High Foxp3 Research Articles

Deciphering CD59: Unveiling Its Role in Immune Microenvironment and Prognostic Significance.

CD59, a GPI-anchored membrane protein, protects cancer cells from complement-dependent cytotoxicity (CDC) by inhibiting the formation of the membrane attack complex (MAC). It has been demonstrated to be overexpressed in most solid tumors, where it facilitates tumor cell escape from complement surveillance. The role of CD59 in cancer growth and interactions between CD59 and immune cells that modulate immune evasion has not been well explored. Using cancer patient database from The Cancer Genome Atlas (TCGA) and other public databases, we analyzed CD59 expression, its prognostic significance, and its association with immune cell infiltration in the tumor microenvironment, identifying associated genomic and functional networks and validating findings with invitro cell-line experimental data. This article describes the abundant expression of CD59 in multiple tumors such as cervical squamous cell carcinoma (CESC), kidney renal cell carcinoma (KIRC), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), and stomach adenocarcinoma (STAD), as well as in pan-cancer, using The Cancer Genome Atlas (TCGA) database and confirmed using multiple cancer cell lines. The expression of CD59 significantly alters the overall survival (OS) of patients with multiple malignancies such as CESC, GBM, HNSC, and STAD. Further, the correlation between CD59 and Treg and/or MDSC in the tumor microenvironment (TME) has shown to be strongly associated with poor outcomes in CESC, GBM, HNSC, and STAD as these tumors express high FOXP3 compared to KIRC. Moreover, unfavorable outcomes were strongly associated with the expression of CD59 and M2 tumor-associated macrophage infiltration in the TME via the IL10/pSTAT3 pathway in CESC and GBM but not in KIRC. In addition, TGFβ1-dominant cancers such as CESC, GBM, and HNSC showed a high correlation between CD59 and TGFβ1, leading to suppression of cytotoxic T cell activity. Overall, the correlation between CD59 and immune cells predicts its prognosis as unfavorable in CESC, GBM, HNSC, and STAD while being favorable in KIRC.

FOXP3+/CD8+ ratio associated with aggressive behavior in RUNX3-methylated diffuse esophagogastric junction tumor.

The tumor immune microenvironment is increasingly becoming a key consideration in developing treatment regimens for aggressive cancers, with evidence that regulatory T cells (Tregs) attenuate the antitumor response by interrupting cytotoxic T cells (CD8+). Here, we hypothesized the prognostic relevance of the proportions of Tregs (marked by forkhead box protein 3 [FOXP3]) and CD8+ cells in diffuse, non-Epstein-Barr virus (EBV)/non-microsatellite instability (MSI)-high gastroesophageal adenocarcinomas (GEAs), which are clinically characterized as more aggressive, immunologically inactive tumors as compared with their intestinal counterparts. Cell-count ratios of FOXP3+/CD8+ expression were calculated at the intratumoral region and invasive margin discretely on digital images from 303 chemo-naive non-EBV/non-MSI-high esophagogastric junction (EGJ) adenocarcinomas. A significant modifying prognostic effect of tumor histology was observed between 5-year EGJ cancer-specific survival and the FOXP3+/CD8+ ratio at the invasive margin in pStage I-III tumors (p for interaction = 0.022; hazard ratio [HR] = 8.47 and 95% confidence interval [CI], 2.04-35.19 for high ratio [vs. low] for diffuse; HR = 1.57 and 95% CI, 0.88-2.83 for high ratio [vs. low] for intestinal). A high FOXP3+/CD8+ ratio at the invasive margin was associated with RUNX3 methylation (p = 0.035) and poor prognosis in RUNX3-methylated diffuse histological subtype (5-year EGJ cancer-specific survival, 52.3% for high and 100% for low, p = 0.015). Multiomics data from The Cancer Genome Atlas linked CCL28 with RUNX3-suppressed diffuse histological subtypes of non-EBV/non-MSI-high GEA. Our data suggest that a high FOXP3+/CD8+ ratio at the invasive margin might indicate tumor immune escape via CCL28, particularly in the RUNX3-methylated diffuse histological subtype.

Tumor-infiltrating immune cell profiles and changes associate with additional trastuzumab in preoperative chemotherapy for patients with HER2-positive gastric cancer

BackgroundHER2(+) gastric cancer (GC) can benefit from trastuzumab. However, the impact of additional trastuzumab in preoperative treatment on immune cells remains largely unknown.MethodsIn cohort I, immune cells were detected by immunohistochemistry in 1321 patients. Then 88 HER2(+) patients received preoperative therapy were collected as cohort II. Immune cell profiles and changes were analyzed in paired pre- and post-operative specimens using multiple immunohistochemistry staining.ResultsIn the treatment-naive GC patients (n = 1002), CD3+ and CD8+ T cell infiltration was significantly lower in the HER2(+) GC patients together with higher FoxP3+ T cells compared with HER2(−). However, FoxP3+ T and CD20+ B cell infiltration was significantly higher in HER2(+) GC after neoadjuvant chemotherapy (n = 319). The trastuzumab-exposed group had higher CD8+ T and lower FoxP3+ T cell infiltration and CD8+ T cell was even more significant in responders. Additionally, tertiary lymphoid structure (TLS) density increased in invasion margin of residual tumors. Patients with lower TLS in the tumor core or lower FoxP3+ T cells had better overall survival in the trastuzumab-exposed group.ConclusionAddition of trastuzumab modulates the immune microenvironment, suggesting the potential mechanism of the favorable outcome of anti-HER2 therapy and providing a theoretical rationale for the combinational immunotherapy in resectable HER2(+) GC patients.

