T-cell expression of CXCL13 is associated with immunotherapy response in a sex-dependent manner in patients with lung cancer.

Emerging evidence in preclinical models demonstrates that antitumor immunity is not equivalent between males and females. However, more investigation in patients and across a wider range of cancer types is needed to fully understand sex as a variable in tumor immune responses. We investigated differences in T-cell responses between male and female patients with lung cancer by performing sex-based analysis of single cell transcriptomic datasets. We found that the transcript encoding CXC motif chemokine ligand 13 (CXCL13), which has recently been shown to correlate with T-cell tumor specificity, is expressed at greater levels in T cells isolated from female compared to male patients. Furthermore, increased expression of CXCL13 was associated with response to PD-1-targeting immunotherapy in female but not male patients. These findings suggest that there are sex-based differences in T-cell function required for response to anti-PD-1 therapy in lung cancer that may need to be considered during patient treatment decisions.

Obesity-related T cell dysfunction impairs immunosurveillance and increases cancer risk.

Obesity is a well-established risk factor for human cancer, yet the underlying mechanisms remain elusive. Immune dysfunction is commonly associated with obesity but whether compromised immune surveillance contributes to cancer susceptibility in individuals with obesity is unclear. Here we use a mouse model of diet-induced obesity to investigate tumor-infiltrating CD8 + T cell responses in lean, obese, and previously obese hosts that lost weight through either dietary restriction or treatment with semaglutide. While both strategies reduce body mass, only dietary intervention restores T cell function and improves responses to immunotherapy. In mice exposed to a chemical carcinogen, obesity-related immune dysfunction leads to higher incidence of sarcoma development. However, impaired immunoediting in the obese environment enhances tumor immunogenicity, making the malignancies highly sensitive to immunotherapy. These findings offer insight into the complex interplay between obesity, immunity and cancer, and provide explanation for the obesity paradox observed in clinical immunotherapy settings.

Momordicine-I Suppresses Head and Neck Cancer Growth by Reprogrammimg Immunosuppressive Effect of the Tumor-Infiltrating Macrophages and B Lymphocytes.

Head and neck cancer (HNC) is prevalent worldwide, and treatment options are limited. Momordicine-I (M-I), a natural component from bitter melon, shows antitumor activity against these cancers, but its mechanism of action, especially in the tumor microenvironment (TME), remains unclear. In this study, we establish that M-I reduces HNC tumor growth in two different immunocompetent mouse models using MOC2 and SCC VII cells. We demonstrate that the anticancer activity results from modulating several molecules in the monocyte/macrophage clusters in CD45+ populations in MOC2 tumors by single-cell RNA sequencing. Tumor-associated macrophages (TAM) often pose a barrier to antitumor effects, but following M-I treatment, we observe a significant reduction in the expression of Sfln4, a myeloid cell differentiation factor, and Cxcl3, a neutrophil chemoattractant, in the monocyte/macrophage populations. We further find that the macrophages must be in close contact with the tumor cells to inhibit Sfln4 and Cxcl3, suggesting that these TAMs are impacted by M-I treatment. Coculturing macrophages with tumor cells shows inhibition of Agr1 expression following M-I treatment, which is indicative of switching from M2 to M1 phenotype. Furthermore, the total B-cell population in M-I-treated tumors is significantly lower, whereas spleen cells also show similar results when cocultured with MOC2 cells. M-I treatment also inhibits PD1, PD-L1, and FoxP3 expression in tumors. Collectively, these results uncover the potential mechanism of M-I by modulating immune cells, and this new insight can help to develop M-I as a promising candidate to treat HNCs, either alone or as adjuvant therapy.

SOX9 Governs Gastric Mucous Neck Cell Identity and Is Required for Injury-Induced Metaplasia.

