Tregs In Tumor Tissues Research Articles

ScRNA-seq revealed targeting regulator of G protein signaling 1 to mediate regulatory T cells in Hepatocellular carcinoma.

Regulatory T cells (Tregs) are central to determine immune response, thus targeting Tregs for immunotherapy is a promising strategy against tumor development and metastasis. The objective of this study was to identify genes for targeting Tregs to improve the outcome of HCC. We integrated expression data from different samples to remove batch effects and further applied embedding function in Scanpy to conduct sub-clustering of CD4+ T cells in HCC for each of two independent scRNA-seq data. The activity of transcription factors (TFs) was inferred by DoRothEA. Gene expression network analysis was performed in WGCNA R package. We finally used R packages (survminer and survival) to conduct survival analysis. Multiplex immunofluorescence analysis was performed to validate the result from bioinformatic analyses. We found that regulator of G protein signaling 1 (RGS1) expression was significantly elevated in Tregs compared to other CD4+ T cells in two independent public scRNA-seq datasets, and increased RGS1 predicted inferior clinical outcome of HCC patients. Multiplex immunofluorescence analysis supported that the higher expression of RGS1 in HCC Tregs in tumor tissue compared to it in adjacent tissue. Moreover, RGS1 expression in Tregs was positively correlated with the expression of marker genes of Tregs, C-X-C chemokine receptor 4 (CXCR4), and three CXCR4-dependent genes in both scRNA-seq and bulk RNA-seq data. We further identified that these three genes were selectively expressed in Tregs as compared to other CD4+ T cells. The activities of two transcription factors, recombination signal binding protein for immunoglobulin kappa J region (RBPJ) and yin yang 1 (YY1), were significantly different in HCC Tregs with RGS1 high and RGS1 low. Our findings suggested that RGS1 may regulate Treg function possibly through CXCR4 signaling and RGS1 could be a potential target to improve responses for immunotherapy in HCC.

Blockage of the IDO1 pathway by charge-switchable nanoparticles amplifies immunogenic cell death for enhanced cancer immunotherapy

Immunosuppressive tumor microenvironment (ITM), poor immunogenicity, and low tumor penetration markedly reduce the capability of tumor immunotherapy. To address these challenges, we successfully engineered acidity-triggered nanoparticles (NPs) with size reduction and charge switchable features to boost tumor immunotherapy based on indoleamine 2,3-dioxygenase 1 siRNA (IDO1 siRNA) and immunogenic cell death (ICD). The NPs significantly augmented tumor penetrating ability and improved cellular uptake via the detachment of 2,3-dimethylmaleic anhydride-grafted poly(ethylene glycol)-poly(L-lysine) copolymer (mPEG-PLL-DMA, PLM) from large-sized NPs with a negative charge. Subsequently, the NPs with a positive charge and small size rapidly escaped from the lysosomes and released mitoxantrone (MIT) and IDO1 siRNA. The antitumor immune response of IDO1 siRNA and MIT provided good antitumor capability by enhancing DC maturation, improving the number of CTLs, and downregulating the level of Tregs in tumor tissues. In summary, the results demonstrated that charge-switchable NPs based on the blockage of the IDO1 pathway and ICD activation induce an efficient antitumor immune response, thus showing high potential for treating primary/distant tumors and reducing metastasis. Statement of significanceAcidity-triggered nanoparticles (NPs) with size reduction and charge reversal to boost tumor immunotherapy based on indoleamine 2,3-dioxygenase 1 siRNA (IDO1 siRNA) and immunogenic cell death (ICD) were engineered. NPs augmented tumor penetrating ability and improved cellular uptake through the detachment of mPEG-PLL-DMA (PLM) from the large-sized MIT/siR-PLM/PPA NPs with negative charge to expose miniature and positively charged MIT/siR-PPA NPs. The NPs rapidly escaped from the lysosome and sequentially released mitoxantrone (MIT) and IDO1 siRNA. The antitumor synergistic effect of inhibiting the IDO1 pathway by IDO1 siRNA and inducing ICD by MIT provided good antitumor capability by enhancing DC maturation, improving the number of CTLs, and downregulating the level of Tregs in tumor tissues. Thus, the NPs showed a promising pathway against aggressive and difficult-to-treat cancers.