MCT4 is an independent prognostic factor and affects immune cell infiltration in patients with colorectal liver oligometastases.

Monocarboxylate transporter 4 (MCT4) is a novel biomarker related to the level of immune cell infiltration, but its impact on tumor immune microenvironment (TIME) of colorectal liver oligometastases (CLO) remains unclear. The aim of this study was to assess MCT4 expression in primary tumor and liver oligometastases, investigate its impact on immune cell infiltration and its prognostic value for CLO patients undergoing liver resection. We retrospectively selected 135 CLO patients who underwent curative liver resection between June 1999 and December 2016, and samples included 74 primary tumor tissues and 122 liver metastases. Immunohistochemistry (IHC) was performed to detect MCT4 expression in paraffin-embedded specimens and tyramine signal amplification (TSA) was used to detect the density of tumor-infiltrating lymphocytes, including CD3 + , CD8 + and Foxp3 + . Recurrence-free survival (RFS) and overall survival (OS) were analyzed using the Kaplan-Meier method and log-rank test, and independent prognostic factors were identified with Cox regression modeling. Survival analysis indicated that CLO patients with low MCT4 expression had better 3-year RFS and 3-year OS rates than those with high MCT4 expression. Multivariate analysis indicated that high MCT4 expression was independently associated with poor RFS and OS. High MCT4 expression was associated with a lower number of intratumoral CD3 + /CD8 + T cells and was associated with higher Foxp3 + T cells infiltration. Patients with low MCT4 expression and high levels of differential immune infiltration had longer survival. MCT4 overexpression was associated with an unfavorable prognosis in patients with CLO and MCT4 expression level had an impact on intratumoral immune infiltration degree. A novel parameter that combined MCT4 expression level and differential immune infiltration level was constructed to stratify patients with CLO into different risk groups.

CD25+FOXP3+CD45RA− regulatory T-cell infiltration as a prognostic biomarker for endometrial carcinoma

BackgroundRegulatory T (Treg) cells reportedly play crucial roles in tumor angiogenesis as well as antitumor immunity. In order to explore their therapeutic potential, we investigated the precise prognostic impact of Treg markers in endometrial carcinoma.MethodsWe performed multiplexed immunofluorescence and quantitative image analyses of CD25, FOXP3, CTLA4, and CD45RA in tumor specimens from 176 consecutive patients treated at our institution for primary endometrial carcinomas. Bioinformatics analyses were further conducted to corroborate the findings.ResultsHigh CD25+, FOXP3+, and CD25+FOXP3+CD45RA− stromal cell counts correlated with better overall survival (OS) (p = 0.00019, 0.028 and 0.0012) and MSI-high (p = 0.015, 0.016 and 0.047). High CD45RA+ stromal cell count was associated with superficial myometrial invasion (p = 0.0038). Bioinformatics survival analysis by Kaplan-Meier plotter showed that high CD25, FOXP3, CTLA4, and CD45RA mRNA expressions correlated with better OS (p = 0.046, 0.00042, 0.000044, and 0.0022). Univariate and multivariate analyses with various clinicopathologic prognostic factors indicated that high CD25+ or CD25+FOXP3+CD45RA− stromal cell count was significant and independent for favorable OS (p = 0.0053 and 0.0015). We subsequently analyzed the correlations between the multiplexed immunofluorescence results and treatment-free interval (TFI) after primary chemotherapy in recurrent cases, finding no significant associations. Further analysis revealed that high ratio of CD25+ : CD8+ cell count or CD25+FOXP3+CD45RA− : CD8+ cell count correlated with longer TFI (p = 0.021 and 0.021).ConclusionThe current observations suggest that the balance between CD25+ or CD25+FOXP3+CD45RA− cells and CD8+ cells, corresponding to promoting or inhibiting effect on tumor angiogenesis, affect tumor chemosensitivity leading to prognostic significance. CD25+FOXP3+CD45RA− effector Treg tumor infiltration may serve as a useful prognostic biomarker and a potential target for immunotherapeutic manipulation of tumor chemosensitivity by novel management for advanced/recurrent endometrial carcinomas.