BACKGROUND & AIMS: Acute and chronic gastric injury induces alterations in differentiation within the corpus of the stomach called pyloric metaplasia. Pyloric metaplasia is characterized by the death of parietal cells and reprogramming of mitotically quiescent zymogenic chief cells into proliferative, mucin-rich spasmolytic polypeptide-expressing metaplasia (SPEM) cells. Overall, pyloric metaplastic units show increased proliferation and specific expansion of mucous lineages, both by proliferation of normal mucous neck cells and recruitment of SPEM cells. Here, we identify Sox9 as a potential gene of interest in the regulation of mucous neck and SPEM cell identity in the stomach. METHODS: We used immunostaining and electron microscopy to characterize the expression pattern of SRY-box transcription factor 9 (SOX9) during murine gastric development, homeostasis, and injury in homeostasis, after genetic deletion of Sox9 and after targeted genetic misexpression of Sox9 in the gastric epithelium and chief cells. RESULTS: SOX9 is expressed in all early gastric progenitors and strongly expressed in mature mucous neck cells with minor expression in the other principal gastric lineages during adult homeostasis. After injury, strong SOX9 expression was induced in the neck and base of corpus units in SPEM cells. Adult corpus units derived from Sox9-deficient gastric progenitors lacked normal mucous neck cells. Misexpression of Sox9 during postnatal development and adult homeostasis expanded mucous gene expression throughout corpus units including within the chief cell zone in the base. Sox9 deletion specifically in chief cells blunts their reprogramming into SPEM. CONCLUSIONS: Sox9 is a master regulator of mucous neck cell differentiation during gastric development. Sox9 also is required for chief cells to fully reprogram into SPEM after injury.

Single-cell analysis of peripheral CD8+ T cell responses in patients receiving checkpoint blockade immunotherapy for cancer.

Checkpoint blockade immunotherapy has become a first-line treatment option for cancer patients, with success in increasingly diverse cancer types. Still, many patients do not experience durable responses and the reasons for clinical success versus failure remain largely undefined. Investigation of immune responses within the tumor microenvironment can be highly informative but access to tumor tissue is not always available, highlighting the need to identify biomarkers in the blood that correlate with clinical success. Here, we used single-cell RNA sequencing coupled with T cell receptor sequencing to define CD8+ T cell responses in peripheral blood of two patients with melanoma before and after immunotherapy with either anti-PD-1 (nivolumab) alone or the combination of anti-PD-1 and CTLA-4 (ipilimumab). Both treatment regimens increased transcripts associated with cytolytic effector function and decreased transcripts associated with naive T cells. These responses were further evaluated at the protein level and extended to a total of 53 patients with various cancer types. Unexpectedly, the induction of CD8+ T cell responses associated with cytolytic function was variable and did not predict therapeutic success in this larger patient cohort. Rather, a decrease in the frequency of T cells with a naive-like phenotype was consistently observed after immunotherapy and correlated with prolonged patient survival. In contrast, a more detailed clonotypic analysis of emerging and expanding CD8+ T cells in the blood revealed that a majority of individual T cell clones responding to immunotherapy acquired a transcriptional profile consistent with cytolytic effector function. These results suggest that responses to checkpoint blockade immunotherapy are evident and traceable in patients' blood, with outcomes predicted by the simultaneous loss of naive-like CD8+ T cells and the expansion of mostly rare and diverse cytotoxic CD8+ T cell clones.

Discovering Immune-Mediated Mechanisms of Gastric Carcinogenesis Through Single-Cell RNA Sequencing.

Single-cell RNA sequencing (scRNAseq) technology is still relatively new in the field of gastric cancer immunology but gaining significant traction. This technology now provides unprecedented insights into the intratumoral and intertumoral heterogeneities at the immunological, cellular, and molecular levels. Within the last few years, a volume of publications reported the usefulness of scRNAseq technology in identifying thus far elusive immunological mechanisms that may promote and impede gastric cancer development. These studies analyzed datasets generated from primary human gastric cancer tissues, metastatic ascites fluid from gastric cancer patients, and laboratory-generated data from in vitro and in vivo models of gastric diseases. In this review, we overview the exciting findings from scRNAseq datasets that uncovered the role of critical immune cells, including T cells, B cells, myeloid cells, mast cells, ILC2s, and other inflammatory stromal cells, like fibroblasts and endothelial cells. In addition, we also provide a synopsis of the initial scRNAseq findings on the interesting epithelial cell responses to inflammation. In summary, these new studies have implicated roles for T and B cells and subsets like NKT cells in tumor development and progression. The current studies identified diverse subsets of macrophages and mast cells in the tumor microenvironment, however, additional studies to determine their roles in promoting cancer growth are needed. Some groups specifically focus on the less prevalent ILC2 cell type that may contribute to early cancer development. ScRNAseq analysis also reveals that stromal cells, e.g., fibroblasts and endothelial cells, regulate inflammation and promote metastasis, making them key targets for future investigations. While evaluating the outcomes, we also highlight the gaps in the current findings and provide an assessment of what this technology holds for gastric cancer research in the coming years. With scRNAseq technology expanding rapidly, we stress the need for periodic review of the findings and assess the available scRNAseq analytical tools to guide future work on immunological mechanisms of gastric carcinogenesis. .

