CD39 expression defines exhausted CD4+ T cells associated with poor survival and immune evasion in human gastric cancer.

Objectives: CD4+ T cell helper and regulatory function in human cancers has been well characterised. However, the definition of tumor-infiltrating CD4+ T cell exhaustion and how it contributes to the immune response and disease progression in human gastric cancer (GC) remain largely unknown. Methods: A total of 128 GC patients were enrolled in the study. The expression of CD39 and PD-1 on CD4+ T cells in the different samples was analysed by flow cytometry. GC-infiltrating CD4+ T cell subpopulations based on CD39 expression were phenotypically and functionally assessed. The role of CD39 in the immune response of GC-infiltrating T cells was investigated by inhibiting CD39 enzymatic activity. Results: In comparison with CD4+ T cells from the non-tumor tissues, significantly more GC-infiltrating CD4+ T cells expressed CD39. Most GC-infiltrating CD39+CD4+ T cells exhibited CD45RA-CCR7- effector-memory phenotype expressing more exhaustion-associated inhibitory molecules and transcription factors and produced less TNF-α, IFN-γ and cytolytic molecules than their CD39-CD4+ counterparts. Moreover, ex vivo inhibition of CD39 enzymatic activity enhanced their functional potential reflected by TNF-α and IFN-γ production. Finally, increased percentages of GC-infiltrating CD39+CD4+ T cells were positively associated with disease progression and patients' poorer overall survival. Conclusion: Our study demonstrates that CD39 expression defines GC-infiltrating CD4+ T cell exhaustion and their immunosuppressive function. Targeting CD39 may be a promising therapeutic strategy for treating GC patients.

CD39hi identifies an exhausted tumor-reactive CD8+ T cell population associated with tumor progression in human gastric cancer.

The ectonucleotidase CD39 has been regarded as a promising immune checkpoint in solid tumors. However, the expression of CD39 by tumor-infiltrating CD8+ T cells as well as their potential roles and clinical implications in human gastric cancer (GC) remain largely unknown. Here, we found that GC-infiltrating CD8+ T cells contained a fraction of CD39hi cells that constituted about 6.6% of total CD8+ T cells in tumors. These CD39hi cells enriched for GC-infiltrating CD8+ T cells with features of exhaustion in transcriptional, phenotypic, metabolic and functional profiles. Additionally, GC-infiltrating CD39hiCD8+ T cells were also identified for tumor-reactive T cells, as these cells expanded in vitro were able to recognize autologous tumor organoids and induced more tumor cell apoptosis than those of expanded their CD39int and CD39-CD8+ counterparts. Furthermore, CD39 enzymatic activity controlled GC-infiltrating CD39hiCD8+ T cell effector function, and blockade of CD39 efficiently enhanced their production of cytokines IFN-γ and TNF-α. Finally, high percentages of GC-infiltrating CD39hiCD8+ T cells correlated with tumor progression and independently predicted patients' poor overall survival. These findings provide novel insights into the association of CD39 expression level on CD8+ T cells with their features and potential clinical implications in GC, and empowering those exhausted tumor-reactive CD39hiCD8+ T cells through CD39 inhibition to circumvent the suppressor program may be an attractive therapeutic strategy against GC.

Tumor-infiltrating mast cells stimulate ICOS+ regulatory T cells through an IL-33 and IL-2 axis to promote gastric cancer progression.

