Regulatory Foxp3 Research Articles

Amniotic MSCs reduce pulmonary fibrosis by hampering lung B-cell recruitment, retention, and maturation.

Growing evidence suggests a mechanistic link between inflammation and the development and progression of fibrotic processes. Mesenchymal stromal cells derived from the human amniotic membrane (hAMSCs), which display marked immunomodulatory properties, have been shown to reduce bleomycin‐induced lung fibrosis in mice, possibly by creating a microenvironment able to limit the evolution of chronic inflammation to fibrosis. However, the ability of hAMSCs to modulate immune cells involved in bleomycin‐induced pulmonary inflammation has yet to be elucidated. Herein, we conducted a longitudinal study of the effects of hAMSCs on alveolar and lung immune cell populations upon bleomycin challenge. Immune cells collected through bronchoalveolar lavage were examined by flow cytometry, and lung tissues were used to study gene expression of markers associated with different immune cell types. We observed that hAMSCs increased lung expression of T regulatory cell marker Foxp3, increased macrophage polarization toward an anti‐inflammatory phenotype (M2), and reduced the antigen‐presentation potential of macrophages and dendritic cells. For the first time, we demonstrate that hAMSCs markedly reduce pulmonary B‐cell recruitment, retention, and maturation, and counteract the formation and expansion of intrapulmonary lymphoid aggregates. Thus, hAMSCs may hamper the self‐maintaining inflammatory condition promoted by B cells that continuously act as antigen presenting cells for proximal T lymphocytes in injured lungs. By modulating B‐cell response, hAMSCs may contribute to blunting of the chronicization of lung inflammatory processes with a consequent reduction of the progression of the fibrotic lesion.

Genome-wide CRISPR screens revealed a reciprocal role of SWI/SNF complexes in controlling regulatory T cell function

Abstract Regulatory T cells (Tregs) are a specialized immune population that act to suppress overactive inflammatory response and maintain immune homeostasis. There is of great interest in harnessing Treg for controlling autoimmune disease or boosting anti-tumor immunity. Since the transcription factor, Forkhead box P3 (Foxp3), is known to control Treg development and function, understanding the regulation of Foxp3 could potentially lead to the development of therapeutics for treating autoimmune diseases and cancer. Here we performed a genome-wide CRISPR/Cas9 knockout screen to identify the regulators of Foxp3 in mouse primary Tregs. The results showed that Foxp3 regulators are highly enriched in genes encoding chromatin regulators, such as SAGA and SWI/SNF complexes. Among the SWI/SNF complexes, the GBAF complex promoted Foxp3 expression, whereas the PBAF complex repressed its expression. Functional genomic studies revealed that both GBAF and PBAF co-localized with Foxp3 in genome-wide binding sites. Targeting of GBAF reduced Foxp3 binding and altered expression a subset of Foxp3 target genes, indicating that GBAF actively participates in the Foxp3-dependent transcriptional program. Importantly, Treg suppressor function can be manipulated by targeting GBAF or PBAF, demonstrating in in vitro suppression assay, as well as mouse in autoimmune diseases and tumor model. Collectively, we provide an unbiased analysis of genes and networks regulating Foxp3, and reveal SWI/SNF complexes, GBAF and PBAF, as novel targets that could be exploited to manipulate Treg function for therapeutics of autoimmune diseases and cancer.

Heterogeneity of regulatory T cells during lung inflammation

Abstract Pulmonary inflammation is the leading cause of respiratory illness worldwide. Although strong T cell responses are desired for defending the hosts against infections, the cytotoxic effector function of the innate and adaptive immune responses can lead to the development of pulmonary immunopathology, which may lead to death. While T cells produce inflammatory cytokines during infections and allergies, they can also produce the immunomodulatory cytokine IL-10, which is critical for limiting the immunopathology caused by the excessive effector immune responses. However, the composition of IL-10-producing lymphocyte population and their molecular signatures are unclear. Using mouse models that report the production of IL-10 by GFP and expression of T regulatory cell marker Foxp3 by RFP, mouse models of lung inflammation (including allergic asthma, hypersensitivity pneumonitis, and flu), transcriptomic analyses at the population and single cell levels, and transgenic mouse models that are impaired in T cell-derived IL-10 production, we found that, in the disease models used, regulatory T cells in the mouse airway are mainly comprised of CD4+ Foxp3+, CD4+ Foxp3− and CD8+ subsets, and they differ under different disease conditions. Furthermore, within each subset, IL-10-producing T cells exhibit significant molecular heterogeneity. Information gained from this dataset provides insights into the T cell subset heterogeneity and signature markers, and shed light for future strategic design for therapeutic development utilizing the immunomodulatory features of T cells for the treatment of pulmonary immunopathology.

