High Foxp3 Expression Research Articles

PD46-10 SATURATED FATTY ACID PROMOTES PROSTATE CANCER DEVELOPMENT WITH TUMOR INFILTRATING REGULATORY T CELL ALTERATION IN A PTEN-DEFICIENT MOUSE MODEL

You have accessJournal of UrologyCME1 May 2022PD46-10 SATURATED FATTY ACID PROMOTES PROSTATE CANCER DEVELOPMENT WITH TUMOR INFILTRATING REGULATORY T CELL ALTERATION IN A PTEN-DEFICIENT MOUSE MODEL Hiromi Sato, Shintaro Narita, Yoshiko Takahashi, Masanori Ishida, Soki Kasima, Ryohei Yamamoto, Atsushi Koizumi, Taketoshi Nara, Mingguo Huang, Kazuyuki Numakura, Mitsuru Saito, Toshiaki Yoshioka, and Tomonori Habuchi Hiromi SatoHiromi Sato More articles by this author , Shintaro NaritaShintaro Narita More articles by this author , Yoshiko TakahashiYoshiko Takahashi More articles by this author , Masanori IshidaMasanori Ishida More articles by this author , Soki KasimaSoki Kasima More articles by this author , Ryohei YamamotoRyohei Yamamoto More articles by this author , Atsushi KoizumiAtsushi Koizumi More articles by this author , Taketoshi NaraTaketoshi Nara More articles by this author , Mingguo HuangMingguo Huang More articles by this author , Kazuyuki NumakuraKazuyuki Numakura More articles by this author , Mitsuru SaitoMitsuru Saito More articles by this author , Toshiaki YoshiokaToshiaki Yoshioka More articles by this author , and Tomonori HabuchiTomonori Habuchi More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002614.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Recent studies reported that gut microbiota plays an important role in systemic immune response and is involved in cancer development. Herein, using a Pten-deficient mouse model, we assessed the role of systematic and local inflammation, tumor immune microenvironment and gut microbiota profiles on specific-fat diet-induced PCa development. METHODS: Immunocompetent Pten-deficient mice were fed with isocaloric diets that differed in fat composition (lard or fish oil), and were sacrificed at 28 weeks. Using 16S rRNA gene amplicon sequencing, the gut microbiota was profiled for the phylum levels. We performed flow cytometry analyses and quantitative RT-PCR to evaluate the number of prostate infiltrating immune cells and expression of immune cell markers in the prostates of the mice. Antibiotic-treated mice were developed to assess the impact of gut microbiome on fat-diet induced PCa development. RESULTS: The lard diet (LD) -fed mice gained more weight and prostate volume than the fish-oil diet (FOD) -fed mice. We noticed invasive PCa in the LD group (3 of 10 mice) alone, and the Ki67 positivity in the prostates of the LD group was significantly higher than that of the FOD mice (p<0.01). The mean serum LPS level tended to be higher in the LD group (p=0.09), and significantly correlated with mouse weight (p<0.01, r=0.78). The two dietary groups showed markedly different gut microbiota profiles, and the proportion of the Proteobacteria phylum in the LD group was significantly higher than in the FOD group (p=0.02). The mean number of regulatory T cells in the prostates of the LD group was significantly higher than the FOD group (p=0.03). Larger growth of the prostate, higher mean serum LPS level and higher expression of Foxp3 in the prostates of the LD group than those of the FOD group were observed, whereas the microbiota ablation using antibiotics-treated mice abrogated the differences. CONCLUSIONS: LD accelerates PCa development through elevated serum LPS, alteration of gut microbiota and regulatory T cell infiltration in tumor microenvironment in an engineered mouse model of PCa. Immune cell modulation in tumor microenvironment on basis of diet and/or gut microbial changes may be a novel treatment strategy for PCa. Source of Funding: nothing © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e791 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hiromi Sato More articles by this author Shintaro Narita More articles by this author Yoshiko Takahashi More articles by this author Masanori Ishida More articles by this author Soki Kasima More articles by this author Ryohei Yamamoto More articles by this author Atsushi Koizumi More articles by this author Taketoshi Nara More articles by this author Mingguo Huang More articles by this author Kazuyuki Numakura More articles by this author Mitsuru Saito More articles by this author Toshiaki Yoshioka More articles by this author Tomonori Habuchi More articles by this author Expand All Advertisement PDF DownloadLoading ...

Low Activation of CD8+ T Cells in response to Viral Peptides in Mexican Patients with Severe Dengue.

