Treg Cell Infiltration Research Articles

Abstract 2180: Modulation of immune infiltration patterns in inflammation-associated colorectal cancer through rice bran-based dietary strategies

Abstract The correlation between gut microbiota dysbiosis and inflammation-associated colorectal cancer (CRC) has generated considerable interest in dietary interventions targeting immune modulation. Our prior study demonstrated the colon cancer protective effects of rice bran (RB) and ex vivo Bifidobacterium longum fermented rice bran (FRB) diet intervention against azoxymethane/dextran sodium sulfate (AOM/DSS)-induced CRC in gut microbiota-intact specific pathogen-free mice (SPF). RB showed higher levels of protection compared to FRB against CRC, as evidenced by diminished neoplastic lesion size and restored colonic epithelial integrity. The major objective of the present study was to leverage cutting-edge multispectral imaging technique for simultaneous detection of multiple immune cells markers and for a comprehensive analysis of distinct immune landscapes. We also performed spatial correlations within the tumor microenvironment of AOM/DSS-SPF mice undergoing RB and FRB dietary interventions. The primary focus was on T cell subtypes including cytotoxic T cells, helper T cells, regulatory T (Treg) cells, natural killer (NK) cells, NKT cells, B cells and macrophages (MΦ) subtypes (M1 MΦ, and M2 MΦ) to uncover the intricacies of the immune landscape and the interplay for CRC prevention through dietary intervention. The results indicated that RB intervention for 15 weeks led to reductions in overall T cell subtypes in the colonic tissue, particularly Treg cells, which are associated with protecting tumors against immune surveillance in CRC. On the other hand, FRB intervention also resulted in reductions in overall T cell subtypes but exhibited higher infiltration of NK cells and proinflammatory M1 macrophages in the colonic tissue after 15 weeks. While an increased presence of NK cells might be beneficial in countering CRC, the infiltration of M1 macrophages could potentially lessen the impact of FRB compared to RB intervention in CRC protection. The outcomes suggest that the better protective efficacy of non-fermented RB diets is linked to the modulation of Treg cell infiltration, whereas the efficacy of the FRB diet is to some extent limited by the presence of M1 macrophages. Overall, this study provides valuable insights into the complex immune landscape influenced by RB and FRB interventions in a murine model of inflammation-induced CRC, and a novel mechanism for the immunomodulatory effects of dietary RB interactions with gut microbiota. In the future, observing immune modulation throughout diet intervention would enhance our understanding of the role played by these immune cells. Citation Format: Robin Kumar, Lakshmi Sai Pratyusha Bugata, Md. Imtiazul Kabir, Rajesh Agarwal, Elizabeth P. Ryan, Komal Raina. Modulation of immune infiltration patterns in inflammation-associated colorectal cancer through rice bran-based dietary strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2180.

Lymphatic endothelial cell-mediated accumulation of CD177+Treg cells suppresses antitumor immunity in human esophageal squamous cell carcinoma

ABSTRACT Regulatory T (Treg) cells are critical in shaping an immunosuppressive microenvironment to favor tumor progression and resistance to therapies. However, the heterogeneity and function of Treg cells in esophageal squamous cell carcinoma (ESCC) remain underexplored. We identified CD177 as a tumor-infiltrating Treg cell marker in ESCC. Interestingly, expression levels of CD177 and PD-1 were mutually exclusive in tumor Treg cells. CD177+ Treg cells expressed high levels of IL35, in association with CD8+ T cell exhaustion, whereas PD-1+ Treg cells expressed high levels of IL10. Pan-cancer analysis revealed that CD177+ Treg cells display increased clonal expansion compared to PD-1+ and double-negative (DN) Treg cells, and CD177+ and PD-1+ Treg cells develop from the same DN Treg cell origin. Importantly, we found CD177+ Treg cell infiltration to be associated with poor overall survival and poor response to anti-PD-1 immunotherapy plus chemotherapy in ESCC patients. Finally, we found that lymphatic endothelial cells are associated with CD177+ Treg cell accumulation in ESCC tumors, which are also decreased after anti-PD-1 immunotherapy plus chemotherapy. Our work identifies CD177+ Treg cell as a tumor-specific Treg cell subset and highlights their potential value as a prognostic marker of survival and response to immunotherapy and a therapeutic target in ESCC.

Identification of ANKRD13D as a potential target in renal cell carcinomas.

The correlation of the expression of ankyrin repeat domain (ANKRD) family members with renal cell carcinoma prognosis was investigated. The GEPIA2, GEO2R, UALCAN, GDC, OncoLnc, TIMER, PanglaoDB, CancerSEA, and Tabula Muris databases were used. Twelve ANKRD family members were identified as having overexpressed renal cell carcinoma samples. The ANKRD13D was identified as a renal cell carcinoma-specific target by cross-referencing the multiple survival databases. To clarify the role of ANKRD13D, the expression of NAKRD13D was analyzed at the single-cell level. ANKRD13D was mainly expressed in immune cells and positively correlated with Treg cell infiltration. The expression of ANKRD13D was also positively correlated with PDCD1, CTLA4, LAG3, TNFSF14, and ISG20. The overexpression of ANKRD13D in Treg was confirmed using reverse transcription-quantitative polymerase chain reaction. The structure of ANKRD13D was predicted using AlphaFold. In conclusion, we identified ANKRD13D as a key immune regulator, and targeting ANKRD13D with immune checkpoints blockade may be a promoting strategy for renal cell carcinoma immunotherapy.