INTRODUCTION: In solid tumors, regulatory T cell (Treg) and mast cell perform different roles depending on the microenvironment. Nevertheless, mast cell and Treg-mediated interactions in gastric cancer (GC) are unclear, as are their regulation, function, and clinical significance. OBJECTIVE: The present study demonstrated the mechanism of tumor-infiltrating mast cells stimulating ICOS+ regulatory T cells via the IL-33/IL-2 axis to promote the growth of gastric cancer. METHODS: Analyses of 98 patients with GC were conducted to examine mast cell counts, ICOS+ Tregs, and the levels of IL-33 or IL-2. Isolated ICOS+ Treg and CD8+ T cell were stimulated, cultured and tested for their functional abilities in vitro and in vivo. RESULTS: GC patients exhibited a significantly more production of IL-33 in tumors. Mast cell stimulated by tumor-derived IL-33 exhibited a prolonged lifespan through IL-33 mediated inhibition of apoptosis. Moreover, mast cells stimulated by tumor-derived IL-33 secreted IL-2, which induced Treg expansion. These inducible Tregs displayed an activated immunosuppressive phenotype with positive expression for the inducible T cell co-stimulator (ICOS). In vitro, IL-2 from IL to 33-stimulated mast cells induced increased numbers of ICOS+ Tregs with increased immunosuppressive activity against proliferation and effector function of CD8+ T cell. In vivo, ICOS+ Tregs were treated with anti-IL-2 neutralizing antibody followed by co-injection with CD8+ T cells in GC mouse model, which showed an increased CD8+ T cell infiltration and effector molecules production, meanwhile tumor growth and progression were inhibited. Besides, reduction in GC patient survival was associated with tumor-derived ICOS+ Tregs. CONCLUSION: Our results highlight a crosstalk between GC-infiltrating mast cells and ICOS+ Tregs and provide a novel mechanism describing ICOS+ Treg expansion and induction by an IL-33/mast cell/IL-2 signaling axis in GC, and also provide functional evidence that the modulation of this immunosuppressive pathway can attenuate GC-mediated immune tolerance.

Extracellular vesicles as next generation immunotherapeutics.

Extracellular vesicles (EVs) function as a mode of intercellular communication and molecular transfer to elicit diverse biological/functional response. Accumulating evidence has highlighted that EVs from immune, tumour, stromal cells and even bacteria and parasites mediate the communication of various immune cell types to dynamically regulate host immune response. EVs have an innate capacity to evade recognition, transport and transfer functional components to target cells, with subsequent removal by the immune system, where the immunological activities of EVs impact immunoregulation including modulation of antigen presentation and cross-dressing, immune activation, immune suppression, and immune surveillance, impacting the tumour immune microenvironment. In this review, we outline the recent progress of EVs in immunorecognition and therapeutic intervention in cancer, including vaccine and targeted drug delivery and summarise their utility towards clinical translation. We highlight the strategies where EVs (natural and engineered) are being employed as a therapeutic approach for immunogenicity, tumoricidal function, and vaccine development, termed immuno-EVs. With seminal studies providing significant progress in the sequential development of engineered EVs as therapeutic anti-tumour platforms, we now require direct assessment to tune and improve the efficacy of resulting immune responses - essential in their translation into the clinic. We believe such a review could strengthen our understanding of the progress in EV immunobiology and facilitate advances in engineering EVs for the development of novel EV-based immunotherapeutics as a platform for cancer treatment.

HLA-A*11:01-restricted CD8+ T cell immunity against influenza A and influenza B viruses in Indigenous and non-Indigenous people.

HLA-A*11:01 is one of the most prevalent human leukocyte antigens (HLAs), especially in East Asian and Oceanian populations. It is also highly expressed in Indigenous people who are at high risk of severe influenza disease. As CD8+ T cells can provide broadly cross-reactive immunity to distinct influenza strains and subtypes, including influenza A, B and C viruses, understanding CD8+ T cell immunity to influenza viruses across prominent HLA types is needed to rationally design a universal influenza vaccine and generate protective immunity especially for high-risk populations. As only a handful of HLA-A*11:01-restricted CD8+ T cell epitopes have been described for influenza A viruses (IAVs) and epitopes for influenza B viruses (IBVs) were still unknown, we embarked on an epitope discovery study to define a CD8+ T cell landscape for HLA-A*11:01-expressing Indigenous and non-Indigenous Australian people. Using mass-spectrometry, we identified IAV- and IBV-derived peptides presented by HLA-A*11:01 during infection. 79 IAV and 57 IBV peptides were subsequently screened for immunogenicity in vitro with peripheral blood mononuclear cells from HLA-A*11:01-expressing Indigenous and non-Indigenous Australian donors. CD8+ T cell immunogenicity screening revealed two immunogenic IAV epitopes (A11/PB2320-331 and A11/PB2323-331) and the first HLA-A*11:01-restricted IBV epitopes (A11/M41-49, A11/NS1186-195 and A11/NP511-520). The immunogenic IAV- and IBV-derived peptides were >90% conserved among their respective influenza viruses. Identification of novel immunogenic HLA-A*11:01-restricted CD8+ T cell epitopes has implications for understanding how CD8+ T cell immunity is generated towards IAVs and IBVs. These findings can inform the development of rationally designed, broadly cross-reactive influenza vaccines to ensure protection from severe influenza disease in HLA-A*11:01-expressing individuals.