USP22 stabilizes FoxP3 to maintain Regulatory T cell function through a dual mechanism resulting in tumor immune evasion

Abstract Aggressive cancers are less sensitive to standard cancer treatments available, reducing overall survival rates and increasing relapse percentage in cancer patients. As a result, there is an increasing need for alternative therapeutics that will prolong the survival of patients. Within the last decade, methods to attenuate tumor immune evasion have become a centerpiece of tumor therapies. However, a major hurdle in tumor immunotherapy is the immunosuppression mediated by Regulatory T (Treg) cells, which can promote tumor progression through the suppression of tumor immune evasion. Therefore, the ability to modulate Treg function in the context of cancer could lead to more effective therapies. Here, we identify the ubiquitin-specific peptidase 22 (USP22), a member of the deubiquitination module of the SAGA chromatin modifying complex, as a specific regulator of Foxp3, the lineage-specifying transcription factor of Tregs. Treg-specific ablation of Usp22 in mice reduced Foxp3 at both the transcriptional and post-translational level, creating defects in their suppressive function and leading to spontaneous autoimmunity. Importantly, USP22 ablation protected against tumor growth in multiple cancer models. Collectively, our findings reveal a previously underappreciated physiological function of USP22 in maintaining Treg stability and identify USP22 as a potential target for cancer immunotherapy.

CRISPR screen in regulatory T cells reveals modulators of Foxp3.

Regulatory T cells (Tregs) are required to control immune responses and maintain homeostasis, but are a significant barrier to anti-tumor immunity1. Conversely, Treg instability, characterized by loss of the master transcription factor Foxp3 and acquisition of pro-inflammatory properties2, can promote autoimmunity and/or facilitate more effective tumor immunity3,4. A comprehensive understanding of the pathways that regulate Foxp3 could lead to more effective Treg therapies for autoimmune disease and cancer. Despite improved functional genetic tools that now allow for systematic interrogation, dissection of the gene regulatory programs that modulate Foxp3 expression has not yet been reported. In this study, we developed a CRISPR-based pooled screening platform for phenotypes in primary mouse Tregs and applied this technology to perform a targeted loss-of-function screen of ~490 nuclear factors to identify gene regulatory programs that promote or disrupt Foxp3 expression. We discovered several novel modulators including ubiquitin-specific peptidase 22 (Usp22) and ring finger protein 20 (Rnf20). Usp22, a member of the deubiquitination module of the SAGA chromatin modifying complex, was discovered to be a positive regulator that stabilized Foxp3 expression; whereas the screen suggested Rnf20, an E3 ubiquitin ligase, can serve as a negative regulator of Foxp3. Treg-specific ablation of Usp22 in mice reduced Foxp3 protein and created defects in their suppressive function that led to spontaneous autoimmunity but protected against tumor growth in multiple cancer models. Foxp3 destabilization in Usp22-deficient Tregs could be rescued by ablation of Rnf20, revealing a reciprocal ubiquitin switch in Tregs. These results reveal novel modulators of Foxp3 and demonstrate a screening method that can be broadly applied to discover new targets for Treg immunotherapies for cancer and autoimmune disease.

Abstract IA06: Tumor and host factors regulating antitumor immunity and immunotherapy efficacy