IL13 Acts Directly on Gastric Epithelial Cells to Promote Metaplasia Development During Chronic Gastritis.

BACKGROUND & AIMS: It is well established that chronic inflammation promotes gastric cancer-associated metaplasia, but little is known regarding the mechanisms by which immune cells and cytokines regulate metaplastic cellular changes. The goals of this study were to identify interleukin 13 (IL13)-producing immune cells, determine the gastric epithelial cell response(s) to IL13, and establish the role(s) of IL13 in metaplasia development. METHODS: Experiments used an established mouse model of autoimmune gastritis (TxA23), TxA23×Il4ra-/- mice, which develop gastritis but do not express the IL4/IL13-receptor subunit IL4Rα, and TxA23×Il13-Yfp mice, which express yellow fluorescent protein in IL13-producing cells. Flow cytometry was used to measure IL13 secretion and identify IL13-producing immune cells. Mouse and human gastric organoids were cultured with IL13 to determine epithelial cell response(s) to IL13. Single-cell RNA sequencing was performed on gastric epithelial cells from healthy and inflamed mouse stomachs. Mice with gastritis were administered IL13-neutralizing antibodies and stomachs were analyzed by histopathology and immunofluorescence. RESULTS: We identified 6 unique subsets of IL13-producing immune cells in the inflamed stomach. Organoid cultures showed that IL13 acts directly on gastric epithelium to induce a metaplastic phenotype. IL4Rα-deficient mice did not progress to metaplasia. Single-cell RNA sequencing determined that gastric epithelial cells from IL4Rα-deficient mice up-regulated inflammatory genes but failed to up-regulate metaplasia-associated transcripts. Neutralization of IL13 significantly reduced and reversed metaplasia development in mice with gastritis. CONCLUSIONS: IL13 is made by a variety of immune cell subsets during chronic gastritis and promotes gastric cancer-associated metaplastic epithelial cell changes. Neutralization of IL13 reduces metaplasia severity during chronic gastritis.

Two Distinct Etiologies of Gastric Cancer: Infection and Autoimmunity.

Gastric cancer is a leading cause of mortality worldwide. The risk of developing gastric adenocarcinoma, which comprises >90% of gastric cancers, is multifactorial, but most associated with Helicobacter pylori infection. Autoimmune gastritis is a chronic autoinflammatory syndrome where self-reactive immune cells are activated by gastric epithelial cell autoantigens. This cause of gastritis is more so associated with the development of neuroendocrine tumors. However, in both autoimmune and infection-induced gastritis, high risk metaplastic lesions develop within the gastric mucosa. This warrants concern for carcinogenesis in both inflammatory settings. There are many similarities and differences in disease progression between these two etiologies of chronic gastritis. Both diseases have an increased risk of gastric adenocarcinoma development, but each have their own unique comorbidities. Autoimmune gastritis is a primary cause of pernicious anemia, whereas chronic infection typically causes gastrointestinal ulceration. Both immune responses are driven by T cells, primarily CD4+ T cells of the IFN-γ producing, Th1 phenotype. Neutrophilic infiltrates help clear H. pylori infection, but neutrophils are not necessarily recruited in the autoimmune setting. There have also been hypotheses that infection with H. pylori initiates autoimmune gastritis, but the literature is far from definitive with evidence of infection-independent autoimmune gastric disease. Gastric cancer incidence is increasing among young women in the United States, a population at higher risk of developing autoimmune disease, and H. pylori infection rates are falling. Therefore, a better understanding of these two chronic inflammatory diseases is needed to identify their roles in initiating gastric cancer.