Distribution, phenotype, functional and clinical relevance of CD8+CD103+ tissue-resident memory T cells in human gastric cancer.

CD8+CD103+ tissue-resident memory T cells (TRMs) are involved in tumor immune response and linked to favorable clinical outcome in human cancer. However, the distribution, phenotype, functional properties and clinical relevance of these cells in gastric cancer (GC) remain elusive. Here, our data show that, in comparison to non-tumor tissues, the percentages of CD8+CD103+ TRMs in tumors are significantly decreased. Most tumor-infiltrating CD8+CD103+ TRMs are CD45RA-CCR7- effector-memory cells with higher PD-1 and 4-1BB expression than those from non-tumor tissues. Further, tumor-infiltrating CD8+CD103+ TRMs show impaired cytolytic capacity due to decreased granzyme B and perforin expression. Moreover, ex vivo PD-1 blockade could restore the cytolytic capacity of tumor-infiltrating CD8+CD103+ TRMs, and such anti-PD-1-mediated reinvigoration of CD8+CD103+ TRMs could be further enhanced by 4-1BB co-stimulation. Finally, lower levels of Tumor-infiltrating CD8+CD103+ TRMs are positively correlated with GC progression and poor patients' survival. Our data suggest that restoring CD8+CD103+ TRM function by combining PD-1 blockade and 4-1BB co-stimulation may be a promising strategy for treating GC.

The Antimalaria Drug Artesunate Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication by Activating AMPK and Nrf2/HO-1 Signaling Pathways.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the pork industry worldwide. Currently, vaccine strategies provide limited protection against PRRSV transmission, and no effective drug is commercially available. Therefore, there is an urgent need to develop novel antiviral strategies to prevent PRRSV pandemics. This study showed that artesunate (AS), one of the antimalarial drugs, potently suppressed PRRSV replication in Marc-145 cells and ex vivo primary porcine alveolar macrophages (PAMs) at micromolar concentrations. Furthermore, we demonstrated that this suppression was closely associated with AS-activated AMPK (energy homeostasis) and Nrf2/HO-1 (inflammation) signaling pathways. AS treatment promoted p-AMPK, Nrf2, and HO-1 expression and, thus, inhibited PRRSV replication in Marc-145 and PAM cells in a time- and dose-dependent manner. These effects of AS were reversed when the AMPK or HO-1 gene was silenced by short interfering RNA. In addition, we demonstrated that AMPK works upstream of Nrf2/HO-1, as its activation by AS is AMPK dependent. Adenosine phosphate analysis showed that AS activates AMPK via improving the AMP/ADP-to-ATP ratio rather than direct interaction with AMPK. Altogether, our findings indicate that AS is a promising novel therapeutic for controlling PRRSV and that its anti-PRRSV mechanism, which involves the functional link between energy homeostasis and inflammation suppression pathways, may provide opportunities for developing novel antiviral agents. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) infections have continuously threatened the pork industry worldwide. Vaccination strategies provide very limited protection against PRRSV infection, and no effective drug is commercially available. We show that artesunate (AS), one of the antimalarial drugs, is a potent inhibitor against PRRSV replication in Marc-145 cells and ex vivo primary porcine alveolar macrophages (PAMs). Furthermore, we demonstrate that AS inhibits PRRSV replication via activation of AMPK-dependent Nrf2/HO-1 signaling pathways, revealing a novel link between energy homeostasis (AMPK) and inflammation suppression (Nrf2/HO-1) during viral infection. Therefore, we believe that AS may be a promising novel therapeutics for controlling PRRSV, and its anti-PRRSV mechanism may provide a strategy to develop novel antiviral agents.

FasL+ PD-L2+ Identifies a Novel Immunosuppressive Neutrophil Population in Human Gastric Cancer That Promotes Disease Progression.

