequent site for invasive disease. Further studies are needed to investigate the epidemiology of Actinomyces species in this population., Competing Interests: Potential conflicts of interest. The authors: No reported conflicts of interest., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2024.)

Intra-organism biodiversity is thought to arise from epigenetic modification of constituent genes and post-translational modifications of translated proteins. Here, we show that post-transcriptional modifications, like RNA editing, may also contribute. RNA editing enzymes APOBEC3A and APOBEC3G catalyze the deamination of cytosine to uracil. RNAsee (RNA site editing evaluation) is a computational tool developed to predict the cytosines edited by these enzymes. We find that 4.5% of non-synonymous DNA single nucleotide polymorphisms that result in cytosine to uracil changes in RNA are probable sites for APOBEC3A/G RNA editing; the variant proteins created by such polymorphisms may also result from transient RNA editing. These polymorphisms are associated with over 20% of Medical Subject Headings across ten categories of disease, including nutritional and metabolic, neoplastic, cardiovascular, and nervous system diseases. Because RNA editing is transient and not organism-wide, future work is necessary to confirm the extent and effects of such editing in humans., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

Ribotyping was performed on Clostridioides difficile isolates from patients with malignancies. Thirty-one (27.9%) isolates from 111 episodes of colitis were recovered representing 14 ribotypes with 25 (80.6%) belonging to 6 ribotypes (014/020, 1/VPI/077/087, 05/015, 015/046, 05/053, 106/174). We identified three novel ribotypes with 1 carrying gene encoding for binary toxin., Competing Interests: Research funding: C.-A.D. Burnham has received grants or contracts from Cepheid, bioMerieux, BioFire Diagnostics, Luminex, ThermoFisher, CDC, AHRQ, NIH, Selux, and Qiagen to Washington University School of Medicine (WUSM). B.H.S. has received research funding from Apellis and NextCure. Consultant or Advisory Role: C.-A.D. Burnham has received consulting fees from Cepheid, BioFire Diagnostics, Shionogi, and Pattern Bioscience paid to herself. Honoraria: C.-A.D. Burnham has received payment or honoraria for lectures, presentations, speakers, bureaus, manuscript writing, or educational events from Roche, Cepheid, BD, BioFire Diagnostics, Medavera, and Medscape paid to herself. B.H.S. is a member of Scientific Advisory Board for NextCure. Employment or Leadership: C.-A.D. Burnham has leadership or fiduciary role in the American Academy of Microbiology (ASM) and Clinical Laboratory Standards Institute (CLSI) for which she or her institution has not been compensated. Stock Ownership: None declared. Expert Testimony: None declared. Patents: None declared. Other Remuneration: None declared., (© The Author(s) 2023.)

Background: Immune checkpoint inhibitors (ICIs) such as programmed cell death protein-1 (PD-1) inhibitors or PD-1 ligand-1 (PD-L1) inhibitors have led to remarkable improvement in outcomes of non-small cell lung cancer (NSCLC). Unfortunately, the significant benefits of ICI therapy are frequently limited by resistance to treatment and adverse effects, and the predictive value of pre-treatment tumor tissue PD-L1 expression is limited. Development of less invasive biomarkers that could identify responders and non-responders in early on-treatment could markedly improve the treatment regimen. Accumulating evidence suggests that baseline gut microbiota profile is associated with response to PD-1/PD-L1 blockade therapy. However, change in the gut microbiome composition during PD-1/PD-L1 blockade therapy and its relation to response remain unclear., Methods: Here, we analyzed pre- and on-treatment fecal samples from five NSCLC patients receiving anti-PD-1 immunotherapy, alone or in tandem with chemotherapy, and performed 16S rRNA sequencing., Results: The overall alpha diversity of the baseline gut microbiome was similar between three responders and two non-responders. While the gut microbiome composition remained stable overall during treatment (R2 = 0.145), responders showed significant changes in microbiome diversity between pre- and on-treatment samples during anti-PD-1 therapy compared to non-responders (P = 0.0274). Within the diverse microbiota, responders showed decreases in the abundance of genera Odoribacter , Gordonibacter , Candidatus Stoquefichus , Escherichia-Shigella , and Collinsella , and increase in abundance of Clostridium sensu stricto 1 . In contrast, non-responders demonstrated on-treatment increases in genera Prevotella , Porphyromonas , Streptococcus , and Escherichia-Shigella , and decrease in abundance of Akkermansia ., Conclusions: This pilot study identified a substantial change in gut microbiome diversity between pre- and on-treatment samples in NSCLC patients responding to anti-PD-1 therapy compared to non-responders. Our findings highlight the potential utility of gut microbiota dynamics as a noninvasive biomarker to predict response to PD-1/PD-L1 blockade therapy for a wide variety of malignancies, which sets a path for future investigation in larger prospective studies., Competing Interests: The authors declare no potential conflicts of interest., (Copyright 2023, Sarkar et al.)