Abstract Two major phenotypes of human melanoma metastases have been observed based on gene expression profiling and confirmatory assays. One subgroup of patients has a T cell-inflamed phenotype that includes expression of chemokines, T cell markers, and a type I IFN signature. In contrast, the other major subset lacks this phenotype and appears to display immune “exclusion.” The mechanisms of immune escape are likely distinct in these two subsets, and therefore the optimal immunotherapeutic interventions necessary to promote clinical responses may be different. The T cell-inflamed tumor microenvironment subset shows the highest expression of negative regulatory factors, including PD-L1, IDO, and FoxP3+ Tregs. Deep analysis of tumor antigen-specific T cells in the tumor microenvironment has identified additional mechanisms of immune dysfunction and new potential therapeutic targets. Treatment strategies targeting several pathways have been translated back into the clinic, with anti-PD-1/PD-L1 agents being FDA approved for 11 different cancer entities. Combinations with new agents based on the biology of T-cell dysfunction are ongoing. In contrast to the T cell-inflamed melanomas, non-T cell-inflamed tumors are largely immunotherapy resistant with current approaches. Natural innate immune sensing of tumors appears to occur via the host STING pathway, type I IFN production, and cross-priming of T cells via CD8+ DCs, and these factors are absent in non-T cell-inflamed tumors. New strategies are being developed to engage or mimic this pathway as a therapeutic endeavor, including STING agonists. The molecular mechanisms that mediate the absence of the T cell-inflamed tumor microenvironment in patients are being elucidated using parallel genomics platforms. The first oncogene pathway identified that mediates immune exclusion is the Wnt/β-catenin pathway, which argues that new pharmacologic strategies to target this pathway should be developed to restore immune access to the tumor microenvironment. Recent evidence has indicated that host factors, including the intestinal microbiota, are also critical. We recently have identified commensal bacteria in mouse models that augment spontaneous antitumor immunity and increase efficacy of anti-PD-L1 therapy. Similar analyses in human melanoma patients revealed commensal bacteria associated with anti-PD-1 efficacy. These results have prompted the pursuit of new probiotics that may improve spontaneous immune infiltration and expand immunotherapy efficacy in the clinic. Citation Format: Thomas F. Gajewski. Tumor and host factors regulating antitumor immunity and immunotherapy efficacy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr IA06.

Carbonic anhydrase 9 (CA9) expression in non-small-cell lung cancer: correlation with regulatory FOXP3+T-cell tumour stroma infiltration

BackgroundLow pH suppresses the proliferation and cytotoxic activity of CD8+ cytotoxic and natural killer lymphocytes. The hypoxia-regulated transmembrane protein, carbonic anhydrase CA9, converts carbon dioxide produced by the Krebs cycle to bicarbonate and protons that acidify the extracellular milieu. We examined whether CA9 is also involved in intratumoural immunosuppression pathways.MethodsA series of 98 tissue samples of primary non-small-cell lung carcinomas (NSCLC) from patients treated with surgery were analysed for the expression of CA9 and programmed-death ligand PD-L1 by cancer cells, and of FOXP3 by tumour-infiltrating lymphocytes (TILs).ResultsThere was no direct association of CA9 with PD-L1 expression or the density of TILs in the tumour stroma, but CA9 was directly related to the extent of FOXP3+ TIL density (p = 0.008). Double-stratification survival analysis showed that patients with high CA9 expression and low TIL score had significantly poorer survival compared with all other groups (p < 0.04). In a multivariate analysis stage (p < 0.0001, HR 1.95, 95% CI: 1.3–2.7), TIL score (p = 0.05, HR 0.55, 95% CI: 0.2–1.0) was an independent prognostic variable of death events. CA9 expression by cancer cells is associated significantly with FOXP3+ regulatory T-cell abundance in the tumour stroma of NSCLC.ConclusionThe study provides a basis for testing CA9 as a marker of resistance to immune-checkpoint inhibitors and as a therapeutic target to enhance the efficacy of immunotherapy.

Regulatory T Cell Suppressive Activity Predicts Disease Relapse During Disease-Modifying Anti-rheumatic Drug Dose Reduction in Rheumatoid Arthritis: A Prospective Cohort Study.