Clinical efficacy and immune response of neoadjuvant camrelizumab plus chemotherapy in resectable locally advanced oesophageal squamous cell carcinoma: a phase 2 trial

BackgroundNeoadjuvant immunotherapy is under intensive investigation for esophageal squamous cell carcinoma (ESCC). This study assesses the efficacy and immune response of neoadjuvant immunochemotherapy (nICT) in ESCC.MethodsIn this phase II trial (ChiCTR2100045722), locally advanced ESCC patients receiving nICT were enrolled. The primary endpoint was the pathological complete response (pCR) rate. Multiplexed immunofluorescence, RNA-seq and TCR-seq were conducted to explore the immune response underlying nICT.ResultsTotally 42 patients were enrolled, achieving a 27.0% pCR rate. The 1-year, 2-year DFS and OS rates were 89.2%, 64.4% and 97.3%, 89.2%, respectively. RNA-seq analysis highlighted T-cell activation as the most significantly enriched pathway. The tumour immune microenvironment (TIME) was characterised by high CD4, CD8, Foxp3, and PD-L1 levels, associating with better pathological regression (TRS0/1). TIME was categorised into immune-infiltrating, immune-tolerant, and immune-desert types. Notably, the immune-infiltrating type and tertiary lymphoid structures correlated with improved outcomes. In the context of nICT, TIM-3 negatively influenced treatment efficacy, while elevated TIGIT/PD-1 expression post-nICT correlated positively with CD8+ T cell levels. TCR-seq identified three TCR rearrangements, underscoring the specificity of T-cell responses.ConclusionsNeoadjuvant camrelizumab plus chemotherapy is effective for locally advanced, resectable ESCC, eliciting profound immune response that closely associated with clinical outcomes.

Frequency of CD4+CD25 + (High) Foxp3+ Regulatory T cells (Tregs) in Peripheral Blood of Pediatric Patients with Autism

Abstract Background Immune system dysfunctions have been observed in autistic spectrum of disorders (ASD). Autoimmunity may have a role in ASD. In addition, skewed cytokine responses, differences in total numbers and frequencies of immune cells and their subsets, neuroinflammation, both adaptive and innate immune dysfunction, altered levels of immunoglobulins and the presence of autoantibodies in some autistic children were reported. Objective To measure the frequency of CD4+CD25 + (High) Foxp3+ Tregs in peripheral blood of children with autism in relation to the disease severity. Patients and Methods This cross-sectional study involved 30 ASD patients were enrolled from the Psychiatry and Development clinic, Children’s Hospital, Ain Shams University, after excluding children with concomitant allergic diseases or infections. Another 30 healthy children were included as a control group. Severity of ASD and associated convulsions or impaired intelligence were demonstrated. The frequency of CD4+CD25 + (High) Foxp3+ Tregs was assessed using flow cytometry. Data collected were coded, tabulated and analyzed using Microsoft Excel software Statistical analysis (SPSS) (version 21, Chicago, IL, USA). Results Thirty patients with primary ASD were recruited, including 24 males (80%) and 6 females (20%) with mean age of 6.62 ± 2.11 years. Mild illness was diagnosed in 20 patients (66.7%) and severe illness was defined in 10 patients (33.3%)%). CD4+CD25 + (High) Foxp3+ Tregs were significantly lower among autistic children compared to healthy control (mean percentage in patients 15.41 ± 32.1%, mean percentage in control 49.2 ± 42.6%, P-value <0.001). The markers were not correlated with disease severity, epilepsy status or IQ level (P = 0.716, 0.729, 0.472, respectively). However,CD4+CD25 + (High) Foxp3+ Tregs were significantly lower among male patients compared to females(Median in males 1.25 (0-96), Median in females 2.25 (1.5-84.2). CD4+CD25 + (High) lymphocytes less than 3.15 cells were found to be 80% sensitive and 73.5% specific for ASD probability (AUC was 0.816). Whereas percentage of FOXP3 less than 2.55% showed 73.3% and 76.7% sensitivity and specificity, respectively (AUC was 0.753). Conclusion CD4+CD25 + (High) FOXP3, the master promotor of Tregs, was detected in lower levels in ASD Egyptian children. A level of expression lower than 2.5% is both sensitive and specific for ASD diagnosis. We recommend considering CD4+CD25 + (High) FOXP3 as a future therapeutic target in children with ASD to achieve early, efficient management.

Regulatory and memory T lymphocytes infiltrating prostate tumors predict long term clinical outcomes.

The localization, density but mostly the phenotype of tumor infiltrating lymphocytes (TIL) provide important information on the initial interaction between the host immune system and the tumor. Our objective was to assess the prognostic significance of T (CD3+), T regulatory (Treg) (FoxP3+) and T memory (Tmem) (CD45RO+) infiltrating lymphocytes and of genes associated with TIL in prostate cancer (PCa). Immunohistochemistry (IHC) was used to assess the infiltration of CD3+, FoxP3+ and CD45RO+ cells in the tumor area, tumor margin and adjacent normal-like epithelium of a series of 98 PCa samples with long clinical follow-up. Expression of a panel of 31 TIL-associated genes was analyzed by Taqman Low-Density Array (TLDA) technology in another series of 50 tumors with long clinical follow-up. Kaplan-Meier and Cox proportional hazards regression analyses were performed to determine association of these markers with biochemical recurrence (BCR), need for definitive androgen deprivation therapy (ADT) or lethal PCa. TIL subtypes were present at different densities in the tumor, tumor margin and adjacent normal-like epithelium, but their density and phenotype in the tumor area were the most predictive of clinical outcomes. In multivariate analyses, a high density of Treg (high FoxP3+/CD3+ cell ratio) predicted a higher risk for need of definitive ADT (HR=7.69, p=0.001) and lethal PCa (HR=4.37, p=0.04). Conversely, a high density of Tmem (high CD45RO+/CD3+ cell ratio) predicted a reduced risk of lethal PCa (HR=0.06, p=0.04). TLDA analyses showed that a high expression of FoxP3 was associated with a higher risk of lethal PCa (HR=5.26, p=0.02). Expression of CTLA-4, PD-1, TIM-3 and LAG-3 were correlated with that of FoxP3. Amongst these, only a high expression of TIM-3 was associated with a significant higher risk for definitive ADT in univariate Cox regression analysis (HR=3.11, p=0.01). These results show that the proportion of Treg and Tmem found within the tumor area is a strong and independent predictor of late systemic progression of PCa. Our results also suggest that inhibition of TIM-3 might be a potential approach to counter the immunosuppressive functions of Treg in order to improve the anti-tumor immune response against PCa.