Nlrp3 inflammasome drives regulatory T cell depletion to accelerate periapical bone erosion.

Apical periodontitis is an inflammatory disorder triggered by an immune response to bacterial infection, leading to the periapical tissue damage and alveolar resorption. However, the underlying mechanisms driving this process remain elusive, due to the complex and interconnected immune microenvironment within the local lesion site. In this study, the influence of Nlrp3 inflammasome-mediated immune response on the apical periodontitis was investigated. RNA sequencing, immunohistochemistry and ELISA assay were performed to investigate the activation of Nlrp3 inflammasome signalling pathways in the human periapical tissues, including radicular cysts, periapical granulomas and healthy oral mucosa. A mouse model of apical periodontitis was established to study the role of Nlrp3 knockout in periapical bone resorption and Treg cell stability, and the underlying mechanism was explored through invitro experiments. Invivo Treg cell adoptive transfer was performed to investigate the effects of Treg cells on the progression of apical periodontitis. Our findings find that the hyperactivated Nlrp3 inflammasome is present in human periapical lesions and plays a vital role in the immune-related periapical bone loss. Using a mouse model of apical periodontitis, we observe that Nlrp3 deficiency is resistant to bone resorption. This protection was accompanied by elevated generation and infiltration of local Treg cells that displayed a notable ability to suppress RANKL-dependent osteoclast differentiation. In terms of the mechanism of action, Nlrp3 deficiency directly inhibits the osteoclast differentiation and bone loss through JNK/MAPK and NF-κB pathways. In addition, Nlrp3 induces pyroptosis in the stem cells from apical papilla (SCAPs), and the subsequent release of cytokines affects the stability of Treg cell in periapical lesions, leading indirectly to enhanced bone resorption. In turn, adoptive transfer of both Nlrp3-deficient and wild-type Treg cells effectively prevent the bone erosion during apical periodontitis. Together, our data identify that the Nlrp3 inflammasome modulates the Treg cell stability and osteoclastogenesis in the periapical inflammatory microenvironment, thus determining the progression of bone erosion.

Costimulatory Molecule CD226 Regulates Atopic Dermatitis in a Mouse Model

This study investigated the role of CD226 in a 2,4-dinitrochlorobenzene (DNCB)-induced mouse model of atopic dermatitis. The results showed that the lack of CD226 (global and CD4+ T-cell specific) significantly increased ear thickness, reddening, swelling, and scaling of the skin as well as inflammatory cell and mast cell infiltration. RT-qPCR results demonstrated that the mRNA expressions of atopic dermatitis-related inflammatory cytokines and chemokines were markedly increased in the draining lymph nodes and lesioned ear skin tissues of global and CD4+ T-cell-specific CD226-deficient mice compared with that in control mice. Invitro assessment revealed that CD226 directly modulates TGFβ-mediated regulatory T (Treg) cell differentiation and proliferation. Notably, Treg cell-specific deletion of CD226 (Cd226fl/flFoxp3cre mice) resulted in more severe dermatitis and epidermal thickening than those observed in littermate mice upon DNCB treatment. Subsequent analysis showed that the infiltration of Treg cells in ear lesions and the number of Tregs in the spleen were significantly reduced in Cd226fl/flFoxp3cre mice after DNCB treatment. In addition, the lack of CD226 induced apoptosis of Treg cells through the activation of caspase 3. Therefore, these results suggest that CD226 has potential efficacy in atopic dermatitis, correlating with Treg cell inhibition.

Evaluation of regulatory T-cells in cancer immunotherapy: therapeutic relevance of immune checkpoint inhibition.

The evolution of the complex immune system is equipped to defend against perilous intruders and concurrently negatively regulate the deleterious effect of immune-mediated inflammation caused by self and nonself antigens. Regulatory T-cells (Tregs) are specialized cells that minimize immune-mediated inflammation, but in malignancies, this feature has been exploited toward cancer progression by keeping the antitumor immune response in check. The modulation of Treg cell infiltration and their induction in the TME (tumor microenvironment) alongside associated inhibitory molecules, both soluble or membranes tethered in the TME, have proven clinically beneficial in boosting the tumoricidal activity of the immune system. Moreover, Treg-associated immune checkpoints pose a greater obstruction in cancer immunotherapy. Inhibiting or blocking active immune checkpoint signaling in combination with other therapies has proven clinically beneficial. This review summarizes the ontogeny of Treg cells and their migration, stability, and function in the TME. We also elucidate the Treg-associated checkpoint moieties that impede effective antitumor activity and harness these molecules for effective and targeted immunotherapy against cancer nuisance.