Mast Cells as Important Regulators in Autoimmunity and Cancer Development.

Mast cells are an essential part of the immune system and are best known as important modulators of allergic and anaphylactic immune responses. Upon activation, mast cells release a multitude of inflammatory mediators with various effector functions that can be both protective and damage-inducing. Mast cells can have an anti-inflammatory or pro-inflammatory immunological effect and play important roles in regulating autoimmune diseases including rheumatoid arthritis, type 1 diabetes, and multiple sclerosis. Importantly, chronic inflammation and autoimmunity are linked to the development of specific cancers including pancreatic cancer, prostate cancer, colorectal cancer, and gastric cancer. Inflammatory mediators released from activated mast cells regulate immune responses and promote vascular permeability and the recruitment of immune cells to the site of inflammation. Mast cells are present in increased numbers in tissues affected by autoimmune diseases as well as in tumor microenvironments where they co-localize with T regulatory cells and T effector cells. Mast cells can regulate immune responses by expressing immune checkpoint molecules on their surface, releasing anti-inflammatory cytokines, and promoting vascularization of solid tumor sites. As a result of these immune modulating activities, mast cells have disease-modifying roles in specific autoimmune diseases and cancers. Therefore, determining how to regulate the activities of mast cells in different inflammatory and tumor microenvironments may be critical to discovering potential therapeutic targets to treat autoimmune diseases and cancer.

Th17 Cells Provide Mucosal Protection against Gastric Trypanosoma cruzi Infection.

Trypanosoma cruzi is the intracellular parasite of Chagas disease, a chronic condition characterized by cardiac and gastrointestinal morbidity. Protective immunity requires CD4+ T cells, and Th1 cells and gamma interferon (IFN-γ) are important players in host defense. More recently, Th17 cells and interleukin 17A (IL-17A) have been shown to exert protective functions in systemic T. cruzi infection. However, it remains unclear whether Th17 cells and IL-17A protect in the mucosa, the initial site of parasite invasion in many human cases. We found that IL-17RA knockout (KO) mice are highly susceptible to orogastric infection, indicating an important function for this cytokine in mucosal immunity to T. cruzi. To investigate the specific role of Th17 cells for mucosal immunity, we reconstituted RAG1 KO mice with T. cruzi-specific T cell receptor transgenic Th17 cells prior to orogastric T. cruzi challenges. We found that Th17 cells provided protection against gastric mucosal T. cruzi infection, indicated by significantly lower stomach parasite burdens. In vitro macrophage infection assays revealed that protection by Th17 cells is reduced with IL-17A neutralization or reversed by loss of macrophage NADPH oxidase activity. Consistently with this, mice lacking functional NADPH oxidase were not protected by Th17 cell transfer. These data are the first report that Th17 cells protect against mucosal T. cruzi infection and identify a novel protective mechanism involving the induction of NADPH oxidase activity by IL-17A. These studies provide important insights for Chagas vaccine development and, more broadly, increase our understanding of the diverse roles of Th17 cells in host defense.

Fructose Promotes Cytoprotection in Melanoma Tumors and Resistance to Immunotherapy.

Checkpoint blockade immunotherapy relies on the empowerment of the immune system to fight cancer. Why some patients fail to achieve durable clinical responses is not well understood, but unique individual factors such as diet, obesity, and related metabolic syndrome could play a role. The link between obesity and patient outcomes remains controversial and has been mired by conflicting reports and limited mechanistic insight. We addressed this in a C57BL/6 mouse model of diet-induced obesity using a Western diet high in both fats and sugars. Obese mice bearing B16 melanoma or MC38 carcinoma tumors had impaired immune responses to immunotherapy and a reduced capacity to control tumor progression. Unexpectedly, these compromised therapeutic outcomes were independent of body mass and, instead, were directly attributed to dietary fructose. Melanoma tumors in mice on the high-fructose diet were resistant to immunotherapy and showed increased expression of the cytoprotective enzyme heme oxygenase-1 (HO-1). This increase in HO-1 protein was recapitulated in human A375 melanoma cells exposed to fructose in culture. Induced expression of HO-1 shielded tumor cells from immune-mediated killing and was critical for resistance to checkpoint blockade immunotherapy, which could be overcome in vivo using a small-molecule inhibitor of HO-1. This study reveals dietary fructose as a driver of tumor immune evasion, identifying HO-1 expression as a mechanism of resistance and a promising molecular target for combination cancer immunotherapy.See article by Khojandi et al., p. 214.