Pre-Transplant Frequencies of FoxP3+CD25+ in CD3+CD8+ T Cells as Potential Predictors for CMV in CMV-Intermediate Risk Kidney Transplant Recipients.

Cytomegalovirus (CMV) infection detrimentally influences graft survival in kidney transplant recipients, with the risk primarily determined by recipient and donor serostatus. However, recipient CD8+ T cells play a crucial role in CMV control. The optimal preventive strategy (prophylaxis vs. pre-emptive treatment), particularly for seropositive (intermediate risk) recipients, remains uncertain. We investigated CD8+ T cell subpopulation dynamics and CMV occurrence (DNAemia ≥ 100IU/mL) in 65 kidney transplant recipients, collecting peripheral blood mononuclear cells before (T1) and 1year after transplantation (T2). Comparing the two timepoints, we found an increase in granulocyte, monocyte and CD3+CD8+ T cells numbers, while FoxP3+CD25+, LAG-3+ and PD-1+ frequencies were reduced at T2. CMV DNAemia occurred in 33 recipients (55.8%) during the first year. Intermediate risk patients were disproportionally affected by posttransplant CMV (N = 29/45, 64.4%). Intermediate risk recipients developing CMV after transplantation exhibited lower leukocyte, monocyte, and granulocyte counts and higher FoxP3+CD25+ frequencies in CD3+CD8+ T cells pre-transplantation compared to patients staying CMV negative. Pre-transplant FoxP3+CD25+ in CD3+CD8+ T cells had the best discriminatory potential for CMV infection prediction within the first year after transplantation (AUC: 0.746). The FoxP3+CD25+ CD3+CD8+ T cell subset may aid in selecting intermediate risk kidney transplant recipients for CMV prophylaxis.

OP02 IL23R-CAR-Tregs: creating a therapeutic breakthrough for Crohn’s Disease

Abstract Background Regulatory T cells (Tregs) are key immunomodulators to maintain immune homeostasis and self-tolerance, including in the intestine. Disrupted immune homeostasis is associated with Crohn’s Disease (CD) pathogenesis. As a living drug, Treg therapy is a promising treatment for restoring immune balance and achieving durable tissue tolerance in the diseased intestine. By targeting a disease-relevant protein, Interleukin-23 receptor (IL23R), we engineered Tregs expressing IL23R-chimeric antigen receptor (CAR) to create IL23R-CAR-Tregs for treating CD. Methods IL23R protein expression in CD intestine was verified by immunohistochemical analysis. Tonic signaling and CAR-specific activation were quantified during the IL23R-CAR screening. The IL23R-CAR-Treg phenotype was assessed by monitoring the expression of FoxP3 and Helios transcription factors, and the demethylation status of FoxP3 Treg-specific demethylated region (TSDR). The suppressive function of IL23R-CAR-Tregs was evaluated using in vitro assays and a colitis mouse model. The functionality of IL23R-CAR-Tregs were further assessed using intestinal biopsy-derived cells from CD patients in an in vitro activation assay. Transcriptomic and proteomic analyses were performed to associate molecular profiles with IL23R-CAR-Treg activation. Results Our highly potent IL23R-CAR lead candidate displays negligible tonic signalling and a strong signal-to-noise ratio. The resulting IL23R-CAR-Tregs maintain their regulatory phenotype with high FoxP3 and Helios expression and TSDR demethylation during in vitro expansion, CAR-specific stimulation, and in the presence of proinflammatory cytokines. In accordance with their regulatory phenotype, IL23R-CAR-Tregs exhibit CAR-dependent suppressive activity, promote a tolerogenic environment, and protect mice from colitis. The expression of IL23R was also confirmed in the intestinal mucosa from CD patients. Importantly, IL23R-CAR-Tregs elicit activation following exposure to intestinal biopsy-derived cells from CD, demonstrating the engagement and potency of IL23R-CAR towards their target in CD. We also identified molecular profiles that correlated with IL23R-CAR activation and, more broadly, showed their potential to monitor IL23R-CAR-Treg activity in CD patients. Conclusion Our results demonstrate that an IL23R-CAR-Treg product could be a promising therapy for CD patients, with the potential long-term benefits of immune tolerance.