It is acknowledged that antiviral immune response contributes to dengue immunopathogenesis. To identify immunological markers that distinguish dengue fever (DF) and dengue hemorrhagic fever (DHF), 113 patients with confirmed dengue infection were analyzed at 6 or 7 days after fever onset. Peripheral blood mononuclear cells (PBMC) were isolated, lymphocyte subsets and activation biomarkers were identified by flow cytometry, and differentiation of T helper (Th) lymphocytes was achieved by the relative expression analysis of T-bet (Th1), GATA-3 (Th2), ROR-γ (Th17), and FOXP-3 (T regulatory) transcription factors quantified by real-time PCR. CD8+, CD40L+, and CD45+ cells show higher numbers in DF compared to DHF patients, whereas CD4+, CD19+, and CD25+ cells show higher numbers in DHF than DF patients. High expression of GATA-3 accompanied by low expression of T-bet indicates predominance of Th2 response. In addition, higher expression of FOXP-3 and reduced functional cytotoxic T cells (CD8+perforin+) were observed in DHF patients. In further experiments, PBMC were stimulated ex vivo with dengue virus E, NS3, NS4, and NS5 peptides, and proliferating T cell subsets were determined. Lower proliferative responses to NS3 and NS4 peptides and reduced CD8+ cytotoxic T cells were observed in DHF patients. Our results suggest that immune response to dengue is dysregulated with predominance of CD4+ T cells, low activation of Th1 cells, and downregulation of the antiviral cytotoxic activity during severe dengue, likely induced by regulatory T cells.

Negative Immune Checkpoint Protein, VISTA, Regulates the CD4+ Treg Population During Sepsis Progression to Promote Acute Sepsis Recovery and Survival.

Sepsis is a systemic immune response to infection that is responsible for ~35% of in-hospital deaths and over 24 billion dollars in annual treatment costs. Strategic targeting of non-redundant negative immune checkpoint protein pathways can cater therapeutics to the individual septic patient and improve prognosis. B7-CD28 superfamily member V-domain Immunoglobulin Suppressor of T cell Activation (VISTA) is an ideal candidate for strategic targeting in sepsis. We hypothesized that immune checkpoint regulator, VISTA, controls T-regulatory cells (Treg), in response to septic challenge, thus playing a protective role/reducing septic morbidity/mortality. Further, we investigated if changes in morbidity/mortality are due to a Treg-mediated effect during the acute response to septic challenge. To test this, we used the cecal ligation and puncture model as a proxy for polymicrobial sepsis and assessed the phenotype of CD4+ Tregs in VISTA-gene deficient (VISTA-/-) and wild-type mice. We also measured changes in survival, soluble indices of tissue injury, and circulating cytokines in the VISTA-/- and wild-type mice. We found that in wild-type mice, CD4+ Tregs exhibit a significant upregulation of VISTA which correlates with higher Treg abundance in the spleen and small intestine following septic insult. However, VISTA-/- mice have reduced Treg abundance in these compartments met with a higher expression of Foxp3, CTLA4, and CD25 compared to wild-type mice. VISTA-/- mice also have a significant survival deficit, higher levels of soluble indicators of liver injury (i.e., ALT, AST, bilirubin), and increased circulating proinflammatory cytokines (i.e., IL-6, IL-10, TNFα, IL-17F, IL-23, and MCP-1) following septic challenge. To elucidate the role of Tregs in VISTA-/- sepsis mortality, we adoptively transferred VISTA-expressing Tregs into VISTA-/- mice. This adoptive transfer rescued VISTA-/- survival to wild-type levels. Taken together, we propose a protective Treg-mediated role for VISTA by which inflammation-induced tissue injury is suppressed and improves survival in early-stage murine sepsis. Thus, enhancing VISTA expression or adoptively transferring VISTA+ Tregs in early-stage sepsis may provide a novel therapeutic approach to ameliorate inflammation-induced death.

Identification of immune-related cells and genes in the breast invasive carcinoma microenvironment.

The clinical prognosis of breast cancer is closely related to its infiltrating immune status. The study sought to explore tumor-infiltrating immune cells (TILs) and immune-associated genes in the tumor microenvironment of breast invasive carcinoma (BRCA). The ESTIMATE algorithm was used to evaluate the microenvironment of breast cancer patients in TCGA database. The tumor's matrix score and immune score were obtained. The median was divided into two sub groups according to the median of the score, and the correlation between the score and prognosis was also discussed. Differentially expressed genes were screened from two subgroups with high and low score of breast cancer, and the differentially expressed genes were analyzed by GO and KEGG enrichment to explore their possible molecular functions, biological processes, cellular components and signal pathways involved in gene enrichment. It was found that there was a significant correlation between immune score and five-year survival rate, and the high score group had a better prognosis. Macrophage M1 and T cell CD8+ cells were positively related to 5-year overall survival in patients with breast invasive carcinoma. However, Macrophage M2 was negatively related to 5-year overall survival. We also observed that the low expression of four genes (CLEC3A, MCTS1, PDP1 and TCP1,) was related to favorable survival outcomes. High expression of FOXP3, CXCL9, CCR5, CXCR3, and CD37 was related to a high overall survival rate in BRCA. We identified a list of immune – related cells and genes that are useful for Prognostic evaluation and individualized treatment of BRCA.