Targeting PLCG2 Suppresses Tumor Progression, Orchestrates the Tumor Immune Microenvironment and Potentiates Immune Checkpoint Blockade Therapy for Colorectal Cancer.

Background: Tumor progression and limited benefits of immune checkpoint blockade (ICB) therapy have been two major challenges in the clinical management of colorectal cancer (CRC). The objective of our research was to explore the role of PLCG2 in CRC progression, tumor microenvironment, and potentiating ICB therapy. Methods: Based on bioinformatics analysis and a prospective clinical observational study, the expression, prognostic significance, and clinical relevance of PLCG2 in CRC were unveiled. The single-cell and spatial transcriptome revealed the role of PLCG2 in shaping the heterogeneity of the CRC tumor microenvironment. The biological function of PLCG2 was validated by in vivo and in vitro experiments. The underlying mechanisms were elucidated by RNA-seq, western blotting, qRT-PCR, and multicolor immunofluorescence. The multiplex immunohistochemistry and flow cytometry were adopted to clarify the immunomodulatory role of PLCG2 in facilitating CRC immune escape. The translational value of targeting PLCG2 to potentiate the efficacy of ICB therapy and synergistic therapy to improve prognosis was explored in the preclinical animal models. Results: In CRC, PLCG2 exhibited high expression levels and was strongly associated with poor prognosis and advanced clinicopathological characteristics of patients. The single-cell transcriptome shed light on its important role in cell communication and the development and differentiation of immune cells. The spatial transcriptome described the spatial distribution of PLCG2 in CRC tissues. Further mechanistic analysis demonstrated that PLCG2 could promote proliferation, invasion, metastasis, epithelial-mesenchymal transition, and cell cycle regulation and inhibit apoptosis of CRC cells via the Akt-mTOR pathway activation. Furthermore, PLCG2 was found to contribute greatly to the immunosuppressive microenvironment and enhanced immune escape as it significantly suppressed the infiltration and functional activation of CD8+ T cells and promoted the infiltration of Treg cells as well as PD-1 and PD-L1 expression. Meanwhile, knockdown of PLCG2 could potentiate the efficacy of ICB therapy. Conclusion: In summary, we have identified for the first time that PLCG2 could be considered a precise biomarker and promising therapeutic target for predicting CRC prognosis, optimizing individualized treatment, reversing CRC immune escape, and overcoming resistance to ICB therapy.

Exendin-4-enriched exosomes from hUCMSCs alleviate diabetic nephropathy via gut microbiota and immune modulation.

Diabetic nephropathy (DN) presents a significant therapeutic challenge, compounded by complex pathophysiological mechanisms. Recent studies suggest Exendin-4 (Ex-4) as a potential ameliorative agent for DN, albeit with unclear mechanisms. This research investigates the effects and underlying mechanisms of Ex-4-enriched exosomes derived from human umbilical cord mesenchymal stem cells (hUCMSCs) on DN, focusing on their renoprotective properties and interactions with gut microbiota. Exosomes from hUCMSCs (hUCMSCs-Exo) were loaded with Ex-4 via electroporation. A streptozotocin (STZ) -induced DN mouse model was employed to assess the therapeutic impact of these engineered exosomes. The study further explored immune cell dynamics, mainly CD4+ regulatory T (Treg) cells, using bioinformatics, flow cytometry, and the influence of gut microbiota through antibiotic treatment and specific bacterial reintroduction. Treatment with hUCMSCs-Exo@Ex-4 significantly improved key DN markers, including blood glucose and proteinuria, alleviating kidney damage. A notable decrease in natural Treg cell infiltration in DN was observed, while Ex-4-loaded exosomes promoted CD4+ Treg cell induction. The therapeutic benefits of hUCMSCs-Exo@Ex-4 were diminished upon CD4+ Treg cell depletion, underscoring their role in this context. Notably, CD4+ Treg cell induction correlated with the presence of Prevotella species, and disruption of gut microbiota adversely affected these cells and the therapeutic efficacy of the treatment. However, the reintroduction of Prevotella strains counteracted these adverse effects. This study elucidates a novel therapeutic mechanism of Ex-4-loaded hUCMSCs exosomes in DN, highlighting the induction of CD4+ Treg cells mediated by specific gut microbiota components. These findings underscore the potential of leveraging gut microbiota and immune cell interplay in developing effective DN treatments.

Interleukin-17 directly stimulates tumor infiltrating Tregs to prevent cancer development.