Stimulation Strength Determined by Superantigen Dose Controls Subcellular Localization of FOXP3 Isoforms and Suppressive Function of CD4+CD25+FOXP3+ T Cells.

Staphylococcal superantigens induce massive activation of T cells and inflammation, leading to toxic shock syndrome. Paradoxically, increasing evidence indicates that superantigens can also induce immunosuppression by promoting regulatory T cell (Treg) development. In this study, we demonstrate that stimulation strength plays a critical role in superantigen-mediated induction of immunosuppressive human CD4+CD25+FOXP3+ T cells. Suboptimal stimulation by a low dose (1 ng/ml) of staphylococcal enterotoxin C1 (SEC1) led to de novo generation of Treg-like CD4+CD25+FOXP3+ T cells with strong suppressive activity. In contrast, CD4+CD25+ T cells induced by optimal stimulation with high-dose SEC1 (1 µg/ml) were not immunosuppressive, despite high FOXP3 expression. Signal transduction pathway analysis revealed differential activation of the PI3K signaling pathway and expression of PTEN in optimal and suboptimal stimulation with SEC1. Additionally, we identified that FOXP3 isoforms in Treg-like cells from the suboptimal condition were located in the nucleus, whereas FOXP3 in nonsuppressive cells from the optimal condition localized in cytoplasm. Sequencing analysis of FOXP3 isoform transcripts identified five isoforms, including a FOXP3 isoform lacking partial exon 3. Overexpression of FOXP3 isoforms confirmed that both an exon 2-lacking isoform and a partial exon 3-lacking isoform confer suppressive activity. Furthermore, blockade of PI3K in optimal stimulation conditions led to induction of suppressive Treg-like cells with nuclear translocation of FOXP3, suggesting that PI3K signaling impairs induction of Tregs in a SEC1 dose-dependent manner. Taken together, these data demonstrate that the strength of activation signals determined by superantigen dose regulates subcellular localization of FOXP3 isoforms, which confers suppressive functionality.

The prognostic value of Forkhead box P3 regulatory T cells in biliary tract cancer: A systematic review and meta-analysis.

This study aimed to explore the value of tumor-infiltrating Forkhead box P3(FoxP3+) regulatory T cells (Tregs) in evaluating the prognosis of biliary tract cancer. Four electronic databases were searched using 2 computers: PubMed, Embase, Web of Science, and Cochrane Library. The vocabulary and syntax were adapted according to the database. Two researchers independently selected the studies, collected information, and assessed the risk of bias. The Meta-analysis was performed using STATA 17.0, and HR and its corresponding 95% CI were used to evaluate the correlation between FoxP3+ Tregs and the overall survival of patients with biliary tract cancer. In addition, the quality of the included studies was evaluated. Ten articles were included in this study. The results of the meta-analysis showed that patients with high FoxP3+ Tregs infiltration had worse overall survival (OS) (HR = 1.34,95% CI 1.16 to 1.71; P < .001). Subgroup analysis of gallbladder carcinoma and cholangiocarcinoma showed that the high infiltration of FoxP3+ Tregs was significantly correlated with the OS of the former (HR = 1.55,95% CI 1.11 to 2.00; P < .001), but not with the OS of the latter (HR = 1.00,95% CI 0.62 to 1.38; P > .05). Our meta-analysis reveals that high infiltration of FoxP3 + Tregs is significantly associated with reduced overall survival in gallbladder carcinoma, endorsing their use as a prognostic biomarker for this subtype. In contrast, no significant prognostic correlation was identified for FoxP3+ Tregs in cholangiocarcinoma, indicating the need for subtype-specific evaluation of their prognostic relevance in biliary tract cancers.

Spatially Resolved Transcriptomics Unveils Unique T-Cell Dysfunctional States and Prognostic Gene Expression Signatures in TP53-Mutated Acute Myeloid Leukemia