Neutrophils constitute abundant cellular components in human gastric cancer (GC) tissues, but their protumorigenic subset in pathogenesis of GC progression is unclear. Here, it is found that patients with GC show significantly higher neutrophil infiltration in tumors that is regulated by CXCL12-CXCR4 chemotaxis. These tumor-infiltrating neutrophils express high level immunosuppressive molecules FasL and PD-L2, and this FasL+ PD-L2+ neutrophil subset with a unique phenotype constitutes at least 20% of all neutrophils in advanced GC and predicts poor patient survival. Tumor induces neutrophils to express FasL and PD-L2 proteins with similar phenotype to those in GC tumors in both time-dependent and dose-dependent manners. Mechanistically, Th17 cell-derived IL-17A and tumor cell-derived G-CSF can significantly induce neutrophil FasL and PD-L2 expression via activating ERK-NF-κB and JAK-STAT3 signaling pathway, respectively. Importantly, upon over-expressing FasL and PD-L2, neutrophils acquire immunosuppressive functions on tumor-specific CD8+ T-cells and promote the growth and progression of human GC tumors in vitro and in vivo, which can be reversed by blocking FasL and PD-L2 on these neutrophils. Thus, the work identifies a novel protumorigenic FasL+ PD-L2+ neutrophil subset in GC and provides new insights for human cancer immunosuppression and anti-cancer therapies targeting these pathogenic cells.

CD8+ T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope cross-react with selective seasonal coronaviruses.

Efforts are being made worldwide to understand the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the coronavirus disease 2019 (COVID-19) pandemic, including the impact of T cell immunity and cross-recognition with seasonal coronaviruses. Screening of SARS-CoV-2 peptide pools revealed that the nucleocapsid (N) protein induced an immunodominant response in HLA-B7+ COVID-19-recovered individuals that was also detectable in unexposed donors. A single N-encoded epitope that was highly conserved across circulating coronaviruses drove this immunodominant response. In vitro peptide stimulation and crystal structure analyses revealed T cell-mediated cross-reactivity toward circulating OC43 and HKU-1 betacoronaviruses but not 229E or NL63 alphacoronaviruses because of different peptide conformations. T cell receptor (TCR) sequencing indicated that cross-reactivity was driven by private TCR repertoires with a bias for TRBV27 and a long CDR3ß loop. Our findings demonstrate the basis of selective T cell cross-reactivity for an immunodominant SARS-CoV-2 epitope and its homologs from seasonal coronaviruses, suggesting long-lasting protective immunity.

Influenza A virus infection-induced macroautophagy facilitates MHC class II-restricted endogenous presentation of an immunodominant viral epitope.

CD4+ T cells recognize peptides presented by major histocompatibility complex class II molecules (MHC-II). These peptides are generally derived from exogenous antigens. Macroautophagy has been reported to promote endogenous antigen presentation in viral infections. However, whether influenza A virus (IAV) infection-induced macroautophagy also leads to endogenous antigen presentation through MHC-II is still debated. In this study, we show that IAV infection leads to endogenous presentation of an immunodominant viral epitope NP311-325 by MHC-II to CD4+ T cells. Mechanistically, such MHC-II-restricted endogenous IAV antigen presentation requires de novo protein synthesis as it is inhibited by the protein synthesis inhibitor cycloheximide, and a functional ER-Golgi network as it is totally blocked by Brefeldin A. These results indicate that MHC-II-restricted endogenous IAV antigen presentation is dependent on de novo antigen and/or MHC-II synthesis, and transportation through the ER-Golgi network. Furthermore, such endogenous IAV antigen presentation by MHC-II is enhanced by TAP deficiency, indicating some antigenic peptides are of cytosolic origin. Most importantly, the bulk of such MHC-II-restricted endogenous IAV antigen presentation is blocked by autophagy inhibitors (3-MA and E64d) and deletion of autophagy-related genes, such as Beclin1 and Atg7. We have further demonstrated that in dendritic cells, IAV infection prevents autophagosome-lysosome fusion and promotes autophagosome fusion with MHC class II compartment (MIIC), which likely promotes endogenous IAV antigen presentation by MHC-II. Our results provide strong evidence that IAV infection-induced autophagosome formation facilitates endogenous IAV antigen presentation by MHC-II to CD4+ T cells. The implication for influenza vaccine design is discussed.

L-Plastin Promotes Gastric Cancer Growth and Metastasis in a Helicobacter pylori cagA-ERK-SP1-Dependent Manner.