Interleukin-17 (IL-17) family cytokines promote protective inflammation for pathogen resistance, but also facilitate autoimmunity and tumor development. A direct signal of IL-17 to regulatory T cells (Tregs) has not been reported and may help explain these dichotomous responses. We generated a conditional knockout of Il17ra in Tregs by crossing Foxp3-YFP-Cre mice to Il17ra-flox mice (Il17ra ΔTreg mice). Subsequently, we adoptively transferred bone marrow cells from Il17ra ΔTreg mice to a mouse model of sporadic colorectal cancer (Cdx2-Cre +/Apc F/+), to selectively ablate IL-17 direct signaling on Tregs in colorectal cancer. Single cell RNA sequencing and bulk RNA sequencing were performed on purified Tregs from mouse colorectal tumors, and compared to those of human tumor infiltrating Treg cells. IL-17 Receptor A (IL-17RA) is expressed in Tregs that reside in mouse mesenteric lymph nodes and colon tumors. Ablation of IL-17RA, specifically in Tregs, resulted in increased Th17 cells, and exacerbated tumor development. Mechanistically, tumor-infiltrating Tregs exhibit a unique gene signature that is linked to their activation, maturation, and suppression function, and this signature is in part supported by the direct signaling of IL-17 to Tregs. To study pathways of Treg programming, we found that loss of IL-17RA in tumor Tregs resulted in reduced RNA splicing, and downregulation of several RNA binding proteins that are known to regulate alternative splicing and promote Treg function. IL-17 directly signals to Tregs and promotes their maturation and function. This signaling pathway constitutes a negative feedback loop that controls cancer-promoting inflammation in CRC.

A new immune-related gene signature predicts the prognosis and immune escape of bladder cancer.

The biological roles of immune-related genes (IRGs) in bladder cancer (BC) need to be further elucidated. To elucidate the predictive value of IRGs for prognosis and immune escape in BC. We comprehensively analyzed the transcriptomic and clinical information of 430 cases, including 19 normal and 411 BC patients from the TCGA database, and verified 165 BC cases in the GSE13507 dataset. The risk model was constructed based on IRGs by applying LASSO Cox regression and exploring the relationship between the risk score and prognosis, gene mutations, and immune escape in BC patients. We identified 4 survival-related genes (PSMC1, RAC3, ROBO2 and ITGB3) among 6,196 IRGs in both the TCGA and GES13507 datasets,, which were used to establish a gene risk model by applying LASSO Cox regression. The results showed that the high-risk (HR) group was closely associated with poor survival or advanced pathological stage of BC. Furthermore, the risk score was found to be an independent risk factor for prognosis of BC patients. In addition, high-risk individuals showed a greater prevalence of TP53 mutations lower CD8+ T-cell and NK cell infiltration, higher Treg cell infiltration, higher expression of PD-L1, and higher immune exclusion scores than those in the low-risk (LR) group. Finally, the experimental verification shows that the model construction gene, especially PMSC1, plays an important role in the growth and metastasis of bladder cancer. These evidences revealed the vital role of IRGs in predicting prognosis, TP53 mutation and immune escape in BC patients.

HOXC6: A promising biomarker linked to an immunoevasive microenvironment in colorectal cancer based on TCGA analysis and cohort validation

HOXC6 plays an essential part of the carcinogenesis of solid tumors, but its functional relevance within the immune contexture in patients with colorectal cancer (CRC) is still uncertain. We intended to investigate the predictive value of HOXC6 expression for survival outcomes and its correlation with immune contexture in CRC patients by utilizing the Cancer Genome Atlas database (n = 619). Validation was performed in cohorts from Zhongshan Hospital (n = 200) and Shanghai Cancer Center (n = 300). Immunohistochemical (IHC) staining was utilized to compare the levels of immunocytes infiltrating the tumor between the groups with high and low expression of HOXC6. Elevated levels of HOXC6 expression in CRC tissues were linked to malignant progression and poor prognosis. HOXC6 as a risk factor for survival of CRC patients was confirmed. Receiver operating characteristic analysis confirmed its diagnostic value, and a reliable prognostic nomogram was constructed. KEGG analysis and GSEA showed that HOXC6 participated in immune regulation, and its expression was tightly linked to the abundance of infiltrating immunocytes. HOXC6 was upregulated in patients diagnosed with CRC within the two cohorts, and high HOXC6 levels were correlated with a worse prognosis. The high-HOXC6 expression group showed increased infiltration of Treg cells, CD68+ macrophages, CD66b+ neutrophils, and CD8+ T-cells and elevated levels of PD-L1 and PD-1, but decreased levels of granzyme B and perforin. These findings suggest that HOXC6 abundance in patients with CRC determines a poor prognosis, promotes an immunoevasive environment, and directs CD8+ T-cell dysfunction. HOXC6 is expected to become a prospective biomarker for the outcome of CRC.

PBRM1 mutation and WDR72 expression as potential combinatorial biomarker for predicting the response to Nivolumab in patients with ccRCC: a tumor marker prognostic study.