IL-6 and TNFα Drive Extensive Proliferation of Human Tregs Without Compromising Their Lineage Stability or Function.

Treg therapies are being tested in clinical trials in transplantation and autoimmune diseases, however, the impact of inflammation on Tregs remains controversial. We challenged human Tregs ex-vivo with pro-inflammatory cytokines IL-6 and TNFα and observed greatly enhanced proliferation stimulated by anti-CD3 and anti-CD28 (aCD3/28) beads or CD28 superagonist (CD28SA). The cytokine-exposed Tregs maintained high expression of FOXP3 and HELIOS, demethylated FOXP3 enhancer, and low IFNγ, IL-4, and IL-17 secretion. Blocking TNF receptor using etanercept or deletion of TNF receptor 2 using CRISPR/Cas9 blunted Treg proliferation and attenuated FOXP3 and HELIOS expression. These results prompted us to consider using CD28SA together with IL-6 and TNFα without aCD3/28 beads (beadless) as an alternative protocol for therapeutic Treg manufacturing. Metabolomics profiling revealed more active glycolysis and oxidative phosphorylation, increased energy production, and higher antioxidant potential during beadless Treg expansion. Finally, beadless expanded Tregs maintained suppressive functions in vitro and in vivo. These results demonstrate that human Tregs positively respond to proinflammatory cytokines with enhanced proliferation without compromising their lineage identity or function. This property can be harnessed for therapeutic Treg manufacturing.

Combination Biomarker of Immune Checkpoints Predict Prognosis of Urothelial Carcinoma.

In contrast to Western counties, the incidence of urothelial carcinoma (UC) remains mar-edly elevated in Taiwan. Regulatory T cells (Tregs) play a crucial role in limiting immune responses within the tumor microenvironment. To elucidate the relationship between immune checkpoints in the tumor immune microenvironment and UC progression, we utilize the Gene Expression Omnibus (GEO) to analyze a microarray obtained from 308 patients with UC. We observed that the expression level of CD276 or TIM-3 was positively correlated with late-stage UC and poor prognosis. Patients with simultaneously high CD276 and TIM-3 expression in tumors have significantly reduced both univariate and multivariate survival, indicating that mRNA levels of these immune checkpoints could be independent prognostic biomarkers for UC overall survival and recurrence. Our cohort study showed rare CD8+ cytotoxic T-cells and Tregs infiltration during early-stage UC-known as cold tumors. Approximately 30% of late-stage tumors exhibited highly infiltrated cytotoxic T cells with high PD-1 and FOXP3 expression, which implied that cytotoxic T cells were inhibited in the advanced UC microenvironment. Collectively, our findings provide a better prognosis prediction by combined immune checkpoint biomarkers and a basis for early-stage UC standard treatment to convert cold tumors into hot tumors, followed by immune checkpoint therapy.

AMP-activated protein kinase alpha1 promotes tumor development via FOXP3 elevation in tumor-infiltrating Treg cells

SummaryOverwhelming evidence indicates that infiltration of tumors by Treg cells with elevated levels of FOXP3 suppresses the host antitumor immune response. However, the molecular mechanisms that maintain high expression of FOXP3 in tumor-infiltrating Treg cells remain elusive. Here, we report that AMP-activated protein kinase alpha1 (AMPKα1) enables high FOXP3 expression in tumor-infiltrating Treg cells. Mice with Treg-specific AMPKα1 deletion showed delayed tumor progression and enhanced antitumor T cell immunity. Further experiments showed that AMPKα1 maintains the functional integrity of Treg cells and prevents interferon-γ production in tumor-infiltrating Treg cells. Mechanistically, AMPKα1 maintains the protein stability of FOXP3 in Treg cells by downregulating the expression of E3 ligase CHIP (STUB1). Our results suggest that AMPKα1 activation promotes tumor growth by maintaining FOXP3 stability in tumor-infiltrating Treg cells and that selective inhibition of AMPK in Treg cells might be an effective anti-tumor therapy.

Epigenetic Alteration and its Association With Downregulated FOXP3 Gene in Indian Breast Cancer Patients.

Background: FOXP3 gene, known to be a potential tumor suppressor, has been identified to interact with HER2 in mammary cancer. Moreover, the high expression of FOXP3 serves as a good predictor of the survival of patients in breast cancer, prostate cancer, and gastric cancer. The expression and epigenetic alterations were evaluated in female breast cancer patients. Material and Methods: Expression studies at the mRNA level and protein level were conducted in 140 breast cancer cases by real-time PCR and immunohistochemistry, respectively. Epigenetic studies were also conducted by analyzing the methylation status at the promoter region of the gene using MS-PCR. Results: FOXP3 mRNA expression and protein expression were downregulated in breast cancer patients. The absence of FOXP3 protein expression is significantly associated with promoter methylation, where 70 methylated cases exhibited protein loss (70/95, 73.6%). Statistically, we also found a significant correlation between FOXP3 protein expression and TNM stage, promoter methylation, and histological grade. The methylated FOXP3 cases that did not express protein were also significantly associated with positive lymph node metastasis and HER-2 status. Conclusion: The expression profile of FOXP3 may serve as a prognostic factor. In short, FOXP3 may stand in the most crucial list of biomarkers for breast cancer, bringing compelling results in terms of treatment and management of the disease.