Lack of reliable predictive biomarkers is a major limitation of combination therapy with chemotherapy and anti-programmed cell death protein 1/programmed death-ligand 1 (anti-PD-1/PD-L1) therapy (chemo-immunotherapy). We previously observed that the increase of peripheral blood CD8 + T cells expressing CX3CR1, a marker of differentiation, correlates with response to anti-PD-1 therapy; however, the predictive and prognostic value of T-cell CX3CR1 expression during chemo-immunotherapy is unknown. Here, we evaluated the utility of circulating CX3CR1 + CD8 + T cells as a predictive correlate of response to chemo-immunotherapy in patients with non-small cell lung cancer (NSCLC). At least 10% increase of the CX3CR1 + subset in circulating CD8 + T cells from baseline (CX3CR1 score) was associated with response to chemo-immunotherapy as early as 4 weeks with 85.7% overall accuracy of predicting response at 6 weeks. Furthermore, at least 10% increase of the CX3CR1 score correlated with substantially better progression-free ( P = 0.0051) and overall survival ( P = 0.0138) on Kaplan-Meier analysis. Combined single-cell RNA/T-cell receptor (TCR) sequencing of circulating T cells from longitudinally obtained blood samples and TCR sequencing of tumor tissue from the same patient who received a long-term benefit from the treatment demonstrated remarkable changes in genomic and transcriptomic signatures of T cells as well as evolution of TCR clonotypes in peripheral blood containing highly frequent tumor-infiltrating lymphocyte repertoires overexpressing CX3CR1 early after initiation of the treatment despite stable findings of the imaging study. Collectively, these findings highlight the potential utility of T-cell CX3CR1 expression as a dynamic blood-based biomarker during the early course of chemo-immunotherapy and a marker to identify frequent circulating tumor-infiltrating lymphocyte repertoires., Significance: Current approaches to combined chemotherapy and anti-PD-1/PD-L1 therapy (chemo-immunotherapy) for patients with NSCLC are limited by the lack of reliable predictive biomarkers. This study shows the utility of T-cell differentiation marker, CX3CR1, as an early on-treatment predictor of response and changes in genomic/transcriptomic signatures of circulating tumor-infiltrating lymphocyte repertoires in patients with NSCLC undergoing chemo-immunotherapy., (© 2023 The Authors; Published by the American Association for Cancer Research.)