Immune checkpoint therapy (ICT) provides a new idea for the treatment of advanced clear cell renal cell carcinoma (ccRCC), which can bring significant benefits to patients. However, the clinical application of ICT is limited because of the lack of predictive biomarkers to select potential responders. This study aims to propose a new biomarker to predict the response to Nivolumab in patients with ccRCC. The genes that significantly improve the prognosis of ccRCC were retrieved from The Cancer Genome Atlas (TCGA) database. The genomic and clinical data were from patients that had been registered in prospective clinical trials (CheckMate 009, CheckMate 010 and CheckMate 025). TCGA, Gene Expression Omnibus (GEO), and The Human Protein Atlas database were used to analyze the gene and protein expression of WD repeat-containing protein 72 (WDR72) in ccRCC. Gene Ontology (GO) & The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) were performed to dig relevant mechanisms of WDR72. Single sample gene set enrichment analysis (ssGSEA) was conducted to evaluate the role of WDR72 in immune infiltration. Cell proliferation assay, FAO and ATP quantification were used to explore and verify the molecular mechanisms. The expression of WDR72, FOXP3, CD8, and CPT1A was examined by IHC in 20 advanced ccRCC tissue samples at the Urology Department of our hospital. The MethSurv was used to identify PBRM1 and WDR72 gene methylation and its effect on prognosis of ccRCC. WDR72 is the most significant gene for improving overall survival (OS) in ccRCC. In all three checkmates, OS and progression free survival (PFS) were found to be significantly higher in WDR72 high expression group than that in WDR72 low expression group (P=0.040 and P=0.012, respectively), and similar conclusions could be drawn from the PBRM1-mutation (MUT) compared with the PBRM1-wildtype (WT) (P=0.007 and P=0.006, respectively). What's more, high expression of WDR72 plus PBRM1-MUT as a combinatorial biomarker showed improved OS (HR=0.388, P=0.0026) and PFS (HR=0.39, P=0.0066) compared to low expression of WDR72 plus PBRM1-WT. Functional enrichment analysis showed that WDR72 was closely positively related to fatty acid degradation and fatty acid beta oxidation pathway in ccRCC. In vitro experiments showed that high expression of WDR72 can promote fatty acids oxidation and inhibit the proliferation of ccRCC cells. Immune analysis revealed that WDR72 high expression was associated with decreased infiltration of Treg cells and low ssGSEA score of check-point. IHC results showed that WDR72 was negatively correlated with FOXP3 expression (r=-0.506, P=0.023) and positively correlated with CPT1A expression (r=0.529, P=0.017). The present study indicated that high expression of WDR72 may indicate a good prognosis of patients treated with Nivolumab and WDR72 expression combined with PBRM1 mutation could be more persuasive to predict the response for ICT in ccRCC patients.

Specific PD-1 Deletion on Regulatory T Cells Leads to Enhanced Anti-Tumor Responses

Programmed cell death 1 (PD-1) is a T cell inhibitory receptor and the most extensively exploited therapeutic target of checkpoint immunotherapy in cancer. Ligation of PD-1 by its ligands PD-L1 or PD-L2 leads to inhibition of CD4 + and CD8 + T cell responses. However, very little is known about the specific role of PD-1 in regulatory T (Treg) cells. Previous studies have shown that PD-1 engagement during induction of Treg polarization by TCR/CD28 signaling in the presence of IL-2 and TGF-β promoted the generation and expansion of Treg cells with potent suppressive function. It has also been shown that patients with high numbers of PD-1 + Treg cells have unfavorable responses to PD-1 checkpoint immunotherapy. Moreover, patients who developed hyperprogressive disease after PD-1 blocking immunotherapy exhibited enhanced Treg cell proliferative ability compared to patients with non-hyperprogressive disease. In the present study, we sought to understand the role of PD-1 on Treg cells in the context of cancer. We generated mice with conditional targeting of the Pdcd1 gene (encoding for PD-1) and crossed them with FoxP3Cre-YFP ( Pdcd1 f/fFoxP3Cre) resulting in specific deletion of PD-1 in Treg cells. Under steady-state conditions, the percentage of Treg cells in the spleen and lymph nodes remained comparable between Pdcd1 f/fFoxP3Cre and Pdcd1wt/wtFoxP3Cre control mice. However, PD-1-deficient Treg cells had a more activated phenotype compared to their counterparts in control mice, with higher expression of CD69, ICOS, and PD-L1. Furthermore, Treg cells in Pdcd1 f/fFoxp3Cre mice expressed increased levels of GITR, CTLA-4, ICOS, and PD-L1 compared to endogenous CD4 + T conventional cells (CD4 +Foxp3 -, Tconv) and CD8 + T cells. To investigate the implications of these immunological changes in anti-tumor responses, we used the MC38 colon adenocarcinoma syngeneic mouse model. We found a smaller tumor size in Pdcd1 f/fFoxP3Cre mice compared to their control counterparts. Treg cells from spleens and tumor-draining lymph nodes (tdLNs) showed minimal differences in their activation state, expression of checkpoint markers, and proliferation levels, as determined by direct ex vivo assessment. Similarly, CD4 + Tconv and CD8 + T cells from spleens and tdLNs showed a very low level of activation that was comparable between the two experimental groups. In the tumor site, however, Pdcd1 f/fFoxp3Cre tumor-bearing mice had a significant reduction in the fractions of CD4 + Tconv and Treg cells and a significant increase in the fraction of CD8 + T cells compared to control tumor-bearing mice. Detailed analysis revealed that in tumor-bearing Pdcd1 f/fFoxp3Cre mice intratumoral naive and central memory CD4 + T cells were reduced whereas CD4 + T effector cells were increased. Similarly, in Pdcd1 f/fFoxp3Cre tumor-bearing mice we noticed a reduction in naïve and central memory CD8 + T cells but a significant expansion in the effector CD8 + T cells . No significant differences were detected in the expression of activation markers in tumor infiltrating Treg cells between the two groups. In contrast, in Pdcd1 f/fFoxp3Cre tumor-bearing mice, tumor infiltrating CD4 + Tconv cells displayed an activated phenotype with upregulation of PD-L1, ICOS, TIM3, CD69, and CTLA-4, downregulated PD-1, and increased IFN-γ production. Tumor infiltrating CD8 + T cells also displayed an activated phenotype with upregulation of activation markers such as CD69, ICOS, PD-L1, TIM3, and LAG3 but reduced PD-1 expression. Notably, both CD4 + Tconv and CD8 + T cells proliferated more robustly in the Pdcd1 f/fFoxp3Cre tumor-bearing mice as determined by the high expression of the proliferation marker Ki67. These results indicate that selective ablation of PD-1 in Treg cells confers a less suppressive immunological environment and allows for expansion and activation of CD4 + and CD8 + T cells, leading to anti-tumor responses from CD4 + and CD8 + T effector cells.