Actin cytoskeleton dynamic rearrangement is required for tumor cell metastasis and is a key characteristic of Helicobacter pylori (H. pylori)-infected host cells. Actin cytoskeleton modulation is coordinated by multiple actin-binding proteins (ABP). Through Kyoto encyclopedia of gene and genomes database, GEPIA website, and real-time PCR data, we found that H. pylori infection significantly induced L-plastin, a key ABP, in gastric cancer cells. We further explored the regulation and function of L-plastin in H. pylori-associated gastric cancer and found that, mechanistically, H. pylori infection induced gastric cancer cells to express L-plastin via cagA-activated ERK signaling pathway to mediate SP1 binding to L-plastin promoter. Moreover, this increased L-plastin promoted gastric cancer cell proliferation and migration in vitro and facilitated the growth and metastasis of gastric cancer in vivo. Finally, we detected the expression pattern of L-plastin in gastric cancer tissues, and found that L-plastin was increased in gastric cancer tissues and that this increase of L-plastin positively correlated with cagA + H. pylori infection status. Overall, our results elucidate a novel mechanism of L-plastin expression induced by H. pylori, and a new function of L-plastin-facilitated growth and metastasis of gastric cancer, and thereby implicating L-plastin as a potential therapeutic target against gastric cancer. IMPLICATIONS: Our results elucidate a novel mechanism of L-plastin expression induced by H. pylori in gastric cancer, and a new function of L-plastin-facilitated gastric cancer growth and metastasis, implicating L-plastin as a potential therapeutic target against gastric cancer.

Helicobacter pylori-Induced Rev-erbα Fosters Gastric Bacteria Colonization by Impairing Host Innate and Adaptive Defense.

BACKGROUND & AIMS: Rev-erbα represents a powerful transcriptional repressor involved in immunity. However, the regulation, function, and clinical relevance of Rev-erbα in Helicobacter pylori infection are presently unknown. METHODS: Rev-erbα was examined in gastric samples from H pylori-infected patients and mice. Gastric epithelial cells (GECs) were isolated and infected with H pylori for Rev-erbα regulation assays. Gastric tissues from Rev-erbα-/- and wild-type (littermate control) mice or these mice adoptively transferred with CD4+ T cells from IFN-γ-/- and wild-type mice, bone marrow chimera mice and mice with in vivo pharmacological activation or inhibition of Rev-erbα were examined for bacteria colonization. GECs, CD45+CD11c-Ly6G-CD11b+CD68- myeloid cells and CD4+ T cells were isolated, stimulated and/or cultured for Rev-erbα function assays. RESULTS: Rev-erbα was increased in gastric mucosa of H pylori-infected patients and mice. H pylori induced GECs to express Rev-erbα via the phosphorylated cagA that activated ERK signaling pathway to mediate NF-κB directly binding to Rev-erbα promoter, which resulted in increased bacteria colonization within gastric mucosa. Mechanistically, Rev-erbα in GECs not only directly suppressed Reg3b and ß-defensin-1 expression, which resulted in impaired bactericidal effects against H pylori of these antibacterial proteins in vitro and in vivo; but also directly inhibited chemokine CCL21 expression, which led to decreased gastric influx of CD45+CD11c-Ly6G-CD11b+CD68- myeloid cells by CCL21-CCR7-dependent migration and, as a direct consequence, reduced bacterial clearing capacity of H pylori-specific Th1 cell response. CONCLUSIONS: Overall, this study identifies a model involving Rev-erbα, which collectively ensures gastric bacterial persistence by suppressing host gene expression required for local innate and adaptive defense against H pylori.

Upregulation of Tubulointerstitial nephritis antigen like 1 promotes gastric cancer growth and metastasis by regulating multiple matrix metallopeptidase expression.