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., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Importance: Changes in postsurgical opioid prescribing practices may help reduce chronic opioid use in surgical patients., Objective: To investigate whether postsurgical acute pain across different surgical subspecialties can be managed effectively after hospital discharge with an opioid supply of 3 or fewer days and whether this reduction in prescribed opioids is associated with reduced new, persistent opioid use., Design, Setting, and Participants: In this prospective cohort study with a case-control design, a restrictive opioid prescription protocol (ROPP) specifying an opioid supply of 3 or fewer days after discharge from surgery along with standardized patient education was implemented across all surgical services at a tertiary-care comprehensive cancer center. Participants were all patients who underwent surgery from August 1, 2018, to July 31, 2019., Main Outcomes and Measures: Main outcomes were the rate of compliance with the ROPP in each surgical service, the mean number of prescription days and refill requests, type of opioid prescribed, and rate of conversion to chronic opioid use determined via a state-run opioid prescription program. Postsurgical complications were also measured., Results: A total of 4068 patients (mean [SD] age, 61.0 [13.8] years; 2528 women [62.1%]) were included, with 2017 in the pre-ROPP group (August 1, 2018, to January 31, 2019) and 2051 in the post-ROPP group (February 1, 2019, to July 31, 2019). The rate of compliance with the protocol was 95%. After implementation of the ROPP, mean opioid prescription days decreased from a mean (SD) of 3.9 (4.5) days in the pre-ROPP group to 1.9 (3.6) days in the post-ROPP group (P < .001). The ROPP implementation led to a 45% decrease in prescribed opioids after surgery (mean [SD], 157.22 [338.06] mean morphine milligram equivalents [MME] before ROPP vs 83.54 [395.70] MME after ROPP; P < .001). Patients in the post-ROPP cohort requested fewer refills (367 of 2051 [17.9%] vs 422 of 2017 [20.9%] in the pre-ROPP cohort; P = .02). There was no statistically significant difference in surgical complications. The conversion rate to chronic opioid use decreased following ROPP implementation among both opioid-naive patients with cancer (11.3% [143 of 1267] to 4.5% [118 of 2645]; P < .001) and those without cancer (6.1% [19 of 310] to 2.7% [16 of 600]; P = .02)., Conclusions and Relevance: In this cohort study, prescribing an opioid supply of 3 or fewer days to surgical patients after hospital discharge was feasible for most patients, led to a significant decrease in the number of opioids prescribed after surgery, and was associated with a significantly decreased conversion to long-term opioid use without concomitant increases in refill requests or significant compromises in surgical recovery.

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., (Copyright © 2022 American Association for the Surgery of Trauma.)

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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

T H 2 cells and innate lymphoid cells 2 (ILC2) can stimulate tumor growth by secreting pro-tumorigenic cytokines such as interleukin-4 (IL-4), IL-5, and IL-13. However, the mechanisms by which type 2 immune cells traffic to the tumor microenvironment are unknown. Here, we show that oncogenic Kras G12D increases IL-33 expression in pancreatic ductal adenocarcinoma (PDAC) cells, which recruits and activates T H 2 and ILC2 cells. Correspondingly, cancer-cell-specific deletion of IL-33 reduces T H 2 and ILC2 recruitment and promotes tumor regression. Unexpectedly, IL-33 secretion is dependent on the intratumoral fungal mycobiome. Genetic deletion of IL-33 or anti-fungal treatment decreases T H 2 and ILC2 infiltration and increases survival. Consistently, high IL-33 expression is observed in approximately 20% of human PDAC, and expression is mainly restricted to cancer cells. These data expand our knowledge of the mechanisms driving PDAC tumor progression and identify therapeutically targetable pathways involving intratumoral mycobiome-driven secretion of IL-33., Competing Interests: Declaration of interests P. Dey and A.A. have a patent pending on targeting IL-33 and mycobiome in cancer: US Provisional Patent Application Serial No. 63/238,531. R.A.D. is a co-founder, adviser, and/or director of Tvardi Therapeutics, Asylia Therapeutics, Stellanova Therapeutics, Nirogy Therapeutics, and Sporos Bioventures. Tvardi and Nirogy are developing STAT3 and MCT inhibitors, respectively. A.M. receives royalties from Cosmos Wisdom Biotechnology and Thrive Earlier Detection, an Exact Sciences Company. A.M. is also a consultant for Freenome and Tezcat Biotechnology. The other authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Two resident macrophage subsets reside in peritoneal fluid. Macrophages also reside within mesothelial membranes lining the peritoneal cavity, but they remain poorly characterized. Here, we identified two macrophage populations (LYVE1hi MHC IIlo-hi CX3CR1gfplo/- and LYVE1lo/- MHC IIhi CX3CR1gfphi subsets) in the mesenteric and parietal mesothelial linings of the peritoneum. These macrophages resembled LYVE1+ macrophages within surface membranes of numerous organs. Fate-mapping approaches and analysis of newborn mice showed that LYVE1hi macrophages predominantly originated from embryonic-derived progenitors and were controlled by CSF1 made by Wt1+ stromal cells. Their gene expression profile closely overlapped with ovarian tumor-associated macrophages previously described in the omentum. Indeed, syngeneic epithelial ovarian tumor growth was strongly reduced following in vivo ablation of LYVE1hi macrophages, including in mice that received omentectomy to dissociate the role from omental macrophages. These data reveal that the peritoneal compartment contains at least four resident macrophage populations and that LYVE1hi mesothelial macrophages drive tumor growth independently of the omentum., Competing Interests: Disclosures:   J.W. Williams reported grants from American Heart Association and grants from NIH NHLBI outside the submitted work. No other disclosures were reported., (© 2021 Zhang et al.)