Immunohistochemical expression of FOXP3 in gastric carcinoma; its relation to Ki-67 proliferation marker, HER2/neu expression, and other clinicopathological parameters

ABSTRACT Gastric cancer is common cancer in the world. Contradictory results regarding FOXP3 expression in gastric carcinoma were detected and the role of Ki-67 in prognosis is not completely understood. Furthermore, due to increasing use of anti-HER2 drug trastuzumab for gastric cancer, assessment of HER2 expression becomes important. This study tried to assess the FOXP3 expression in gastric carcinoma and to study the relation between its expression and Ki-67 and HER2/neu expression and relation between their expression and other clinicopathological variables. This retrospective study was carried out on 60 gastric adenocarcinoma cases. Tissue microarrays and immunohistochemical staining for FOXP3, Ki-67 and HER2/neu were done and then assessed and scored. HER2/neu expression showed significant relation to Lauren histological type and lymph node status. High Ki-67 index was related significantly to patients’ age, lympho-vascular tumor emboli, peri-neural invasion, tumor grade, lymph node status, and cancer stage. There was significant relation between high FOXP3 expression and patients’ age, lympho-vascular tumor emboli, peri-neural invasion, tumor grade, lymph node status, and cancer stage. Direct positive significant relationships between HER2/neu, Ki-67, and FOXP3 expression were noticed. Finally, high FOXP3 expression, positive HER2/neu, and high Ki-67 nuclear proliferation index may be an indication of the aggressiveness of gastric carcinoma.

Diversity of Intratumoral Regulatory T Cells in Non-Hodgkin Lymphoma

Introduction: Regulatory T cells (Tregs), a highly immunosuppressive subset of CD4 + T cells, represent a key challenge in the tumor microenvironment by limiting potent antitumor immune responses. While high densities of tumor-infiltrating Tregs are associated with poor prognosis in patients with various types of solid cancers, their prognostic impact in B-cell non-Hodgkin lymphoma (NHL) remains unclear. Emerging studies suggest substantial heterogeneity in the phenotype and suppressive capacities of Tregs, emphasizing the importance of understanding Treg diversity and the need for additional markers to identify highly suppressive Tregs. Our in-depth characterization of Tregs in NHL tumors could open new paths for rational drug design, facilitating selective therapeutic manipulation of Tregs to reduce immunosuppression and improve anti-tumor immunity.Methods: Single-cell suspensions from NHL patients (diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL) and healthy donors (tonsils and peripheral blood)) were analyzed by fluorescent flow- and mass cytometry to characterize Tregs, focusing on their expression of co-stimulatory and co-inhibitory checkpoint receptors. The immunosuppressive capacity of Tregs was measured by in vitro co-culture of FACS-sorted subsets of Tregs together with autologous CellTrace Violet-labelled T effector cells as responder cells, using samples from FL and tonsils. Live CD4 + T cells were obtained by FACS sorting from DLBCL (n = 3), FL (n = 3) and healthy donor tonsils (n = 3) and subjected to single-cell RNA sequencing (scRNA-seq), Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) and scTCR-seq by the 10X Genomics platform. The computational framework of CIBERSORTx was used to generate unique signature matrices for the three Treg subsets identified by scRNA-seq, to facilitate validation in separate scRNA-seq cohorts (King, Sci Immunol 2021; Roider, Nat Cell Biol 2020), and to impute frequencies of the Treg subsets in cohorts with bulk RNA-seq data (Chapuy, Nat Med 2018; Schmitz, NEJM 2018; Pastore, Lancet Oncol 2015).Results: Immunophenotyping by mass cytometry revealed a subset of activated Tregs identified by co-expression of TIGIT, CTLA-4, PD-1, ICOS and OX40, and higher expression of FOXP3, CD25 and CD45RO, that was present in DLBCL and tonsils, but lacking in peripheral blood. This was validated by fluorescent flow cytometry, demonstrating significantly higher frequencies of activated Tregs in NHL tumors compared to PBMCs and tonsils from healthy donors. The phenotypic heterogeneity of intratumoral Tregs reflected different suppressive capacities as activated Tregs more potently suppressed the proliferation of autologous effector CD4 + and CD8 + T cells than naïve Tregs. For global transcriptomic profiling of CD4 + T cells from FL, DLBCL and tonsillar samples, we integrating scRNA-seq and CITE-seq data from 17,774 cells, revealing 13 distinct cellular states including three states of Tregs: naïve, activated and non-conventional LAG3 +FOXP3 - Tregs. Activated Tregs had higher expression of checkpoint receptors (TNFRSF4, TNFRSF18, ICOS), phosphatases (DUSP2, DUSP4), NF-κB pathway (NFKBIA, TNFAIP3, NFKBIZ, REL), chemokine receptors (CXCR4) and transcription factors (JUNB, IRF1, STAT3) as compared to naïve Tregs. We next used a computational approach to develop unique signature matrices for each Treg subset. This approach demonstrated strong concordance between CIBERSORTx estimated cell abundances of the three Treg subsets and the ground truth, and was validated in two external scRNA-seq cohorts. The development of unique signature matrices for Treg subsets facilitated imputation of their frequencies in bulk RNA-seq datasets. These analyses revealed that higher frequency of activated Tregs was enriched in the germinal B cell subtype of DLBCL and was associated with adverse outcome in FL.Conclusion: This study demonstrates that Tregs infiltrating NHL tumors are transcriptionally and functionally diverse and include highly immunosuppressive activated Tregs co-expressing several checkpoint receptors, which distinguish them from peripheral blood Tregs. Activated intratumoral Tregs could hamper clinical responses to checkpoint blockade, and identifying and targeting their vulnerabilities has the potential to improve anti-tumor immune responses. DisclosuresHolte: Gilead: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Nordic: Membership on an entity's Board of Directors or advisory committees; Nanovector: Membership on an entity's Board of Directors or advisory committees, Other: lectures honorarias; Novartis: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Alizadeh: Cibermed: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; CAPP Medical: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Forty Seven: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Foresight Diagnostics: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Roche: Consultancy, Honoraria; Janssen Oncology: Honoraria; Celgene: Consultancy, Research Funding; Gilead: Consultancy; Bristol Myers Squibb: Research Funding.