CCR8-targeted specific depletion of clonally expanded Treg cells in tumor tissues evokes potent tumor immunity with long-lasting memory.

Foxp3-expressing CD25+CD4+ regulatory T cells (Tregs) are abundant in tumor tissues. Here, hypothesizing that tumor Tregs would clonally expand after they are activated by tumor-associated antigens to suppress antitumor immune responses, we performed single-cell analysis on tumor Tregs to characterize them by T cell receptor clonotype and gene-expression profiles. We found that multiclonal Tregs present in tumor tissues predominantly expressed the chemokine receptor CCR8. In mice and humans, CCR8+ Tregs constituted 30 to 80% of tumor Tregs in various cancers and less than 10% of Tregs in other tissues, whereas most tumor-infiltrating conventional T cells (Tconvs) were CCR8- CCR8+ tumor Tregs were highly differentiated and functionally stable. Administration of cell-depleting anti-CCR8 monoclonal antibodies (mAbs) indeed selectively eliminated multiclonal tumor Tregs, leading to cure of established tumors in mice. The treatment resulted in the expansion of CD8+ effector Tconvs, including tumor antigen-specific ones, that were more activated and less exhausted than those induced by PD-1 immune checkpoint blockade. Anti-CCR8 mAb treatment also evoked strong secondary immune responses against the same tumor cell line inoculated several months after tumor eradication, indicating that elimination of tumor-reactive multiclonal Tregs was sufficient to induce memory-type tumor-specific effector Tconvs. Despite induction of such potent tumor immunity, anti-CCR8 mAb treatment elicited minimal autoimmunity in mice, contrasting with systemic Treg depletion, which eradicated tumors but induced severe autoimmune disease. Thus, specific removal of clonally expanding Tregs in tumor tissues for a limited period by cell-depleting anti-CCR8 mAb treatment can generate potent tumor immunity with long-lasting memory and without deleterious autoimmunity.

Abstract 3958: FAK inhibition reduces tumor infiltration of Tregs via restricting CCL5 release and boosts the present therapeutic regimen for BRAFV600E-mutated colorectal carcinoma