FOXP3-positive cell infiltration in the chorionic villi is increased in the placenta accreta and decreased in the placental abruption.

Growing data suggest a role of Treg cells in placentation. The aim of the study was to evaluate Treg cells (FOXP3-positive cells) placental bed infiltration in patients with placenta accrete syndrome (PAS) and patients who experienced placental abruption. The study group included 13 patients with PAS and the control group consisted of 66 women who had caesarean (CD) delivery of whom, 44 patients with elective caesarean (EC) delivery, and 22 patients with urgent caesarean (UC) delivery due to placental abruption. FOXP3 cell infiltration was assessed by means of immunohistochemistry in placental chorionic villous (CV) and in the decidua (D) and cumulatively in the placental bed (PB). We observed significant difference in the degree of FOXP3-positive cell CV infiltration between studied groups (p = 0.04). FOXP3-positive cells were the most commonly observed in PAS patients, while, they were the least frequently presented in patients after UC. The immunoreactivity for FOXP3-positive cells in CV were as follows: PAS 5 (38%), urgent CS 1 (5%) and elective CS 8 (18%) subjects. We found no difference in the presence of FOXP3-positive cells in the D (p = 0.35) and in the PB (p = 0.23) of analyzed groups. FOXP3-cell infiltration was not related with patient age, BMI, gestational age and neonatal birth weight. Our study provides further evidence that abnormal invasive placentation is an associated disturbance of the maternal immune response. Accordingly, we have theorized that alteration of the FOXP3-positive Treg cell infiltration into the placental bed allows trophoblast cell invasion.

Targeting regulatory T cells by E7777 enhances CD8 T-cell-mediated anti-tumor activity and extends survival benefit of anti-PD-1 in solid tumor models.

Regulatory T cell (Treg)-targeting cancer immunotherapy aims to transiently deplete Treg cells in the tumor microenvironment, without affecting effector T cells (Teff), thus both enhancing anti-tumor activity and avoiding autoimmunity. This study evaluated whether adding E7777 (a new formulation of denileukin diftitox [DD]) improved the efficacy of anti-PD-1 antibody therapy. DD is a recombinant protein containing the hydrophobic and catalytic portions of diphtheria toxin fused to full-length human IL-2. E7777 has the same amino acid sequence and brief circulatory half-life as DD, but with greater purity and potency. Subcutaneous syngeneic murine solid tumor models (colon cancer CT-26 and liver cancer H22) were used to evaluate safety, efficacy, and overall survival with E7777 and anti-PD-1 antibodies, each administered as monotherapy or in concurrent or sequential combination. In Experiment 1, treatments were compared to assess anti-tumor activity at various time points, with tumors excised and dissociated and tumor leukocytes characterized. In Experiment 2, tumor growth, response, and overall survival were characterized for 100 days following a 3-week treatment. E7777 administered in combination with anti-PD-1 led to significantly increased anti-tumor activity and durable, extended overall survival compared to either treatment alone. In both tumor models, the Treg cell infiltration induced by anti-PD-1 treatment was counterbalanced by co-treatment with E7777, suggesting potential synergistic activity. Combination therapy showed the most favorable results. Treatment with E7777 was safe and well-tolerated. Combined E7777 and anti-PD-1 therapy was well tolerated and more effective than monotherapy with either drug.