When the dose of conventional disease-modifying anti-rheumatic drugs (cDMARDs) is tapered in rheumatoid arthritis (RA) patients who achieve sustained remission, biomarkers for predicting disease relapse may be needed. A prospective, unblinded cohort study was conducted in nine RA patients with remission. Peripheral blood samples were collected at baseline and at 6, 12, and 24 weeks after cDMARD dose reduction (dose of combination regimens reduced to 50%) to determine the number of regulatory Foxp3+T cells (Tregs) and other T cell subpopulations as well as Treg suppressive activity. Additionally, plasma levels of 14 cytokines at each time-point were measured via flow cytometry. Univariate and multivariate analyses were performed to identify the factor(s) associated with RA relapse during the observational period. In univariate analysis, Treg suppression and DAS28 and VAS scores were associated with RA relapse after cDMARD dose tapering. However, in multivariate analysis, only Treg suppressive activity (<42%) was found to be an independent factor associated with RA relapse after cDMARD dose reduction to 50%. Of all patients who had ≥42% Treg suppressive activity during cDMAD reduction, three-fourth patients remained in the remission stage for 24 weeks. Treg suppressive activity (<42%) in RA patients with remission could be a potential biomarker for predicting RA relapse after cDMARD dose reduction, especially over a short-term period (24 weeks).

Lack of cell movement impairs survival of peripheral blood IL-2-stimulated natural killer cells originating from solid cancer and promotes red blood cells to induce their switch toward a regulatory phenotype

BackgroundRed blood cells (RBCs) can have a modulatory effect on immune cells; so changes in their dynamism could considerably influence their physiology, and consequently the immune activities of neighbouring cells, like natural killer (NK) cells. Herein, we studied the effect of both RBCs and lack of cell movement on the proliferation, survival and regulation of peripheral IL-2-stimulated NK cells from normal and solid malignant conditions. MethodsExperiments were conducted on twelve cell culture groups, including NK cells from patients with solid malignant tumor or healthy controls, cultured alone or with autologous or nonautologous RBCs under shaking or no shaking conditions. ResultsNK cells from neoplastic patients behaved differently depending on the culture conditions including shaking and/or RBCs presence. Therefore, NK cells survival was downregulated in the absence of shaking; whereas, shaking have not only upregulated cell survival, but also downregulated the levels of p53-related apoptosis. Moreover, RBCs enhanced NK cells proliferation; while, this effect was modulated by shaking. Furthermore, RBCs can generate opposite effects on the production and modulation of protumoral or immunosuppressive cytokines, depending on the origin of NK cells, i.e., whether they derive from healthy or solid malignant tumor conditions. Finally, NK cells become able to express Foxp3 regulatory marker when combining three main conditions that include (i) treatment with high dose of IL-2, (ii) presence of RBCs, and (iii) absence of shaking. ConclusionsOur outcomes showed for the first time that cell stagnation would be markedly involved in peripheral NK cell apoptosis, as well as in switching toward a regulatory phenotype-induced Foxp3. Cell movement may be one of ex vivo potential approaches in boosting the activities and survival of such cells during solid cancer.

Safe and Successful Treatment of Acute Cellular Rejection of an Intestine and Abdominal Wall Transplant With Vedolizumab.

Background.Graft survival rates after intestinal transplantation (ITx) are still the lowest in comparison to other solid organ transplants. One of the main reasons is the frequent occurrence of acute cellular rejection (ACR). Vedolizumab is an antibody against α4β7+ integrin involved in gut-homing of T cells which has been approved for inflammatory bowel diseases (IBD). We report its off-label use to treat ACR after ITx.Methods.Following abdominal wall transplantation (AWTx) and ITx, clinical course was followed biochemically. Sequential small intestinal biopsies were taken preceding, during, and after ACR treatment with vedolizumab, following the standard therapy regime for IBD. Rejection was diagnosed histologically, and proinflammatory (α4β7+, interleukin-17+) and regulatory (FoxP3+) T cells were analyzed by immunohistochemistry.Results.ACR in both the ITx and AWTx resolved upon vedolizumab treatment, which was safe, evidenced by clearing an astrovirus and primary cytomegalovirus infection. Only a slight reduction of α4β7+ cells in the mucosa was observed, and α4β7+ and regulatory T cells could still move into the lamina propria upon infection.Conclusions.Vedolizumab is a safe treatment option for ACR after ITx but its mechanism is probably not only based on inhibition of gut-selective T-cell homing.

Local immune response in colon cancer: Indicative of good or poor prognosis?