Clinical Impact of X-Ray Repair Cross-Complementary 1 (XRCC1) and the Immune Environment in Colorectal Adenoma-Carcinoma Pathway Progression.

PurposeColorectal cancer (CRC) can develop via a hypermutagenic pathway characterized by frequent somatic DNA base-pair mutations. Alternatively, the immunogenicity of tumor cells themselves may influence the anticancer activity of the immune effector cells. Impaired DNA repair mechanisms drive mutagenicity, which then increase the neoantigen load and immunogenicity. However, no studies have analyzed immune checkpoint protein expression, particularly programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1), in adenoma–carcinoma progression and its relationship with the emergence of other DNA repair gene mutation.Materials and MethodsWe investigated mutations of 10 genes involved in DNA repair function: XRCC1, TP53, MLH1, MSH, KRAS, GSTP, UMP, MTHF, DPYD, and ABCC2. We performed sequencing to determine mutations and immunohistochemistry of immune checkpoints in clinical samples and determined changes in XRCC1 expression during progression through the adenoma–carcinoma pathway. We further investigated the prognostic associations of gene XRCC1 according to the expression, mutational profile, and immune profile using The Cancer Genome Atlas-colon adenocarcinoma (TCGA-COAD) dataset.ResultsFrom clinical samples, XRCC1 mutation demonstrated the strongest association with adenomas with a mutation frequency of 56.2% in adenomas and 34% in CRCs (p =0.016). XRCC1 was abnormally expressed and altered by mutations contributing to adenoma carcinogenesis. High expression of XRCC1, CD4, FOXP3, and PD-1/PD-L1 showed an overall upward trend with increased lesion severity (all p < 0.01). PD-1/PD-L1 expression and CD4+ intraepithelial lymphocytes (IELs) correlated with cytological dysplasia progression, specifically in patients with wild-type XRCC1 (all p < 0.01), whereas FOXP3 expression was independently associated with adenoma–carcinoma progression. From TCGA-COAD analysis, XRCC1 expression was associated with patients survival, tumor-infiltrating lymphocytes and immune marker expression.ConclusionIncreased IEL density and PD-1/PD-L1 expression correlate with cytological dysplasia progression and specifically with the XRCC1 mutation status in CRC. Our findings support a stepwise dysplasia-carcinoma sequence of adenoma carcinogenesis and an XRCC1 hypermutated phenotypic mechanism of lesions.