Introduction Somatic TP53 mutations and 17p deletions with genomic loss of TP53 occur in 8% to 10% of AML and correlate with primary induction failure, high risk of relapse, and dismal prognosis. We have previously shown that TP53 abnormalities are associated with a pro-inflammatory and immunosuppressive tumor microenvironment (TME), including high CD274 and FoxP3 expression, with dysfunctional natural killer (NK)/CD8 + T-cell states and with response to flotetuzumab, a CD123 × CD3 bispecific T-cell engager. In the current study, we explore the cellular neighborhoods of TP53-mutated ( TP53-m) AML using spatially resolved transcriptomics and we employ machine learning to identify prognostic RNA signatures. Methods We analyzed the expression of &amp;gt;1,800 immune and TME genes in bone marrow (BM) formalin-fixed paraffin-embedded (FFPE) sections (5-µm) from a discovery cohort of 11 patients with chemotherapy-naïve AML (N=5 TP53-m and N=6 TP53-wt cases) using the GeoMx™ Digital Spatial Profiling (DSP) platform (Cancer Transcriptome Atlas with next generation sequencing read-out; NanoString Technologies, Seattle, WA). Imaging was performed at 20× magnification and a total of 151 circular regions of interest (ROIs) with a 300-600 µm diameter were selected (Fig. 1A). We then generated a ROI-by-gene count matrix for deconvolution and downstream pathway analysis. For each ROI, enrichment scores reflecting immune senescence/dysfunction and leukemia stemness were calculated with gene set variation analysis (GSVA), using a manually curated compendium of published gene signatures as input. We also employed public bulk and single-cell RNA-sequencing (sc-RNA-seq) AML cohorts for in silico modeling and validation. Results We initially captured transcripts from ROIs of TP53-m and TP53-wt AML with varying degrees of BM infiltration with T cells (CD3 hotspots) and AML blasts, and we found that TP53-m is enriched in T cell-specific, type I/II interferon (IFN) and pro-inflammatory genes ( IL1B), as well as leukemia stem cell (LSC)-related gene programs (LSC17). After dichotomizing ROIs based on a median split of CD3D RNA expression, we observed that T-cell low ROIs express higher levels of monocyte-macrophage and myeloid derived suppressor cell (MDSC)-related genes, including S100A9 and S100A8. These findings point to previously unappreciated links between BM infiltration with immune-modulatory myeloid cell types and leukemia stemness / T-cell abundance. We then tested for differential cell density in neighborhoods (regions of transcriptionally similar cells) across TP53-m and TP53-wt AML using MiloR in a sc-RNA-seq AML dataset annotated by cell type (N=42 cases, of which N=3 with TP53-m; Lasry A, et al. Nat. Cancer 2023). This orthogonal approach highlighted a significantly higher frequency (FDR &amp;lt;0.05) of multipotent and granulocyte-macrophage progenitor neighborhoods in TP53-m AML, alongside suppression of adaptive immune system pathways (Fig. 1B). We next inferred active receptor-ligand pairs mediating the crosstalk between T cells and leukemic blasts in the TME of TP53-m AML, and we uncovered decreased MHC class I/II-mediated signaling, curbed CD8 +/CD4 + T cell-to-AML interactions and enhanced CD6/ CDCP1( CD318) signaling (Fig. 1C). We also found that CD8 + and CD4 + T cells from TP53-m AML are enriched in effector memory (T EM) / terminally differentiated effector memory (T EMRA) and regulatory T-cell gene programs, respectively, compared with their cellular counterparts from TP53-wt patients (Fig. 1D). In particular, T EM cells from TP53-m AML expressed genes known to regulate T-cell dysfunctionality, foremost PRDM1, and transcription factors ( ZEB2) expected to drive the differentiation of killer cell lectin-like receptor (KLR) + exhausted T-cell states with residual cytolytic potential. Finally, we applied the least absolute shrinkage and selection operator (LASSO) statistical method for feature selection to our discovery AML dataset, and we constructed a TP53-m-related ‘spatial metagene’ that predicted poor overall survival in TCGA-LAML (N=170) and Beat-AML2 cases (N=444; Fig. 1E). Conclusions Spatial gene programs of leukemia stemness, T-cell dysfunction and immune suppression are enriched in TP53-m AML. It remains to be determined whether TP53-m AML with a pre-existing but ineffective T-cell response may be amenable to T cell-targeting immunotherapies.

Investigating the T regulatory cells and Sirtuin-I serum level in immunotherapy treated house dust mite allergic asthma patients

Objectives House dust mite aeroallergens are predominant triggers of frequent asthma attacks among adults and children. The intensity of asthma and immune reaction necessitates treatment alternatives based on adjusting chosen immunity biomarkers to control the exacerbation of symptoms and establish long-term immune tolerance. In this study, we selected CD4+CD25+Foxp3+ regulatory T cells (Tregs), FOXP3, and Sirtuin-1 as they are known to have a potential role in the immune reaction in different allergic diseases. We investigated their interplay during HDM allergic asthma and its respective immunotherapy. Methods Eighty-four subjects were divided into 3 groups; healthy controls (CT), HDM asthma patients without immunotherapy (WOIT), and HDM asthma patients treated with subcutaneous immunotherapy for 6 months before recruitment (WIT). They were enrolled according to the pulmonary function, skin prick tests, and HDM-specific IgE. CD4+ CD25+ and CD4+ CD25+ FOXP3+hi T cells Cell percentages, FOXP3 gene expression, and Sirtuin-1 (Sirt1) serum level were analyzed. Results We found that there is a significant difference between WOIT and WIT groups in the CD4+ CD25+ and CD4+ CD25+ FOXP3+hi T cell percentages. While there is no statistically significant difference between WOIT and WIT groups in FOXP3 level. On the controversy, the SIRT1 level in the CT group (4.53 ± 3.880) significantly decreased in the WOIT and WIT groups. Conclusion This study revealed that both CD4 CD25 and CD4 CD25 high FOXP3 cell percentages increased in the WIT group and declined in the WOIT group. While, FOXP3 gene expression increased in both groups. In addition, the Sirt1 serum level showed some improvement in WIT group after a serious drop in the WOIT group comparing with the CT group. The modulation of these biomarkers for the remission and control of allergic asthma can be a prognostic outcome of immunotherapy which needs to be confirmed by larger scale studies.

Regulatory T cells control Staphylococcus aureus and disease severity of cutaneous leishmaniasis.