213 Background: The tumour microenvironment is an important determinant of survival in patients with colon cancer. Although the generalised inflammatory infiltrate as assessed by Klintrup-Mäkinen (KM) grade is a stage-independent prognostic marker, immunohistochemical staining for specific immune cell populations, such as T-cells, may have greater prognostic value. The present study examines the clinical utility of combined assessment of KM grade in addition to cytotoxic (CD8) and regulatory (FoxP3) T-cells. Methods: KM, CD8 and FoxP3 were assessed retrospectively in a cohort of 520 patients with stage I-III colon cancers. The relationship between KM grade, T-cell density and cancer-specific survival was examined. Results: KM was high in 33% of patients, of which: 16% had high FoxP3, 12% high CD8 and 56% both FoxP3 and CD8, while the remaining 15% were high KM alone. Conversely, 67% had low KM and of these: 47% had high CD8 alone or with FoxP3; 27% were high for FoxP3 alone and 25% had no inflammatory infiltrate. KM in the presence of other T-cells resulted in good outcome (Table), whereas CD8 with low KM with or without FoxP3 resulted in intermediate outcome. FoxP3 alone with low KM resulted in poor outcome. Similarly, KM alone without T-cells resulted in poor outcome. When no inflammatory cells were present, outcomes were comparable to KM or FoxP3 alone. Conclusions: A co-ordinated immune response results in good outcome and this can be assessed using a combination of a simple H&amp;E based method (KM) in addition to T-cell markers. Where KM is low, despite the presence of cytotoxic T-cells, survival outcome is worse. Conversely, high KM in the absence of T-cells, or FoxP3 alone, are associated with poor outcome, comparable to no inflammatory response. [Table: see text]

Single-cell RNA-seq unveils critical regulators of human FOXP3+ regulatory T cell stability

The heterogeneity and plasticity of T lymphocytes is critical for determining immune response outcomes. Functional regulatory T (Treg) cells are commonly characterized by stable FOXP3 expression and have reported to exhibit heterogeneous phenotypes under inflammatory conditions. However, the interplay between inflammation and Treg cell suppressive activity still remains elusive. Here, we utilized single-cell RNA sequencing to investigate how human Treg cells respond to the pro-inflammatory cytokine interleukin-6 (IL-6). We observed that Treg cells divided into two subpopulations after IL-6 stimulation. TIGIT− unstable Treg cells lost FOXP3 expression and gained an effector-like T cell phenotype, whereas TIGIT+ Treg cells retained robust suppressive function. Single cell transcriptome analysis revealed a spectrum of cellular states of IL-6-stimulated Treg cells and how cytochrome P450 family 1 subfamily A member 1 (CYP1A1) is a crucial regulator of Treg cell suppressive capability and stability. CYP1A1-deficient human Treg cells developed a Th17-like phenotype after IL-6 stimulation. Our findings implicate CYP1A1 as a previously unidentified regulator of Treg cells that may have target potential for clinical application for biotherapies.

Abstract C132: Fusobacterium nucleatum in colorectal carcinoma and expression of multiple immune checkpoint molecules on T cells

Abstract Background: Fusobacterium nucleatum (Fn) appears to play a role in colorectal cancer carcinogenesis through suppression of antitumor immune response. This study aims to characterize immune-related signatures, including the expression of multiple immune checkpoint molecules in tumor-infiltrating T cells. Methods: We measured Fn DNA by quantitative PCR in a total of 155 colorectal cancer tissues. Gene expression profile was studied using microarray technology to determine the consensus molecular subtypes (CMS) and immune-related signatures using CIBERSORT and Gene Set Enrichment Analysis (GSEA), respectively. Expression of immune checkpoint molecules (PD-1, TIM-3, and TIGIT) on CD8+ cytotoxic T cells and the ratio of FOXP3-CD4-/FOXP3+CD4 were examined using multicolor flow cytometric analysis. Results: All patients had stage III disease underwent curative surgery. Fn infection was observed in 43% of patients and was associated with recurrence (Fisher’ exact test, P=0.002) and decreased overall survival (log-rank test, P&amp;lt;0.001). The distribution of CMS subtypes was CMS1/2/3/4/mixed (14%/24%/15%/27%/20%), which is comparable to previous data. Fn infection was associated with the platinum-resistant signature, hypoxia pathway, and stem cell pathways in the GSEA. Interestingly, Patients with Fn infection showed a trend toward enrichment in CMS4, mesenchymal subtype with the inflamed immune phenotype (9% vs. 17% with Fn-infection, P=0.24). The number of cytotoxic CD8+ T cells showed a trend toward decreased expression in patients with Fn infection. Furthermore, higher expression of inhibitory immune checkpoints (PD-1, P&amp;lt;0.01; TIM-3, P&amp;lt;0.05; TIGIT, P&amp;lt;0.05) was observed on CD8+ cytotoxic T cells of patients with Fn infection, compared to that of Fn negative cases. Besides, the regulatory FOXP3+CD4+ cells were increased in patients with Fn infection, supporting the suppressive role of Fn infection in the tumor immune microenvironment (P&amp;lt;0.05). Conclusions: The adverse prognostic impact of Fn infection depends on the suppression of T cell-mediated immune response by increased expression of inhibitory immune checkpoints on cytotoxic T cell and by increased regulatory T cells. Citation Format: Han Sang Kim, Wonkyu Kim, Chang Gon Kim, Jinseon Yoo, Soonmyung Paik, Eui-Cheol Shin, Tae-Min Kim, Hoguen Kim, Joong Bae Ahn. Fusobacterium nucleatum in colorectal carcinoma and expression of multiple immune checkpoint molecules on T cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C132. doi:10.1158/1535-7163.TARG-19-C132