The association of immunosurveillance and distant metastases in colorectal cancer

BackgroundColorectal cancer (CRC) is the third most common malignancy worldwide, but the key driver to distant metastases is still unknown. This study aimed to elucidate the link between immunosurveillance and organotropism of metastases in CRC by evaluating different gene signatures and pathways.Material and methodsCRC patients undergoing surgery at the Department of General, Visceral and Transplantation Surgery at the Ludwig-Maximilian University Hospital Munich (Munich, Germany) were screened and categorized into M0 (no distant metastases), HEP (liver metastases) and PER (peritoneal carcinomatosis) after a 5-year follow-up. Six patients of each group were randomly selected to conduct a NanoString analysis, which includes 770 genes. Subsequently, all genes were further analyzed by gene set enrichment analysis (GSEA) based on seven main cancer-associated databases.ResultsComparing HEP vs. M0, the gene set associated with the Toll-like receptor (TLR) cascade defined by the Reactome database was significantly overrepresented in HEP. HSP90B1, MAPKAPK3, PPP2CB, PPP2R1A were identified as the core enrichment genes. The immunologic signature pathway GSE6875_TCONV_VS_FOXP3_KO_TREG_DN with FOXP3 as downstream target was significantly overexpressed in M0. RB1, TMEM 100, CFP, ZKSCAN5, DDX50 were the core enrichment genes. Comparing PER vs. M0 no significantly differentially expressed gene signatures were identified.ConclusionChronic inflammation might enhance local tumor growth. This is the first study identifying immune related gene sets differentially expressed between patients with either liver or peritoneal metastases. The present findings suggest that the formation of liver metastases might be associated with TLR-associated pathways. In M0, a high expression of FOXP3 + tumor infiltrating lymphocytes (TILs) seemed to prevent at least in part metastases. Thus, these correlative findings lay the cornerstone to further studies elucidating the underlying mechanisms of organotropism of metastases.

Intratumoral regulatory T cells from colon cancer patients comprise several activated effector populations

BackgroundIntratumoral regulatory T cells (Treg) in colon cancer are a heterogeneous cell population, with potential impact on patient outcome. Generally, a high Treg infiltration has been correlated to a worse patient outcome, but it is still unclear how the composition of different Treg subsets affects patient relapse and survival. In this study, we used mass and flow cytometry to characterize Treg in colon tumors and corresponding unaffected tissue, followed by a correlation to clinical parameters and patient outcome.ResultsUsing mass cytometry, we defined 13 clusters of intestinal Treg, three of which were enriched in the tumors. The two most enriched clusters were defined by their expression of the proliferation marker Ki67 and CD56, respectively. The Treg accumulating in the tumors expressed inducible T-cell co-stimulator (ICOS), OX-40, and CD39, indicating that they were effector Treg (eTreg). Intratumoral CD39+ Treg also had a higher expression of Foxp3, suggesting a higher suppressive activity, and we subsequently used CD39 as a marker for eTreg. Our further studies showed that colon tumors can be divided into two tumor groups, based on the proportion of CD39+ putative eTreg in the tumors. This property was independent of both tumor microsatellite status and tumor stage, which are important factors in predicting cancer disease progression. In a prospective study of forty-four colon cancer patients, we also showed that patients with a high CD39 expression on tumor-infiltrating Treg have a tendency towards a less favorable patient outcome in terms of cumulative cancer-specific survival.ConclusionsThis study uncovers novel subsets of tumor-infiltrating Treg in colon cancer, and suggests that CD39 may be a potential therapeutic target in patients with microsatellite stable colon tumors, which are usually refractory to checkpoint blockade therapy.

Identification of circulating regulatory T lymphocytes with membrane markers - a new multiparameter flow cytometry protocol.

Regulatory T cells (Tregs) are a unique CD4+ T cell subset involved in the regulation of immune responses. The traditional immunophenotype used to define Tregs includes CD4+CD25high and the expression of the transcription factor Forkhead box protein 3 (FoxP3). A complex technique of intracellular staining, transient upregulation of FoxP3 in activated conventional T lymphocytes (Tcons), and the omission of naïve CD45RA+ Tregs with downregulated FoxP3 activity but a demethylated FOXP3 promoter region may lead to inaccurate quantification. In an attempt to meet the need for a reliable and simplified enumeration strategy, we investigated different membrane markers to capture the entire Treg compartment and to identify subpopulations of Tregs. Analyses were performed on whole blood. Tested gating strategies were based on the expression of the following membrane antigens: CD45, CD3, CD4, CD25, CD127, CD26, CD6, CD39, CD71, HLA-DR, CD45RA and CD31. Double controls with FoxP3 were performed. The final enumeration panel consisted of the membrane markers CD45, CD3, CD4, CD25, CD127, CD26, CD39, CD45RA and CD31. A deep analysis of T cells with the CD4+CD25+CD127low/-CD26low/-CD45RAimmunophenotype revealed high expression of FoxP3 and/or CD39, while cells with the naïve immunophenotype, CD4+CD25+CD127low/-CD26low/-CD45RA+, presented lower expression of suppressor markers. Antigen CD31 is considered to be a valuable membrane marker of thymus-derived Tregs. The presented 9-color panel that can be easily applied in laboratories enables reliable enumeration of Tregs with additional information about the functionality, maturity and origin of T regulatory cells.