GPC3-IL7-CCL19-CAR-T primes immune microenvironment reconstitution for hepatocellular carcinoma therapy.

Chimeric antigen receptor (CAR)-T-cell therapy is a revolutionary treatment that has become a mainstay of advanced cancer treatment. Conventional glypican-3 (GPC3)-CAR-T cells have not produced ideal clinical outcomes in advanced hepatocellular carcinoma (HCC), and the mechanism is unclear. This study aims to investigate the clinical utility of novel GPC3-7-19-CAR-T cells constructed by our team and to explore the mechanisms underlying their antitumor effects. We engineered a novel GPC3-targeting CAR including an anti-GPC3 scFv, CD3ζ, CD28 and 4-1BB that induces co-expression of IL-7 at a moderate level (500pg/mL) and CCL19 at a high level (15000pg /mL) and transduced it into human T cells. In vitro, cell killing efficacy was validated by the xCELLigence RTCA system, LDH nonradioactive cytotoxicity assay and was confirmed in primary HCC organoid models employing a 3D microfluid chip. In vivo, the antitumor capacity was assessed in a humanized NSG mouse xenograft model. Finally, we initiated a phase I clinical trial to evaluate the safety and effect of GPC3-7-19-CAR-T cells in the clinic. GPC3-7-19-CAR-T cells had 1.5-2 times higher killing efficiency than GPC3-CAR-T cells. The tumor formation rates in GPC3-7-19-CAR-T cells treated model were reduced (3/5vs.5/5), and the average tumor volumes were 0.74 cm3 ± 1.17 vs. 0.34 cm3 ± 0.25. Of note, increased proportion of CD4+ TEM and CD8+ TCM cells was infiltrated in GPC3-7-19-CAR-T cells group. GPC3-7-19-CAR-T cells obviously reversed the immunosuppressive tumor microenvironment (TME) by reducing polymorphonuclear (PMN)-myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells infiltration and recruiting more dendritic cells (DCs) to HCC xenograft tumor tissues. In one patient with advanced HCC, GPC3-7-19-CAR-T-cell treatment resulted in tumor reduction 56days after intravenous infusion. In conclusion, GPC3-7-19-CAR-T cells achieved antitumor effects superior to those of conventional GPC3-CAR-T cells by reconstructing the TME induced by the dominant CD4+ TEM and CD8+ TCM cell subsets. Most importantly, GPC3-7-19-CAR-T cells exhibited good safety and antitumor efficacy in HCC patients in the clinic. ► Novel GPC3-7-19-CAR-T cells designed with mediate level of IL-7 secretion and high level of CCL19 secretion, which could recruit more mature DCs to assist killing on GPC3+HCCs. ►DC cells recruited by CCL19 could interact with CD4+ T cells and promote the differentiation of CD4+TEFF cells into CD4+TEM and CD8+TCM subsets, leading a better anti-tumor effect on GPC3+HCCs. ►Compared with conventional GPC3-CAR-T, GPC3-7-CCL19-CAR-T cells could reverse tumor immunosuppressive microenvironment by reducing PMN-MDSC and Treg cell infiltration.

Treg cells-derived exosomes promote blood-spinal cord barrier repair and motor function recovery after spinal cord injury by delivering miR-2861

The blood-spinal cord barrier (BSCB) is a physical barrier between the blood and the spinal cord parenchyma. Current evidence suggests that the disruption of BSCB integrity after spinal cord injury can lead to secondary injuries such as spinal cord edema and excessive inflammatory response. Regulatory T (Treg) cells are effective anti-inflammatory cells that can inhibit neuroinflammation after spinal cord injury, and their infiltration after spinal cord injury exhibits the same temporal and spatial characteristics as the automatic repair of BSCB. However, few studies have assessed the relationship between Treg cells and spinal cord injury, emphasizing BSCB integrity. This study explored whether Treg affects the recovery of BSCB after SCI and the underlying mechanism. We confirmed that spinal cord angiogenesis and Treg cell infiltration occurred simultaneously after SCI. Furthermore, we observed significant effects on BSCB repair and motor function in mice by Treg cell knockout and overexpression. Subsequently, we demonstrated the presence and function of exosomes in vitro. In addition, we found that Treg cell-derived exosomes encapsulated miR-2861, and miR-2861 regulated the expression of vascular tight junction (TJs) proteins. The luciferase reporter assay confirmed the negative regulation of IRAK1 by miR-2861, and a series of rescue experiments validated the biological function of IRAKI in regulating BSCB. In summary, we demonstrated that Treg cell-derived exosomes could package and deliver miR-2861 and regulate the expression of IRAK1 to affect BSCB integrity and motor function after SCI in mice, which provides novel insights for functional repair and limiting inflammation after SCI.

Plant exosomes fused with engineered mesenchymal stem cell-derived nanovesicles for synergistic therapy of autoimmune skin disorders.