Lamin A/C deficiency in CD4+ T-cells enhances regulatory T-cells and prevents inflammatory bowel disease.

The mechanisms by which lamin A/C in CD4+ T-cells control intestinal homeostasis and can cause inflammatory bowel disease (IBD) are unknown. Here, we explore lamin A/C in a mouse model of IBD. Adoptive transfer to Rag1-/- mice of Lmna-/- CD4+ T-cells, which have enhanced regulatory T-cells (Treg) differentiation and function, induced less severe IBD than wild-type T-cells. Lamin A/C deficiency in CD4+ T-cells enhanced transcription of the Treg master regulator FOXP3, thus promoting Treg differentiation, and reduced Th1 polarization, due to epigenetic changes in the Th1 master regulator T-bet. In mesenteric lymph nodes, retinoic acid (RA) released by CD103+ dendritic cells downregulated lamin A/C in CD4+ T-cells, enhancing Treg differentiation. However, non-RA-producing CD103- dendritic cells predominated in peripheral lymph nodes, facilitating lamin A/C expression in CD4+ T-cells and therefore Th1 differentiation. Our findings establish lamin A/C as a key regulator of Th differentiation in physiological conditions and show it as a potential immune-regulatory target in IBD. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Tumor and Host Factors Regulating Anti-tumor Immunity and Immunotherapy Efficacy

Abstract Characterization of the and tumor microenvironments has generated a framework for identifying immunotherapeutic targets and for understanding mechanisms of efficacy versus resistance. The T cell-inflamed phenotype includes expression of chemokines, T cell markers, and a type I IFN signature, whereas the non-T cell-inflamed phenotype lacks these factors and appears to display immune exclusion. The mechanisms of immune escape are likely distinct in these two subsets, and therefore the optimal immunotherapeutic interventions necessary to promote clinical responses may be different. The T cell-inflamed tumor microenvironment subset shows the highest expression of negative regulatory factors, including PD-L1, IDO, and FoxP3+ Tregs. Deep analysis of tumor antigen-specific T cells in the tumor microenvironment has identified additional mechanisms of immune dysfunction and new potential therapeutic targets, beyond PD-1. In contrast to the T cell-inflamed tumors, non-T cell-inflamed tumors are largely immunotherapy resistant with current approaches. Natural innate immune sensing of tumors appears to occur via the host STING pathway, type I IFN production, and cross-priming of T cells via Batf3-lineage DCs. New strategies are being developed to engage or mimic this pathway, including STING agonists, TLR agonists, and other innate immune activators. The molecular mechanisms that mediate the absence of the T cell-inflamed tumor microenvironment in patients are being elucidated using parallel genomics platforms and include tumor-intrinsic oncogenic events, germline polymorphisms in immune regulatory genes, and the composition of the commensal microbiota. Each of these is giving rise to new therapeutic strategies to improve spontaneous immune infiltration and expand immunotherapy efficacy.