Cutaneous leishmaniasis causes alterations in the skin microbiota, leading to pathologic immune responses and delayed healing. However, it is not known how these microbiota-driven immune responses are regulated. Here, we report that depletion of Foxp3+ regulatory T cells (Tregs) in Staphylococcus aureus-colonized mice resulted in less IL-17 and an IFN-γ-dependent skin inflammation with impaired S. aureus immunity. Similarly, reducing Tregs in S. aureus-colonized and Leishmania braziliensis-infected mice increased IFN-γ, S. aureus, and disease severity. Importantly, analysis of lesions from L. braziliensis patients revealed that low FOXP3 gene expression is associated with high IFNG expression, S. aureus burden, and delayed lesion resolution compared to patients with high FOXP3 expression. Thus, we found a critical role for Tregs in regulating the balance between IL-17 and IFN-γ in the skin, which influences both bacterial burden and disease. These results have clinical ramifications for cutaneous leishmaniasis and other skin diseases associated with a dysregulated microbiome when Tregs are limited or dysfunctional.

Study of immunophenotypic characteristics, clinicopathological parameters and prognosis in gastric cancer microenvironment

Objective: To explore the immunophenotypic characteristics of gastric cancer microenvironment and analyze its correlation with clinicopathological parameters and prognosis of patients. Methods: The expression levels of leukocyte differentiation antigen (CD) 8, CD4, T lymphocyte immunoglobulin mucoprotein 3 (TIM3), human forkhead box protein P3 (Foxp3) and co-localized tumor infiltrating lymphocytes (TILs) were detected in 92 cases of gastric cancer tissue [58 males and 34 females; aged M(Q1, Q3), 70(59, 77) years ] and 84 cases of paracancer tissue [57 males and 27 females, aged 70(59, 77) years] purchased from Shanghai Xinchao Biotechnology Co., Ltd., and the samples were from 28 hospitals in the sample bank. Gastric cancer and adjacent tissues were divided into high expression group and low expression group according to the optimal cut-off value of positive lymphocytepercentage. The expression of immunophenotypes in gastric cancer and adjacent tissues was analyzed. Kaplan-Meier method was used for survival analysis. Cox proportional hazard model was used to explore the prognostic factors of gastric cancer patients. Results: The optimal cut-off values of CD8, CD4, TIM3 and Foxp3 positive cells in gastric cancer were 12.73%, 1.39%, 10.77% and 2.44%, respectively. The expression of Foxp3 in gastric cancer tissues was higher than that in paracancer tissues [M (Q1, Q3), 0.93 (0.45, 2.16) vs 0.31 (0.09, 0.86), P<0.001], and the expression of CD8 [4.92 (2.34, 8.80) vs 8.81 (6.61, 12.17), P<0.001], CD4 [4.79 (1.77, 11.36) vs 8.40 (4.84, 12.77), P=0.022] and TIM3 [5.68 (2.05, 11.58) vs 7.07 (3.13, 11.43), P=0.338] were lower than that in paracancer tissues. There were significant differences in TIM3 expression in gastric cancer patients with different lymph node metastasis and clinical stage (all P<0.05). The 5-year survival rate of patients with high CD4 expression, low TIM3 expression and low Foxp3 expression in gastric cancer tissues was poor, among which the high CD4 expression and low CD4 expression groups were 29.3% and 64.7%, respectively; The high and low TIM3 expression groups were 60.9% and 30.4%, respectively; The high and low Foxp3 expression groups were 64.3% and 33.3%, respectively (all P<0.05). The optimal cut-off values of CD8+TIM3+TILs, CD4+TIM3+TILs, CD8+Foxp3+TILs and CD4+Foxp3+TILs were 3.86%, 0.23%, 0.08% and 0.76%, respectively. Colocalization analysis showed that the expression of CD8+Foxp3+TILs in gastric cancer tissues was higher than that in adjacent tissues(all P<0.05). Multivariate Cox regression analysis showed that high expression of CD4 (HR=3.079, 95%CI: 1.350-7.024,P=0.008), low expression of TIM3 (HR=0.428, 95%CI: 0.208-0.879, P=0.021) and low expression of Foxp3 (HR=0.288, 95%CI: 0.121-0.687, P=0.005) were the influencing factor for the 5-year survival rate of patients with gastric cancer after operation. Conclusions: Gastric cancer tissues have complex immune microenvironment characteristics. The expression of CD4, TIM3 and Foxp3 is closely related to the prognosis of patients.

Immunohistological evaluation of patients treated with intra-arterial chemoradiotherapy and surgery for oral cancer.

Preoperative intra-arterial chemoradiotherapy (IACRT) can improve the outcome and reduce the extent of surgery in patients with advanced oral cancer. However, the response to this regimen varies among patients, which may be related to the immune status of the tumor. We investigated the effects of proteins involved in tumor immunity on the outcomes of combined IACRT and surgery for oral cancer. We examined CD8 + and FoxP3 + tumor-infiltrating lymphocytes (TILs) and programmed death ligand 1 (PD-L1) expression on immune cells and tumor cells in pretreatment biopsy samples from 69 patients diagnosed with oral cancer treated with IACRT at our institution during 2000-2020. Patients with abundant CD8 + TILs had significantly better 5-year disease-specific survival (DSS) compared to that of patients with less infiltration of these cells (P = 0.016). Patients with higher FoxP3 + T-cells invasion had significantly better DSS compared to that of less FoxP3 (P = 0.005). Patients with high PD-L1 expression in tumor cells and immune cells had significantly better DSS than that of patients with low PD-L1 expression in these cells (P = 0.009 and P = 0.025, respectively). Collectively, these results suggest that the tumor immune microenvironment could affect outcomes of IACRT treatment in oral cancer.