Cancer immunotherapies have generated remarkable clinical responses for some patients with advanced/metastatic disease, prompting exploration of rational combination therapies to bolster anti-tumor immunity in patients with limited response or those who experience tumor progression following an initial response to immunotherapy. In contrast to other tumor indications, objective response rates to single-agent PD-1/PD-L1 blockade in ovarian cancer are limited, suggesting a need to identify combinatorial approaches that lead to tumor regression in a setting where checkpoint blockade alone is ineffective. Using a pre-clinical model of aggressive intraperitoneal ovarian cancer, we have previously reported on a heterologous prime/boost cancer vaccine that elicits robust anti-tumor immunity, prolongs survival of tumor-bearing mice, and which is further improved when combined with checkpoint blockade. As tumor control in this model is CD8 + T cell dependent, we reasoned that the prime/boost vaccine platform could be used to explore additional treatment combinations intended to bolster the effects of CD8 + T cells. Using whole tumor transcriptomic data, we identified candidate therapeutic targets anticipated to rationally combine with prime/boost vaccination. In the context of a highly effective cancer vaccine, CD27 agonism or antibody-mediated depletion of granulocytic cells each modestly increased tumor control following vaccination, with anti-PD-1 therapy further improving treatment efficacy. These findings support the use of immunotherapies with well-defined mechanisms(s) of action as a valuable platform for identifying candidate combination approaches for further therapeutic testing in ovarian cancer., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

SARS-CoV-2 vaccines are effective in preventing COVID-19. Patients with cancer are at high risk for severe COVID-19 and are appropriately prioritized for vaccination. Several studies in this issue of Cancer Cell add to our knowledge of the heterogeneity of immune responses to vaccination among patients with cancer and identify important areas for future research., Competing Interests: Declaration of interests E.A.G. declares receipt of honoraria and/or consulting fees from Taiho Oncology, Takeda Pharmaceuticals, Alexion Pharmaceuticals, Abbvie, Celgene/BMS, and Novartis and research funding (to Roswell Park) from Genentech, Celgene/BMS, Celldex Therapeutics, Apellis Pharmaceuticals, Alexion Pharmaceuticals, Imago Biosciences, and Astex Pharmaceuticals. B.H.S. has no interests to declare., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (©2021 American Association for Cancer Research.)

Immune checkpoint inhibitors (ICI) have revolutionized treatment for various cancers; however, durable response is limited to only a subset of patients. Discovery of blood-based biomarkers that reflect dynamic change of the tumor microenvironment, and predict response to ICI, will markedly improve current treatment regimens. Here, we investigate CX3C chemokine receptor 1 (CX3CR1), a marker of T-cell differentiation, as a predictive correlate of response to ICI therapy. Successful treatment of tumor-bearing mice with ICI increases the frequency and T-cell receptor clonality of the peripheral CX3CR1 + CD8 + T-cell subset that includes an enriched repertoire of tumor-specific and tumor-infiltrating CD8 + T cells. Furthermore, an increase in the frequency of the CX3CR1 + subset in circulating CD8 + T cells early after initiation of anti-PD-1 therapy correlates with response and survival in patients with non-small cell lung cancer. Collectively, these data support T-cell CX3CR1 expression as a blood-based dynamic early on-treatment predictor of response to ICI therapy.

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&#95;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-1β 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.