Existing therapeutics for autoimmune diseases remain problematic due to low efficacy, severe side effects, and difficulties to reach target tissues. Herein, we design multifunctional fusion nanovesicles that can target lesions for the treatment of autoimmune skin diseases. The grapefruit-derived exosome-like nanovesicles (GEVs) with anti-inflammatory and antioxidant effects are first encapsulated with CX5461, an immunosuppressant with anti-proliferative properties to form GEV@CX5461. In order to enhance therapeutic efficiency and safety, GEV@CX5461 are then fused with CCR6+ nanovesicles derived from membranes of engineered gingiva-derived mesenchymal stem cells (GMSCs). The resulting FV@CX5461 not only maintain the bioactivity of GEVs, CX5461, and GMSC membranes but also home to inflamed tissues rich in chemokine CCL20 through the chemotaxis function of CCR6 on FVs. Moreover, FV@CX5461 reduce the secretion of inflammatory factors, calm down Th17 cell activation, and induce Treg cell infiltration. Finally, impressive therapeutic efficiency in both psoriasis and atopic dermatitis disease models is demonstrated using FV@CX5461 to reshape the unbalanced immune microenvironment. A nanotherapeutic drug delivery strategy is developed using fusion nanovesicles derived from plant and animal cells with high clinical potential.

Oxamate enhances the efficacy of CAR-T therapy against glioblastoma via suppressing ectonucleotidases and CCR8 lactylation

BackgroundChimeric antigen receptor (CAR)-T immunotherapy fails to treat solid tumors due in part to immunosuppressive microenvironment. Excess lactate produced by tumor glycolysis increases CAR-T immunosuppression. The mechanism of lactate inducing the formation of immunosuppressive microenvironment remains to be further explored.MethodsImmunocyte subpopulations and molecular characteristics were analyzed in the orthotopic xenografts of nude mice using flow cytometry assay and immunohistochemical staining after oxamate, a lactate dehydrogenase A (LDHA) inhibitor, and control T or CAR-T cells injection alone or in combination. RT-qPCR, western blot, flow cytometry, immunofluorescence, luciferase reporter assay, chromatin immunoprecipitation and ELISA were performed to measure the effect of lactate on the regulation of CD39, CD73 and CCR8 in cultured glioma stem cells, CD4 + T cells or macrophages.ResultsOxamate promoted immune activation of tumor-infiltrating CAR-T cells through altering the phenotypes of immune molecules and increasing regulatory T (Treg) cells infiltration in a glioblastoma mouse model. Lactate accumulation within cells upregulated CD39, CD73 and CCR8 expressions in both lactate-treated cells and glioma stem cells-co-cultured CD4 + T cells and macrophages, and intracellular lactate directly elevated the activities of these gene promotors through histone H3K18 lactylation.ConclusionsUtilizing lactate generation inhibitor not only reprogramed glucose metabolism of cancer stem cells, but also alleviated immunosuppression of tumor microenvironment and reduced tumor-infiltrating CAR-Treg cells, which may be a potential strategy to enhance CAR-T function in glioblastoma therapy.

Regulatory T cells are associated with the tumor immune microenvironment and immunotherapy response in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with a high risk of distant metastasis, an extremely poor prognosis, and a high risk of death. Regulatory T cells (Tregs) contribute to the formation of a tumor immunosuppressive microenvironment, which plays an important role in the progression and treatment resistance of TNBC. A public single-cell sequencing dataset demonstrated increased infiltration of Tregs in TNBC tissues relative to normal breast tissue. Weighted gene co-expression network analysis was used to identify Treg infiltration-related modules for METABRIC TNBC samples. Subsequently, we obtained two Treg infiltration-associated clusters of TNBC by applying consensus clustering and further constructed a prognostic model based on this Treg infiltration-associated gene module. The ability of the selected gene in the prognostic model, thymidine kinase-1 (TK1), to promote the progression of TNBC was evaluated in vitro. We concluded that two Treg infiltration-associated clusters had different prognoses and sensitivities to drugs commonly used in breast cancer treatment, and multi-omics analysis revealed that the two clusters had different copy number variations of key tumor progression genes. The 7-gene risk score based on TNBC Treg infiltration was a reliable prognostic indicator both in the training and validation cohorts. Moreover, patients with TNBC with high Treg infiltration-related scores lacked the activation of immune activation pathways and exhibited resistance to anti-PD1 immunotherapy. Knocking down TK1 led to impaired proliferation, migration, and invasion of TNBC cells in vitro. In addition, specimens from patients with TNBC with high TK1 expression showed significantly higher Treg infiltration in tumors. Results of spatial transcriptome analysis showed that TK1 positive cells mainly localize in tumor area, and Treg cell infiltration in TNBC tissues was associated with high expression of TK1. Pan-cancer analysis also demonstrated that TK1 is associated with poor prognosis and activation of proliferation pathways in multiple cancers. We established a prognostic model related to Treg infiltration and this model can be used to establish a clinically relevant classification of TNBC progression. Additionally, our work revealed the underestimable potential of TK1 as a tumor biomarker and immunotherapeutic target.