Spatial Distribution and Densities of CD103+ and FoxP3+ Tumor Infiltrating Lymphocytes by Digital Analysis for Outcome Prediction in Breast Cancer.

The evaluation of tumor-infiltrating lymphocytes (TILs) for breast cancer prognosis is now established. However, the clinical value for their spatial distributions of specific immune subsets, namely CD103+ tissue-resident memory T cells FoxP3+ regulatory T ells, have not been thoroughly examined. Representative whole sections of breast cancers were subjected to CD103 and FoxP3 double staining. Their density, ratio, and spatial features were analyzed in tumor area and tumor-stromal interface. Their associations with clinicopathological parameters and patient's prognosis were analyzed. CD103 TILs were closer to tumor nests than FoxP3 TILs in the tumor-stromal interface. Their densities were associated with high-grade disease, TNBC, and stromal TILs. High stromal FoxP3 (sFoxP3) TILs and close proximity of sCD103 TILs to tumor were independently associated with better survival at multivariate analysis. Subgroup analysis showed the high FoxP3 TILs density associated better survival was seen in HER2-OE and TNBC subtypes while the proximity of CD103 TILs to tumor nests associated better survival was seen in luminal cancers. The prognostic impact of CD103 and FoxP3 TILs in breast cancer depends on their spatial localization. High sFoxP3 TIL density and the lower distance of CD103 TILs from the tumor nests had independent favorable prognostic values.

Effects of ruxolitinib on murine regulatory T cells are immune-context dependent

Aplastic anemia is a bone marrow failure (BMF) disorder characterized by pancytopenia and hypocellular marrow from an immune-mediated etiology. Regulatory T cells (Tregs) prevent autoimmunity by suppressing autoreactive T cells. We recently demonstrated the efficacy of ruxolitinib (RUX), a JAK 1/2 inhibitor, in attenuating murine BMF. Herein, we investigated the changes of Tregs in the context of RUX treatment for murine BMF. Tregs are conventionally identified by surface expression of CD4 and CD25, in addition to intracellular transcription factor FoxP3. RUX promoted the expansion of Tregs in BMF mice defined by increased expression of FoxP3 in CD4 T cells but suppressed expression of activation marker CD25 in CD4 and CD8 T cells. In this context, CD25 is no longer a reliable surface marker for Tregs. We observed strong co-expression of FoxP3 with surface marker GITR instead of CD25 in RUX-treated BMF mice. Fluorescence-activated cell sorting (FACS)-sorted CD4+GITRhi cells showed high FoxP3 expression and intact suppressive function in vitro, suggesting GITR to be a surrogate marker for Tregs. In contrast to its expansive effect on Tregs in BMF, RUX suppressed Tregs in normal and sublethal irradiation conditions, indicating that the effects of RUX on Tregs are immune-context dependent.

Expression of FOXP3 of Tumor-Infiltrating Lymphocytes in Invasive Breast Cancer: Its Relationship to Histopathological Parameters and Overall Survival

BACKGROUND: Forkhead box P3 protein (FOXP3) is expressed in both tumor cells and tumor-infiltrating lymphocytes (TILs) and is reported to be associated with differences in clinical outcomes. Recent literature shows that FOXP3 positive (FOXP3+) T regulator cells (Tregs) influence anti-tumor immunity in solid tumors.&#x0D; АIM: To explore FOXP3+ Tregs expression related to various prognostic factors in breast carcinoma (BC) in the central Indian population. Our study is also helpful in correlating the role of FOXP3+ Tregs in the survival of invasive ductal BC with different histopathological presentations.&#x0D; MATERIALS AND METHODS: This is a retrospective and prospective observational study in which FOXP3+ Tregs was counted in the peritumoral area by immunohistochemistry in 47 consecutive cases of BC operated on and diagnosed already. The patients were followed for 48 to 69 months for disease progression.&#x0D; RESULTS: High-grade tumors are prevalent (n = 30) in the study area irrespective of the stage of clinical presentation. Patients who could adhere to their treatment plan remained free of adverse outcomes until the end of our follow-up period of 69 months (p = 0.001). No molecular subtype in our study showed specific predilection towards a high Tregs count in the peri-tumoral area. No other clinical or pathological parameters significantly correlated with FOXP3 +Treg count, including overall survival and disease-free survival.&#x0D; CONCLUSION: The study shows that luminal human epidermal growth factor receptor 2 negativ and human epidermal growth factor receptor 2 enriched BC show comparatively high FOXP3+ Tregs count. There is no relation with tumor grade, TNM stage, important immune markers, or overall survival and disease-free survival.