Single-Cell Transcriptional Analyses Identify Lineage-Specific Epithelial Responses to Inflammation and Metaplastic Development in the Gastric Corpus.

BACKGROUND & AIMS: Chronic atrophic gastritis can lead to gastric metaplasia and increase risk of gastric adenocarcinoma. Metaplasia is a precancerous lesion associated with an increased risk for carcinogenesis, but the mechanism(s) by which inflammation induces metaplasia are poorly understood. We investigated transcriptional programs in mucous neck cells and chief cells as they progress to metaplasia mice with chronic gastritis. METHODS: We analyzed previously generated single-cell RNA-sequencing (scRNA-seq) data of gastric corpus epithelium to define transcriptomes of individual epithelial cells from healthy BALB/c mice (controls) and TxA23 mice, which have chronically inflamed stomachs with metaplasia. Chronic gastritis was induced in B6 mice by Helicobacter pylori infection. Gastric tissues from mice and human patients were analyzed by immunofluorescence to verify findings at the protein level. Pseudotime trajectory analysis of scRNA-seq data was used to predict differentiation of normal gastric epithelium to metaplastic epithelium in chronically inflamed stomachs. RESULTS: Analyses of gastric epithelial transcriptomes revealed that gastrokine 3 (Gkn3) mRNA is a specific marker of mouse gastric corpus metaplasia (spasmolytic polypeptide expressing metaplasia, SPEM). Gkn3 mRNA was undetectable in healthy gastric corpus; its expression in chronically inflamed stomachs (from TxA23 mice and mice with Helicobacter pylori infection) identified more metaplastic cells throughout the corpus than previously recognized. Staining of healthy and diseased human gastric tissue samples paralleled these results. Although mucous neck cells and chief cells from healthy stomachs each had distinct transcriptomes, in chronically inflamed stomachs, these cells had distinct transcription patterns that converged upon a pre-metaplastic pattern, which lacked the metaplasia-associated transcripts. Finally, pseudotime trajectory analysis confirmed the convergence of mucous neck cells and chief cells into a pre-metaplastic phenotype that ultimately progressed to metaplasia. CONCLUSIONS: In analyses of tissues from chronically inflamed stomachs of mice and humans, we expanded the definition of gastric metaplasia to include Gkn3 mRNA and GKN3-positive cells in the corpus, allowing a more accurate assessment of SPEM. Under conditions of chronic inflammation, chief cells and mucous neck cells are plastic and converge into a pre-metaplastic cell type that progresses to metaplasia.

Interleukin 27 Protects From Gastric Atrophy and Metaplasia During Chronic Autoimmune Gastritis.

BACKGROUND & AIMS: The association between chronic inflammation and gastric carcinogenesis is well established, but it is not clear how immune cells and cytokines regulate this process. We investigated the role of interleukin 27 (IL27) in the development of gastric atrophy, hyperplasia, and metaplasia (preneoplastic lesions associated with inflammation-induced gastric cancer) in mice with autoimmune gastritis. METHODS: We performed studies with TxA23 mice (control mice), which express a T-cell receptor against the H+/K+ adenosine triphosphatase α chain and develop autoimmune gastritis, and TxA23xEbi3-/- mice, which develop gastritis but do not express IL27. In some experiments, mice were given high-dose tamoxifen to induce parietal cell atrophy and spasmolytic polypeptide-expressing metaplasia (SPEM). Recombinant IL27 was administered to mice with mini osmotic pumps. Stomachs were collected and analyzed by histopathology and immunofluorescence; we used flow cytometry to measure IL27 and identify immune cells that secrete IL27 in the gastric mucosa. Single-cell RNA sequencing was performed on immune cells that infiltrated stomach tissues. RESULTS: We identified IL27-secreting macrophages and dendritic cell in the corpus of mice with chronic gastritis (TxA23 mice). Mice deficient in IL27 developed more severe gastritis, atrophy, and SPEM than control mice. Administration of recombinant IL27 significantly reduced the severity of inflammation, atrophy, and SPEM in mice with gastritis. Single-cell RNA sequencing showed that IL27 acted almost exclusively on stomach-infiltrating CD4+ T cells to suppress expression of inflammatory genes. CONCLUSIONS: In studies of mice with autoimmune gastritis, we found that IL27 is an inhibitor of gastritis and SPEM, suppressing CD4+ T-cell-mediated inflammation in the gastric mucosa.