Intersection of DNA Repair Pathways and the Immune Landscape Identifies PD-L2 as a Prognostic Marker in Epithelial Ovarian Cancer

Simple SummaryHere, we examined the interaction between DNA repair proteins and immune biomarkers and their association with survival in 181 cases of epithelial ovarian carcinoma (EOC). We used a panel of 12 antibodies for immunocytochemistry staining of tissue microarray (TMA) consisting of 181 cases. Applying standard statistical methods, we detected that PD-L2 expression was associated with decreased survival in ovarian cancer. This is the first demonstration that increased expression of PD-L2 may serve as a marker for decreased progression-free survival (PFS). Therefore, further investigation into PD-L2 based immunotherapy as a strategy to treat ovarian cancer is warranted.Background: Targeting DNA repair and immune checkpoint pathways has been the focus of multiple clinical trials. In this study, we explore the association between DNA repair proteins, immune response markers, and clinical outcome in women with EOC. Methods: Immunohistochemical analysis of TMA with 181 EOC samples was used to determine expression levels for DNA repair proteins (PARP, PTEN, p53, H2Ax, FANCD2, and ATM) and immune-markers (CD4, CD8, CD68, PD-L2, PD-L1, and FOXP3). Biomarker expression was correlated to clinical data. Prognostic discriminatory ability was assessed per the combination of biomarkers. Results: Tumor immunity biomarkers correlated with HRD biomarkers. High PD-L2 was significantly associated with high expression of CD8 (r = 0.18), CD68 (r = 0.17), and FOXp3 (r = 0.16) (all, p < 0.05). In a multivariate analysis, PD-L2 (hazard ratio (HR) 1.89), PARP (HR 1.75), and PTEN (HR 1.96) expressions were independently associated with decreased progression-free survival (PFS), whereas PD-L1 (HR 0.49) and CD4 (HR 0.67) were associated with improved PFS (all, p < 0.05). In 15 biomarker combinations, six combinations exhibited a discriminatory ability of >20% for the 4.5-year PFS rate, with four based on PD-L2 (PARP, PTEN, CD4, and PD-L1, 20.5–30.0%). Conclusions: Increased PD-L2 expression is a prognostic marker of decreased survival in EOC. Interaction between tumor DNA repair and microenvironment determines tumor progression and survival.

Characterization of the Tumor Immune Microenvironment in Lung Squamous Cell Carcinoma Using Imaging Mass Cytometry.

BackgroundLung squamous cell carcinoma (LUSC) is a major subtype of non-small cell lung cancer. The tumor immune microenvironment (TIME) affects the anti-tumor immune response and the patient’s prognosis, although the TIME in LUSC patients is incompletely understood.MethodsWe retrospectively collected surgical specimens from patients with previously untreated primary LUSC. Histopathological examination was used to identify tumor regions and adjacent regions, and imaging mass cytometry was used to characterize the immune cells in those regions. The results were compared between regions and between patients.ResultsWe identified heterogeneity in the TIME on comparing different patients with LUSC, although the tumor region and adjacent region both exhibited an immune response to the tumor. The TIME typically included a large number of infiltrating and activated T-cells, especially CD8+ T-cells, which closely interacted with the tumor cells in the tumor region. There was limited infiltration of B-cells, NK cells, and NKT cells, while the major immune suppressor cells were CD33+ myeloid-derived cells. We also identified a novel population of CD3−CD4+ cells with high expression of Foxp3 and TNFα, which might modulate the tumor microenvironment and play a proinflammatory role in the TIME.ConclusionsThe TIME of LUSC appears to be immunogenic and heterogenous, with predominant infiltration of activated CD8+ T-cells. The interactions between the tumor cells and T-cells facilitate the anti-tumor activity. A novel subpopulation of CD3−CD4+ cells with high TNFα and Foxp3 expression may modulate the tumor microenvironment and play a proinflammatory role.

Sex Hormones and Gender Influence the Expression of Markers of Regulatory T Cells in SLE Patients.

Regulatory T cells have been implicated in the regulation and maintenance of immune homeostasis. Whether gender and sex hormones differentially influence the expression and function of regulatory T cell phenotype and their influence on FoxP3 expression remains obscure. We provide evidence in this study that the number and percent of human regulatory T cells (Tregs) expressing CD4+ and CD8+ are significantly reduced in healthy females compared to healthy males. In addition, both CD4+CD25+hi and CD8+CD25+hi subsets in healthy males have a 2-3 fold increase in FoxP3 mRNA expression compared to healthy females. Female SLE patients, compared to healthy women, have elevated plasma levels of estradiol and decreased levels of testosterone. Higher levels of testosterone correlate with higher expression of FoxP3 in CD4+CD25hiCD127low putative Tregs in women with SLE. Incubation of CD4+ regulatory T cells with 17β-estradiol at physiological levels generally decreased FoxP3 expression in females with SLE. These data suggest that females may be more susceptible than males to SLE and other autoimmune diseases in part because they have fewer Tregs and reduced FoxP3 expression within those cells due to normal E2 levels which suppress FoxP3 expression. In addition, low levels of plasma testosterone in women may further reduce the ability of the Tregs to express FoxP3. These data suggest that gender and sex hormones can influence susceptibility to SLE via effects on regulatory T cells and FoxP3 expression.