Previous studies have reported a beneficial effect from cytomegalovirus (CMV) reactivation after allogeneic hematopoietic stem cell transplantation (alloHCT) on immune reconstitution. We determined the CMV antigenemia level associated with increased CMV antigen-specific T cells (CASTs) at day +100 and decreased CMV reactivation after day +100. CMV reactivation and CASTs were measured with CMV antigenemia and CMV-specific major histocompatibility complex multimers. The analysis consisted of 775 CAST measurements obtained before and 30, 100, and 365 days post-alloHCT from 327 consecutive patients treated between 2008 and 2016. Detectable CASTs correlated with recipient (P < .0001) and donor (P < .0001) CMV seropositivity pre-alloHCT. CMV reactivation before day +100 was associated with a higher proportion of patients who achieved ≥3 CASTs/µL by day +100 (61% with versus 39% without reactivation, P < .001). In alloHCT recipients at high risk for CMV reactivation (R + D ± ) with a maximum of grade II acute graft-versus-host-disease, reactivating CMV before day +100 and achieving ≥3 versus <3 CASTs/µL at day +100 was associated with reduced CMV reactivation from day +100 to +365 (27% versus 62%, P = .04). This protective effect was observed with low-level but not high-level CMV reactivation (<5 versus ≥5/50,000 polymorphonuclear leukocytes + pp65, respectively). These findings suggest low-level CMV reactivation may be beneficial and that treatment may be delayed until progression. These findings will need validation in prospective clinical trials using CMV PCR and antigenemia assays., (Copyright © 2020 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Neutrophils, the most abundant circulating leukocyte, are critical for host defense. Granulopoiesis is under the control of transcriptional factors and culminates in mature neutrophils with a broad armamentarium of antimicrobial pathways. These pathways include nicotinamide adenine dinucleotide phosphate oxidase, which generates microbicidal reactive oxidants, and nonoxidant pathways that target microbes through several mechanisms. Activated neutrophils can cause or worsen tissue injury, underscoring the need for calibration of activation and resolution of inflammation when infection has been cleared. Acquired neutrophil disorders are typically caused by cytotoxic chemotherapy or immunosuppressive agents. Primary neutrophil disorders typically result from disabling mutations of individual genes that result in impaired neutrophil number or function, and provide insight into basic mechanisms of neutrophil biology. Neutrophils can also be activated by noninfectious causes, including trauma and cellular injury, and can have off-target effects in which pathways that typically defend against infection exacerbate injury and disease. These off-target effects include acute organ injury, autoimmunity, and variable effects on the tumor microenvironment that can limit or worsen tumor progression. A greater understanding of neutrophil plasticity in these conditions is likely to pave the way to new therapeutic approaches., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

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 CD123 high 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., (©2020 American Association for Cancer Research.)

In the age of modern medicine and artificial intelligence, image analysis and machine learning have revolutionized diagnostic pathology, facilitating the development of computer aided diagnostics (CADs) which circumvent prevalent diagnostic challenges. Although CADs will expedite and improve the precision of clinical workflow, their prognostic potential, when paired with clinical outcome data, remains indeterminate. In high impact renal diseases, such as diabetic nephropathy and lupus nephritis (LN), progression often occurs rapidly and without immediate detection, due to the subtlety of structural changes in transient disease states. In such states, exploration of quantifiable image biomarkers, such as Neutrophil Extracellular Traps (NETs), may reveal alternative progression measures which correlate with clinical data. NETs have been implicated in LN as immunogenic cellular structures, whose occurrence and dysregulation results in excessive tissue damage and lesion manifestation. We propose that renal biopsy NET distribution will function as a discriminate, predictive biomarker in LN, and will supplement existing classification schemes. We have developed a computational pipeline for segmenting NET-like structures in LN biopsies. NET-like structures segmented from our biopsies warrant further study as they appear pathologically distinct, and resemble non-lytic, vital NETs. Examination of corresponding H&E regions predominantly placed NET-like structures in glomeruli, including globally and segmentally sclerosed glomeruli, and tubule lumina. Our work continues to explore NET-like structures in LN biopsies by: 1.) revising detection and analytical methods based on evolving NETs definitions, and 2.) cataloguing NET morphology in order to implement supervised classification of NET-like structures in histopathology images.

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.