BACKGROUND AND AIM: Tubulointerstitial nephritis antigen-like 1 (TINAGL1), as a novel matricellular protein, has been demonstrated to participate in cancer progression, whereas the potential function of TINAGL1 in gastric cancer (GC) remains unknown. METHODS: The expression pattern of TINAGL1 in GC was examined by immunohistochemistry, ELISA, real-time polymerase chain reaction, and Western blot. Correlation between TINAGL1 and matrix metalloproteinases (MMPs) was analyzed by the GEPIA website and Kaplan-Meier plots database. The lentivirus-based TINAGL1 knockdown, CCK-8, and transwell assays were used to test the function of TINAGL1 in vitro. The role of TINAGL1 was confirmed by subcutaneous xenograft, abdominal dissemination, and lung metastasis model. Microarray experiments, ELISA, real-time polymerase chain reaction, and Western blot were used to identify molecular mechanism. RESULTS: TINAGL1 was increased in GC tumor tissues and associated with poor patient survival. Moreover, TINAGL1 significantly promoted GC cell proliferation and migration in vitro as well as facilitated GC tumor growth and metastasis in vivo. TINAGL1 expression in GC cells was accompanied with increasing MMPs including MMP2, MMP9, MMP11, MMP14, and MMP16. GEPIA database revealed that these MMPs were correlated with TINAGL1 in GC tumors and that the most highly expressed MMP was MMP2. Mechanically, TINAGL1 regulated MMP2 through the JNK signaling pathway activation. CONCLUSIONS: Our data highlight that TINAGL1 promotes GC growth and metastasis and regulates MMP2 expression, indicating that TINAGL1 may serve as a therapeutic target for GC.

Spliced Peptides and Cytokine-Driven Changes in the Immunopeptidome of Melanoma.

Antigen recognition by CD8+ T cells is governed by the pool of peptide antigens presented on the cell surface in the context of HLA class I complexes. Studies have shown not only a high degree of plasticity in the immunopeptidome, but also that a considerable fraction of all presented peptides is generated through proteasome-mediated splicing of noncontiguous regions of proteins to form novel peptide antigens. Here, we used high-resolution mass spectrometry combined with new bioinformatic approaches to characterize the immunopeptidome of melanoma cells in the presence or absence of IFNγ. In total, we identified more than 60,000 peptides from a single patient-derived cell line (LM-MEL-44) and demonstrated that IFNγ induced changes in the peptidome, with an overlap of only approximately 50% between basal and treated cells. Around 6% to 8% of the peptides were identified as cis-spliced peptides, and 2,213 peptides (1,827 linear and 386 cis-spliced peptides) were derived from known melanoma-associated antigens. These peptide antigens were equally distributed between the constitutive- and IFNγ-induced peptidome. We next examined additional HLA-matched patient-derived cell lines to investigate how frequently these peptides were identified and found that a high proportion of both linear and spliced peptides was conserved between individual patient tumors, drawing on data amassing to more than 100,000 peptide sequences. Several of these peptides showed in vitro immunogenicity across multiple patients with melanoma. These observations highlight the breadth and complexity of the repertoire of immunogenic peptides that can be exploited therapeutically and suggest that spliced peptides are a major class of tumor antigens.

Suboptimal SARS-CoV-2-specific CD8+ T cell response associated with the prominent HLA-A*02:01 phenotype.

An improved understanding of human T cell-mediated immunity in COVID-19 is important for optimizing therapeutic and vaccine strategies. Experience with influenza shows that infection primes CD8+ T cell memory to peptides presented by common HLA types like HLA-A2, which enhances recovery and diminishes clinical severity upon reinfection. Stimulating peripheral blood mononuclear cells from COVID-19 convalescent patients with overlapping peptides from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to the clonal expansion of SARS-CoV-2-specific CD8+ and CD4+ T cells in vitro, with CD4+ T cells being robust. We identified two HLA-A*02:01-restricted SARS-CoV-2-specfic CD8+ T cell epitopes, A2/S269-277 and A2/Orf1ab3183-3191 Using peptide-HLA tetramer enrichment, direct ex vivo assessment of A2/S269+CD8+ and A2/Orf1ab3183+CD8+ populations indicated that A2/S269+CD8+ T cells were detected at comparable frequencies (∼1.3 × 10-5) in acute and convalescent HLA-A*02:01+ patients. These frequencies were higher than those found in uninfected HLA-A*02:01+ donors (∼2.5 × 10-6), but low when compared to frequencies for influenza-specific (A2/M158) and Epstein-Barr virus (EBV)-specific (A2/BMLF1280) (∼1.38 × 10-4) populations. Phenotyping A2/S269+CD8+ T cells from COVID-19 convalescents ex vivo showed that A2/S269+CD8+ T cells were predominantly negative for CD38, HLA-DR, PD-1, and CD71 activation markers, although the majority of total CD8+ T cells expressed granzymes and/or perforin. Furthermore, the bias toward naïve, stem cell memory and central memory A2/S269+CD8+ T cells rather than effector memory populations suggests that SARS-CoV-2 infection may be compromising CD8+ T cell activation. Priming with appropriate vaccines may thus be beneficial for optimizing CD8+ T cell immunity in COVID-19.