The expression of C-MYC in gastric adenocarcinoma is associated with PD-L1 and FOXP3 expression: C-MYC overexpression is a good prognostic factor

BackgroundC-MYC appears to initiate and maintain tumorigenesis through modulation of immune regulatory molecules such as PD-L1. The aim of our research was to evaluate the clinical implication of C-MYC expression in gastric adenocarcinoma in relation to the expression of the immune regulatory molecules PD-L1 and FOXP3. MethodsTissue samples were acquired from 182 cases of gastric adenocarcinoma that were surgically resected at Kyung Hee University Hospital at Gangdong from 2006 to 2012. Immunohistochemical staining for C-MYC, PD-L1, CD8 and FOXP3 was done. ResultsC-MYC overexpression showed a significant correlation with smaller tumor size, lower T category, lower N category, lower recurrence rate, and less lymphatic invasion. And C-MYC overexpression was negatively correlated with PD-L1 expression. The tumoral FOXP3 was positively correlated with C-MYC overexpression and Tregs count. PD-L1 expression was positively correlated with Tregs, CD8 + T cells, and tumor infiltrating lymphocytes (TIL). Tregs count was positively correlated with CD8 + T cells and TIL. CD8 + T cells was positively correlated with TIL. ConclusionWe discovered that the immune regulatory effect of C-MYC and PD-L1, and the tumor suppressor function of tumoral FOXP3 had a significant influence on the tumor microenvironment (Tregs, CD8 + T cells, and tumor infiltrating lymphocytes) in a complex manner. The C-MYC overexpression is a good prognostic factor in gastric adenocarcinoma.

Expression of immune regulatory genes correlate with the abundance of specific Clostridiales and Verrucomicrobia species in the equine ileum and cecum

Billions of bacteria inhabit the gastrointestinal tract. Immune-microbial cross talk is responsible for immunological homeostasis, and symbiotic microbial species induce regulatory immunity, which helps to control the inflammation levels. In this study we aimed to identify species within the equine intestinal microbiota with the potential to induce regulatory immunity. These could be future targets for preventing or treating low-grade chronic inflammation occurring as a result of intestinal microbial changes and disruption of the homeostasis. 16S rRNA gene amplicon sequencing was performed on samples of intestinal microbial content from ileum, cecum, and colon of 24 healthy horses obtained from an abattoir. Expression of genes coding for IL-6, IL-10, IL-12, IL-17, 18 s, TNFα, TGFβ, and Foxp3 in the ileum and mesenteric lymph nodes was measured by qPCR. Intestinal microbiota composition was significantly different in the cecum and colon compared to the ileum, which contains large abundances of Proteobacteria. Especially members of the Clostridiales order correlated positively with the regulatory T-cell transcription factor Foxp3 and so did the phylum Verrucomicrobia. We conclude that Clostridiales and Verrucomicrobia have the potential to induce regulatory immunity and are possible targets for intestinal microbial interventions aiming at regulatory immunity improvement.

The Alzheimer's Disease-Associated Protein BACE1 Modulates T Cell Activation and Th17 Function.

β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) is best known for its role in Alzheimer's disease amyloid plaque formation but also contributes to neurodegenerative processes triggered by CNS injury. In this article, we report that BACE1 is expressed in murine CD4+ T cells and regulates signaling through the TCR. BACE1-deficient T cells have reduced IL-17A expression under Th17 conditions and reduced CD73 expression in Th17 and inducible T regulatory cells. However, induction of the Th17 and T regulatory transcription factors RORγt and Foxp3 was unaffected. BACE1-deficient T cells showed impaired pathogenic function in experimental autoimmune encephalomyelitis. These data identify BACE1 as a novel regulator of T cell signaling pathways that impact autoimmune inflammatory T cell function.

Ultrasound inflammatory parameters and Treg/Th17 cell profiles in established rheumatoid arthritis