Single-cell transcriptional analyses of spasmolytic polypeptide-expressing metaplasia arising from acute drug injury and chronic inflammation in the stomach.

OBJECTIVE: Spasmolytic polypeptide-expressing metaplasia (SPEM) is a regenerative lesion in the gastric mucosa and is a potential precursor to intestinal metaplasia/gastric adenocarcinoma in a chronic inflammatory setting. The goal of these studies was to define the transcriptional changes associated with SPEM at the individual cell level in response to acute drug injury and chronic inflammatory damage in the gastric mucosa. DESIGN: Epithelial cells were isolated from the gastric corpus of healthy stomachs and stomachs with drug-induced and inflammation-induced SPEM lesions. Single cell RNA sequencing (scRNA-seq) was performed on tissue samples from each of these settings. The transcriptomes of individual epithelial cells from healthy, acutely damaged and chronically inflamed stomachs were analysed and compared. RESULTS: scRNA-seq revealed a population Mucin 6 (Muc6)+gastric intrinsic factor (Gif)+ cells in healthy tissue, but these cells did not express transcripts associated with SPEM. Furthermore, analyses of SPEM cells from drug injured and chronically inflamed corpus yielded two major findings: (1) SPEM and neck cell hyperplasia/hypertrophy are nearly identical in the expression of SPEM-associated transcripts and (2) SPEM programmes induced by drug-mediated parietal cell ablation and chronic inflammation are nearly identical, although the induction of transcripts involved in immunomodulation was unique to SPEM cells in the chronic inflammatory setting. CONCLUSIONS: These data necessitate an expansion of the definition of SPEM to include Tff2+Muc6+ cells that do not express mature chief cell transcripts such as Gif. Our data demonstrate that SPEM arises by a highly conserved cellular programme independent of aetiology and develops immunoregulatory capabilities in a setting of chronic inflammation.

Checkpoint blockade immunotherapy enhances the frequency and effector function of murine tumor-infiltrating T cells but does not alter TCRß diversity.

Checkpoint blockade immunotherapy is now a first-line treatment option for patients with melanoma. Despite achieving objective responses in about half of patients, the exact immune mechanisms elicited and those required for therapeutic success have not been clearly identified. Insight into these mechanisms is key for improving outcomes in a broader range of cancer patients. We used a murine melanoma model to track responses by different subsets of tumor-infiltrating lymphocytes (TIL) during checkpoint blockade immunotherapy. Tumors from treated mice had increased frequencies of both CD4+ and CD8+ T cells, which also showed evidence of functional reinvigoration and elevated effector cytokine production after immunotherapy. We predicted that increased T cell numbers and function within tumors reflected either infiltration by new T cells or clonal expansion by a few high-affinity tumor-reactive T cells. To address this, we compared TIL diversity before and after immunotherapy by sequencing the complementarity determining region 3 (CDR3) of all T cell receptor beta (TCRß) genes. While checkpoint blockade effectively slowed tumor progression and increased T cell frequencies, the diversity of intratumoral T cells remained stable. This was true when analyzing total T cells and when focusing on smaller subsets of effector CD4+ and CD8+ TIL as well as regulatory T cells. Our study suggests that checkpoint blockade immunotherapy does not broaden the T cell repertoire within murine melanoma tumors, but rather expands existing T cell populations and enhances effector capabilities.

Interferon-γ directly induces gastric epithelial cell death and is required for progression to metaplasia.