PD-1 does not mark tumor-infiltrating CD8+ T cell dysfunction in human gastric cancer.

BACKGROUND: Overexpression of programmed cell death protein 1 (PD-1) is linked to CD8+ T cell dysfunction and contributes to tumor immune escape. However, the prevalence and functional regulations of PD-1 expression on CD8+ T cells in human gastric cancer (GC) remain largely unknown. METHODS: Flow cytometry was performed to analyze the level, phenotype, functional and clinical relevance of PD-1+CD8+ T cells in GC patients. Peripheral blood CD8+ T cells were purified and subsequently exposed to culture supernatants from digested primary GC tumor tissues (TSN) in vitro for PD-1 expression and functional assays. Tumor responses to adoptively transferred TSN-stimulated CD8+ T cells or to the TSN-stimulated CD8+ T cell transfer combined with an anti-PD-1 antibody injection were measured in an in vivo xenograft mouse model. RESULTS: GC patients' tumors showed a significantly increased PD-1+CD8+ T cell infiltration. However, these GC-infiltrating PD-1+CD8+ T cells showed equivalent function to their PD-1-CD8+ counterparts and they did not predict tumor progression. High level of transforming growth factor-ß1 (TGF-ß1) in tumors was positively correlated with PD-1+CD8+ T cell infiltration, and in vitro GC-derived TGF-ß1 induced PD-1 expression on CD8+ T cells via Smad3 signaling, whereas Smad2 signaling was involved in GC-derived TGF-ß1-mediated CD8+ T cell dysfunction. Furthermore, GC-derived TGF-ß1-mediated CD8+ T cell dysfunction contributed to tumor growth in vivo that could not be attenuated by PD-1 blockade. CONCLUSIONS: Our data highlight that GC-derived TGF-ß1 promotes PD-1 independent CD8+ T cell dysfunction. Therefore, restoring CD8+ T cell function by a combinational PD-1 and TGF-ß1 blockade might benefit future GC immunotherapy.

Expression of ETS1 in gastric epithelial cells positively regulate inflammatory response in Helicobacter pylori-associated gastritis.

Gastric epithelial cells (GECs) provide the first point of contact of the host by Helicobacter pylori (H. pylori), and the interaction between H. pylori and GECs plays a critical role in H. pylori-associated diseases. Aberrant expression of transcription factors (TFs) contributes to the pathogenesis of inflammatory disorders, including H. pylori-associated gastritis. ETS (E26 transformation specific) transcription factor family is one of the largest families of evolutionarily conserved TFs, regulating critical functions during cell homeostasis. We screened ETS family gene expression in H. pylori-infected mouse and human GECs and found that ETS1 (ETS proto-oncogene 1, transcription factor) expression was highly affected by H. pylori infection. Then, we reported that ETS1 was induced in GECs by H. pylori via cagA activated NF-κB pathway. Notably, we demonstrated that proinflammatory cytokines IL-1ß and TNFα have synergistic effects on ETS1 expression during H. pylori infection in an NF-κB-pathway-dependent manner. RNA-seq assay and Gene-ontology functional analysis revealed that ETS1 positively regulate inflammatory response during H. pylori infection. Increased ETS1 is also detected in the gastric mucosa of mice and patients with H. pylori infection. Collectively, these data showed that ETS1 may play an important role in the pathogenesis of H. pylori-associated gastritis.

Arrestin domain containing 3 promotes Helicobacter pylori-associated gastritis by regulating protease-activated receptor 1.