BackgroundImbalance and disfuntion in regulatory T-cells (Tregs) and IL-17 producer lymphocytes (Th17) have been implicated in the pathogenesis of rheumatoid arthritis (RA). Gray scale synovial proliferation (GS), power Doppler signal (pD) and bone erosions seen on high resolution muskuloskeletal ultrasound (MSUS) are hallmarks of destructive articular disease.ObjectiveTo evaluate the association of peripheral Tregs and Th17 with MSUS findings in RA.MethodsRA patients (1987 ACR criteria) treated with disease-modifying antirheumatic drugs (DMARDs) were included. Lymphocytes were isolated and immunophenotyped by flow cytometry to investigate regulatory FoxP3+ T cells and IL-17+ cells. MSUS (MyLab 60, Esaote, Genova, Italy, linear probe 6–18 MHz) was performed on hand joints, and a 10-joint US score was calculated for each patient.ResultsData on lymphocytes subsets were avaiable for 90 patients. The majority of patients were Caucasian women with a median disease duration of 6 years (interquartile range: 2–13 years). Mean DAS28 was 4.28 (SD ± 1.64) and mean HAQ score was 1.11 (SD ± 0.83).There was no significant correlation of 10-joint GS score (rS = 0.122, 95% CI: − 0.124 to 0.336, P = 0.254) and 10-joint pD score (rS = 0.056, 95% CI: − 0.180 to 0.273, P = 0.602) with the mean percentage of peripheral Treg cells. Also, 10-joint GS score (rS = 0.083, 95% CI: − 0.125 to 0.302, P = 0.438) and 10-joint pD score 10 (rS = − 0.060, 95% CI: − 0.271 to 0.150, P = 0.575); did not correlate to Th17 profile. No association of bone erosions on MSUS with Treg and Th17 profiles (P = 0.831 and P = 0.632, respectively) was observed.ConclusionIn this first study addressing MSUS features and lymphocytes subtypes in established RA, data did not support an association of circulating Tregs and Th17 lymphocytes with inflammatory and structural damage findings on MSUS.

Abstract 2356: Estrogen medicates sex specific function of epithelial STAT3 in K-ras mutant lung tumorigenesis by reprogramming lung tumor microenvironment

Abstract Activating mutations of K-ras are one of the most common molecular alterations associated with lung cancer but have not yielded to therapeutic attack so far. We have recently shown a sex-specific role for epithelial Stat3 signaling in the pathogenesis of K-ras mutant lung cancer. We specifically found that deletion of STAT3 in K-ras mutant lung epithelial cells (LR/STAT3Δ/Δ mice) significantly reduced lung tumor burden in female mice which was associated with an enhanced anti-tumor immune response but, surprisingly, caused a dramatic enhancement of lung tumorigenesis and induction of a NF-κB driven pro-tumor immune response in male mice. To further dissect the mechanism of STAT3 dependent sex disparity in the pathogenies of K-ras mutant lung cancer, we performed loss- and gain- of function studies to manipulate estrogen signaling in both female and male mice. In LR/Stat3Δ/Δ males, implantation of estrogen pellet led to significantly reduced lung tumor burden and decreased neutrophils in the lung. This was accompanied by decreased Il6, and Cxcl1 expression explaining reduced neutrophil numbers. We also detected reduced expression of immunosuppressive markers; Arg1, Nos2, Tgfb, Il10, indicating to an attenuated immunosuppressive pro-tumor microenvironment. On the other hand, bilateral oophorectomy in female LR/Stat3Δ/Δ mice led to higher tumor burden, higher lung neutrophil counts, and increased Il6, and Cxcl1 expression. We also found decreased expression of Gzmb, and Ifng, and increased expression of T regulatory markers Foxp3 and Il10, suggesting an attenuated cytotoxic immune response but a stronger immune-inhibitory response. Hormonal replacement therapy by implanting estrogen pellet in oophorectomized LR/Stat3Δ/Δ females rescued the tumor-promoting effect of estrogen depletion characterized by reduced tumor number and neutrophil infiltration, decreased expression of Il6, Cxcl1, Foxp3 and Il10, and increased Gzmb and Ifng expression. Taken together, our study suggests that there is a differential regulation of NF-κB activation in K-ras mutant lung epithelium within sex, and estrogen plays a protective role (anti-tumor) by suppressing the activation of NF-κB pathway and reformatting the lung tumor microenvironment toward an anti-tumor phenotype. Our finding could lead to development of personalized (e.g. sex-based) immunotherapeutic and/or preventive modalities for K-ras mutant lung cancer. Citation Format: Shanshan Deng, Marco Ramos-Castaneda, Walter Velasco Torrez, Oscar Noble, Neha Daga, Mauricio Caetano, Seyed Javad Moghaddam. Estrogen medicates sex specific function of epithelial STAT3 in K-ras mutant lung tumorigenesis by reprogramming lung tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2356.