Background: Protein recoding by RNA editing is required for normal health and evolutionary adaptation. However, de novo induction of RNA editing in response to environmental factors is an uncommon phenomenon. While APOBEC3A edits many mRNAs in monocytes and macrophages in response to hypoxia and interferons, the physiological significance of such editing is unclear., Results: Here, we show that the related cytidine deaminase, APOBEC3G, induces site-specific C-to-U RNA editing in natural killer cells, lymphoma cell lines, and, to a lesser extent, CD8-positive T cells upon cellular crowding and hypoxia. In contrast to expectations from its anti-HIV-1 function, the highest expression of APOBEC3G is shown to be in cytotoxic lymphocytes. RNA-seq analysis of natural killer cells subjected to cellular crowding and hypoxia reveals widespread C-to-U mRNA editing that is enriched for genes involved in mRNA translation and ribosome function. APOBEC3G promotes Warburg-like metabolic remodeling in HuT78 T cells under similar conditions. Hypoxia-induced RNA editing by APOBEC3G can be mimicked by the inhibition of mitochondrial respiration and occurs independently of HIF-1α., Conclusions: APOBEC3G is an endogenous RNA editing enzyme in primary natural killer cells and lymphoma cell lines. This RNA editing is induced by cellular crowding and mitochondrial respiratory inhibition to promote adaptation to hypoxic stress.

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.

Background: We evaluated the overall and site-specific incidence of cancer in subjects with primary immunodeficiency diseases (PIDD) enrolled in the United States Immune Deficiency Network (USIDNET) registry compared with age-adjusted cancer incidence in the Surveillance, Epidemiology and End Results Program (SEER) database., Objective: We hypothesized that subjects with PIDD would have an increased incidence of cancer due to impaired immune function., Methods: Overall and site-specific cancer incidence rates were evaluated in subjects with PIDD (n = 3658) enrolled in the USIDNET registry from 2003 to 2015 and compared with age-adjusted incidence rates in the SEER database., Results: We observed a 1.42-fold excess relative risk of cancer in subjects with PIDD compared with the age-adjusted SEER population (P < .001). Men with PIDD had a 1.91-fold excess relative risk of cancer compared with the age-adjusted male population (P < .001), while women with PIDD had similar overall cancer rates compared with the age-adjusted female population. Of the 4 most common malignancies in men and women in SEER (lung, colon, breast, and prostate cancers), we found no significant increase in these diagnoses in subjects with PIDD. Significant increases in lymphoma in both men (10-fold increase, P < .001) and women (8.34-fold increase, P < .001) with PIDD were observed., Conclusions: Excess incidence of cancer occurred in subjects with PIDD. An excess of lymphoma in specific PIDD populations principally drove this increased incidence, while no increased risk of the most common solid tumor malignancies was observed. These data point to a restricted role of the immune system in protecting from specific cancers., (Copyright © 2017 American Academy of Allergy, Asthma & Immunology. All rights reserved.)

Purpose: Multiple studies have examined the role of anthropometric characteristics in ovarian cancer risk and survival; however, their results have been conflicting. We investigated the associations between weight change, height and height change and risk and outcome of ovarian cancer using data from a large population-based case-control study., Methods: Data from 699 ovarian cancer cases and 1,802 controls who participated in the HOPE study were included. We used unconditional logistic regression adjusted for age, race, number of pregnancies, use of oral contraceptives, and family history of breast or ovarian cancer to examine the associations between self-reported height and weight and height change with ovarian cancer risk. Cox proportional hazards regression models adjusted for age and stage were used to examine the association between the exposure variables and overall and progression-free survival among ovarian cancer cases., Results: We observed an increased risk of ovarian cancer mortality and progression for gaining more than 20 pounds between ages 18-30, HR 1.36; 95% CI 1.05-1.76, and HR 1.31; 95% CI 1.04-1.66, respectively. Losing weight and gaining it back multiple times was inversely associated with both ovarian cancer risk, OR 0.78; 95% CI 0.63-0.97 for 1-4 times and OR 0.73; 95% CI 0.54-0.99 for 5-9 times, and mortality, HR 0.63; 95% CI 0.40-0.99 for 10-14 times. Finally, being taller during adolescence and adulthood was associated with increased risk of mortality. Taller stature and weight gain over lifetime were not related to ovarian cancer risk., Conclusions: Our results suggest that height and weight and their change over time may influence ovarian cancer risk and survival. These findings suggest that biological mechanisms underlying these associations may be hormone driven and may play an important role in relation to ovarian carcinogenesis and tumor progression.