Identification of an immune overdrive high-risk subpopulation with aberrant expression of FOXP3 and CTLA4 in colorectal cancer.

Colorectal cancer (CRC) is characterized by a heterogeneous tumor microenvironment (TME) that regulates cancer progression and therapeutic response. Overexpression of FOXP3 and CTLA4 is associated with immunosuppressive TME and poor prognosis in many cancer types. However, opposite results were reported in CRC. Thus, we performed comprehensive analyses to evaluate the exact prognostic value of FOXP3 and CTLA4 in CRC. Here, the expression levels of FOXP3 and CTLA4 were used to construct a subtyping system based on >1200 CRC patients from multiple independent public datasets. We revealed that, in CRC patients with relatively high expression of FOXP3, there exist two different subpopulations with opposite survival patterns according to CLTA4 expression. We further established a method for evaluating all cohorts and identified a novel FOXP3HighCTLA4High* CRC risk subpopulation that accounts for 5-10% of CRC patients. Moreover, different methods of functional enrichment and immune evaluation were used to analyze the TME characteristics of different FOXP3/CTLA4 subtypes. The FOXP3HighCTLA4High* CRC risk subpopulation was characterized by an immune overdrive TME phenotype, including high immune cell infiltration, low tumor purity, high immune checkpoint levels, and TGF-β activation. Finally, the constructed FOXP3/CTLA4 subtyping system was further validated by quantitative RT-PCR, immunochemistry staining, and multicolor immunofluorescence in an independent CRC cohort we collected. This high-risk subpopulation was also observed in kidney cancers and low-grade glioma patients by a Pan-cancer analysis. Together, our study revealed that the established FOXP3/CTLA4 molecular subtyping system could be used to select treatment and management strategies for CRC and other cancers.

Immunomodulatory Effects of Rhinovirus and Enterovirus Infections During the First Year of Life.

Early childhood infections have been implicated in the development of immune-mediated diseases, such as allergies, asthma, and type 1 diabetes. We set out to investigate the immunomodulatory effects of early viral infections experienced before the age of one year on the peripheral regulatory T cell population (Treg) and circulating cytokines in a birth-cohort study of Estonian and Finnish infants. We show here a temporal association of virus infection with the expression of FOXP3 in regulatory T cells. Infants with rhinovirus infection during the preceding 30 days had a higher FOXP3 expression in Treg cells and decreased levels of several cytokines related to Th1 and Th2 responses in comparison to the children without infections. In contrast, FOXP3 expression was significantly decreased in highly activated (CD4+CD127−/loCD25+FOXP3high) regulatory T cells (TregFOXP3high) in the infants who had enterovirus infection during the preceding 30 or 60 days. After enterovirus infections, the cytokine profile showed an upregulation of Th1- and Th17-related cytokines and a decreased activation of CCL22, which is a chemokine derived from dendritic cells and associated with Th2 deviation. Our results reveal that immunoregulatory mechanisms are up-regulated after rhinovirus infections, while enterovirus infections are associated with activation of proinflammatory pathways and decreased immune regulation.

Molecular Linking of HIPEC (Hyperthermic Intraperitoneal Chemotherapy) and Tregs (Regulatory T- cells) in Advanced Epithelial Ovarian Cancer - A Review

The mainstay in the management of advanced epithelial ovarian cancer is platinum-based chemotherapy and complete cytoreductive surgery. Despite this, about two-thirds of patients have disease recurrence mostly within the peritoneal cavity. HIPEC (Hyperthermic Intraperitoneal Chemotherapy) is a modality that delivers cell-cycle non-specific chemotherapeutic agents along with heat 41°C to 43°C into the peritoneal cavity. HIPEC is done intra-operatively after achieving complete cytoreduction (which means after removal of all the tumor deposits more than 2.5 mm). Ovarian cancer is associated with the frequent finding of tumor-infiltrating lymphocytes in their tissue microenvironment. Especially studies have shown that ovarian cancer evades immune surveillance by higher expression of FOXP3 T cells. HIPEC has been used in the treatment of primary and recurrent tumors. In this review, we discuss how the significance of HIPEC on genetics and immunology of these patients with cancer have provided unique insights into the molecular and cellular basis of Treg cells. Studies of HIPEC and its association with Tregs cells should make it possible to increase the paucity of immuno- therapeutic modalities of most human cancer at an unprecedented level of molecular and cellular precision. The predictive, preventive, and therapeutic implications of these studies of HIPEC in relation to immunity in EOC may extend to patients with other peritoneal carcinomatosis.