Chronic inflammation of the gastric mucosa, often caused by autoimmune gastritis and/or infection with Helicobacter pylori, can lead to atrophy of acid-secreting parietal cells with metaplasia of remaining cells. The histological pattern marks a critical step in the progression from chronic gastritis to gastric cancer, yet underlying mechanism(s) of inflammation-induced cell death of gastric epithelial cells are poorly understood. We investigated direct effects of a type 1 cytokine associated with autoimmunity and infection, interferon-γ (IFN-γ), on gastric epithelial cells. IFN-γ was applied to three-dimensional organoid cultures of gastric epithelial cells derived from gastric corpus gland (gastroids) of control and IFN-γ receptor-deficient mice. Gastroids were also treated with supernatants from activated immune cells isolated from a mouse model of autoimmune-mediated atrophic gastritis (TxA23) with and without IFN-γ expression. Finally, histopathological analysis of atrophy and metaplasia severity was performed in TxA23 mice and compared to TxA23 × Ifng-/- mice. Gastric epithelial cells in gastroid cultures expressed IFN-γ receptor in the basolateral membrane, and gastroids died when treated with IFN-γ in an IFN-γ receptor-dependent manner. Supernatants from immune cells containing high levels of IFN-γ were highly toxic to gastroids, and toxicity was tempered when IFN-γ was either neutralized using a monoclonal antibody or when supernatants from Ifng-/- mouse immune cells were used. Finally, TxA23 × Ifng-/- mice showed near-complete abrogation of pre-cancerous histopathological atrophy and metaplasia versus IFN-γ-sufficient controls. We identify IFN-γ as a critical promoter of parietal cell atrophy with metaplasia during the progression of gastritis to gastric atrophy and metaplasia. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Identifying and Tracking Low-Frequency Virus-Specific TCR Clonotypes Using High-Throughput Sequencing.

Tracking antigen-specific T cell responses over time within individuals is difficult because of lack of knowledge of antigen-specific TCR sequences, limitations in sample size, and assay sensitivities. We hypothesized that analyses of high-throughput sequencing of TCR clonotypes could provide functional readouts of individuals' immunological histories. Using high-throughput TCR sequencing, we develop a database of TCRß sequences from large cohorts of mice before (naive) and after smallpox vaccination. We computationally identify 315 vaccine-associated TCR sequences (VATS) that are used to train a diagnostic classifier that distinguishes naive from vaccinated samples in mice up to 9 months post-vaccination with >99% accuracy. We determine that the VATS library contains virus-responsive TCRs by in vitro expansion assays and virus-specific tetramer sorting. These data outline a platform for advancing our capabilities to identify pathogen-specific TCR sequences, which can be used to identify and quantitate low-frequency pathogen-specific TCR sequences in circulation over time with exceptional sensitivity.

CD4+T cells mediate protection against Zika associated severe disease in a mouse model of infection.

Zika virus (ZIKV) has gained worldwide attention since it emerged, and a global effort is underway to understand the correlates of protection and develop diagnostics to identify rates of infection. As new therapeutics and vaccine approaches are evaluated in clinical trials, additional effort is focused on identifying the adaptive immune correlates of protection against ZIKV disease. To aid in this endeavor we have begun to dissect the role of CD4+T cells in the protection against neuroinvasive ZIKV disease. We have identified an important role for CD4+T cells in protection, demonstrating that in the absence of CD4+T cells mice have more severe neurological sequela and significant increases in viral titers in the central nervous system (CNS). The transfer of CD4+T cells from ZIKV immune mice protect type I interferon receptor deficient animals from a lethal challenge; showing that the CD4+T cell response is necessary and sufficient for control of ZIKV disease. Using a peptide library spanning the complete ZIKV polyprotein, we identified both ZIKV-encoded CD4+T cell epitopes that initiate immune responses, and ZIKV specific CD4+T cell receptors that recognize these epitopes. Within the ZIKV antigen-specific TCRß repertoire, we uncovered a high degree of diversity both in response to a single epitope and among different mice responding to a CD4+T cell epitope. Overall this study identifies a novel role for polyfunctional and polyclonal CD4+T cells in providing protection against ZIKV infection and highlights the need for vaccines to develop robust CD4+T cell responses to prevent ZIKV neuroinvasion and limit replication within the CNS.