Arrestin domain containing 3 (ARRDC3) represents a newly discovered α-arrestin involved in obesity, inflammation, and cancer. Here, we demonstrate a proinflammation role of ARRDC3 in Helicobacter pylori-associated gastritis. Increased ARRDC3 was detected in gastric mucosa of patients and mice infected with H. pylori. ARRDC3 in gastric epithelial cells (GECs) was induced by H. pylori, regulated by ERK and PI3K-AKT pathways in a cagA-dependent manner. Human gastric ARRDC3 correlated with the severity of gastritis, and mouse ARRDC3 from non-BM-derived cells promoted gastric inflammation. This inflammation was characterized by the CXCR2-dependent influx of CD45+CD11b+Ly6C-Ly6G+ neutrophils, whose migration was induced via the ARRDC3-dependent production of CXCL2 by GECs. Importantly, gastric inflammation was attenuated in Arrdc3-/- mice but increased in protease-activated receptor 1-/- (Par1-/-) mice. Mechanistically, ARRDC3 in GECs directly interacted with PAR1 and negatively regulated PAR1 via ARRDC3-mediated lysosomal degradation, which abrogated the suppression of CXCL2 production and following neutrophil chemotaxis by PAR1, thereby contributing to the development of H. pylori-associated gastritis. This study identifies a regulatory network involving H. pylori, GECs, ARRDC3, PAR1, and neutrophils, which collectively exert a proinflammatory effect within the gastric microenvironment. Efforts to inhibit this ARRDC3-dependent pathway may provide valuable strategies in treating of H. pylori-associated gastritis.

Results of a randomized, double-blind phase II clinical trial of NY-ESO-1 vaccine with ISCOMATRIX adjuvant versus ISCOMATRIX alone in participants with high-risk resected melanoma.

BACKGROUND: To compare the clinical efficacy of New York Esophageal squamous cell carcinoma-1 (NY-ESO-1) vaccine with ISCOMATRIX adjuvant versus ISCOMATRIX alone in a randomized, double-blind phase II study in participants with fully resected melanoma at high risk of recurrence. METHODS: Participants with resected stage IIc, IIIb, IIIc and IV melanoma expressing NY-ESO-1 were randomized to treatment with three doses of NY-ESO-1/ISCOMATRIX or ISCOMATRIX adjuvant administered intramuscularly at 4-week intervals, followed by a further dose at 6 months. Primary endpoint was the proportion free of relapse at 18 months in the intention-to-treat (ITT) population and two per-protocol populations. Secondary endpoints included relapse-free survival (RFS) and overall survival (OS), safety and NY-ESO-1 immunity. RESULTS: The ITT population comprised 110 participants, with 56 randomized to NY-ESO-1/ISCOMATRIX and 54 to ISCOMATRIX alone. No significant toxicities were observed. There were no differences between the study arms in relapses at 18 months or for median time to relapse; 139 vs 176 days (p=0.296), or relapse rate, 27 (48.2%) vs 26 (48.1%) (HR 0.913; 95% CI 0.402 to 2.231), respectively. RFS and OS were similar between the study arms. Vaccine recipients developed strong positive antibody responses to NY-ESO-1 (p≤0.0001) and NY-ESO-1-specific CD4+ and CD8+ responses. Biopsies following relapse did not demonstrate differences in NY-ESO-1 expression between the study populations although an exploratory study demonstrated reduced (NY-ESO-1)+/Human Leukocyte Antigen (HLA) class I+ double-positive cells in biopsies from vaccine recipients performed on relapse in 19 participants. CONCLUSIONS: The vaccine was well tolerated, however, despite inducing antigen-specific immunity, it did not affect survival endpoints. Immune escape through the downregulation of NY-ESO-1 and/or HLA class I molecules on tumor may have contributed to relapse.

Upexpression of BHLHE40 in gastric epithelial cells increases CXCL12 production through interaction with p-STAT3 in Helicobacter pylori-associated gastritis.

BHLHE40, a member of the basic helix-loop-helix transcription factor family, has been reported to play an important role in inflammatory diseases. However, the regulation and function of BHLHE40 in Helicobacter pylori (H pylori)-associated gastritis is unknown. We observed that gastric BHLHE40 was significantly elevated in patients and mice with H pylori infection. Then, we demonstrate that H pylori-infected GECs express BHLHE40 via cagA-ERK pathway. BHLHE40 translocates to cell nucleus, and then binds to cagA protein-activated p-STAT3 (Tyr705). The complex increases chemotactic factor CXCL12 expression (production). Release of CXCL12 from GECs fosters CD4+ T cell infiltration in the gastric mucosa. Our results identify the cagA-BHLHE40-CXCL12 axis that contributes to inflammatory response in gastric mucosa during H pylori infection.