Abstract Background: BRAFV600E-mutated colorectal carcinoma (CRC) is a refractory disease with poor prognosis and limited therapeutic approaches. Recently, researches have indicated that the recruitment of Tregs in tumor tissue promoted by BRAFV600E is responsible for the weakened antitumor immunity. Whereas, as the main targeting therapy for this intractable carcinoma, BRAFV600E inhibitor has little response. In addition, FAK, being the novel therapeutic target in CRC, is considered to have an impact on the release of chemokines. In this study, we sought to explore the capacity of FAK inhibitor restricting Treg infiltration in tumor and its mechanism, as well as the synergistic antitumor effect with the present therapeutic strategy for BRAFV600E-mutated CRC. Methods: Cell proliferation was determined by CCK-8 assay and colony formation assay. Migration ability was detected by transwell assay. Cell cycle and cell group analysis were assessed by flow cytometry. Western blot, Elisa and RT-qPCR analysis were conducted for mechanism exploration. Immunohistochemistry and immunofluorescence analysis were performed to confirm the link between BRAFV600E, FAK and Tregs. CRC xenograft models were established to detect the Treg recruitment and evaluate the synergistic antitumor effect in vivo. Results: BRAFV600E-mutated CRC was of higher expression of FAK, and hence more sensitive to FAK inhibitor APG-2449. Besides, BRAFV600E-mutated in CRC was linked with more Treg infiltration in tumor site, therefore contributed to the attenuated antitumor immunity. What’s more, despite directly inhibiting tumor proliferation and migration, one of the important mechanisms of FAK blockade restricting the growth of BRAFV600E-mutated CRC, was by downregulating the release of Treg chemokine CCL5 from tumor cells through FAK-IL33 axis. With the recruitment of Tregs decreasing induced by FAK inhibitor APG-2449, the number of CD8+T cells relatively elevated in tumor tissue. In BRAFV600E-mutated CRC xenotransplantation models, we revealed that the combination of FAK inhibitor APG-2449 and Vemurafenib was of no significant difference with the combination of Vemurafenib and cetuximab. While adding APG-2449 to the current combination strategy achieved greater tumor suppressing effect than not adding. Conclusions: BRAFV600E-mutated CRC is associated with a higher expression level of FAK, as well as the tumor infiltration of Tregs. FAK inhibitor APG-2449 can promote antitumor immunity by restricting Treg number in tumor tissue, and shows a synergistic effect with the present therapeutic strategy for BRAFV600E-mutated CRC treatment. Citation Format: Luping Yuan, Wentao Pan, Suna Zhou, Qiuyun Luo, Xianglei Yan, Yuxin Zhang, Mengxian Pan, Lin Zhang, Miaozhen Qiu, Dajun Yang. FAK inhibition reduces tumor infiltration of Tregs via restricting CCL5 release and boosts the present therapeutic regimen for BRAFV600E-mutated colorectal carcinoma [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 3958.

FAK Inhibition Reduces Tumor Infiltration of Tregs via Restricting CCL5 Release and Boosts the Combination Therapy for BRAF V600E Mutant Colorectal Carcinoma

Background: BRAFV600E mutant colorectal carcinoma (CRC) is a refractory disease with poor prognosis and has little response to regular chemotherapy and targeted therapy. Recruitment of Tregs in tumor tissue promoted by BRAFV600E is responsible for the weakened antitumor immunity. FAK, a therapeutic target, has an impact on the release of Treg chemokines. Methods: Transwell assay, flow cytometry, western blot, Elisa and RT-qPCR analysis were conducted for mechanism exploration. Fluorescent multiplex immunohistochemistry analysis was performed to confirm the connection of BRAFV600E, FAK and Tregs. CRC xenograft models were established to detect the Treg recruitment and evaluate the synergistic antitumor effect in vivo. Results: CRC cells harboring BRAFV600E mutation was of higher expression of FAK, and hence more sensitive to the tumor inhibitory effect of FAK blockade. FAK inhibitor APG-2449 inhibited the proliferation via inducing G1 phase arrest and suppressed the migration of CRC cells, and APG-2449 reduced the tumor infiltrated Tregs through FAK-IL33-CCL5 axis. Overexpressed FAK in BRAFV600E mutant CRC led to larger amount of tumor infiltrated Tregs. BRAFV600E mutant CRC can benefit from FAK inhibition through downregulating Tregs in tumor site. Moreover, a synergistic antitumor effect of APG-2449 with Vemurafenib and/or Cetuximab in BRAFV600E mutant CRC was observed. Conclusions: BRAFV600E mutation in CRC is associated with higher expression level of FAK, and more Tregs infiltrated in tumors. FAK inhibitor APG-2449 has an impact on antitumor immunity of CRC by decreasing infiltrated Treg numbers, and boosts the targeted combination therapy of BRAF inhibitor and/or EGFR inhibitor for BRAFV600E mutant CRC. Funding Statement: This work was supported by National Natural Science Foundation of China (Grant number. 81602066 and 81772587); Guangdong Esophageal Cancer Institute Science and Technology Program (grant number M201809); CSCO-HengRui Oncology Research Fund (grant number Y-HR2018-184); the third outstanding young talents training plan and Medical Scientist program of Sun Yat-sen University cancer center; Science and Technology Planning Project of Guangzhou, China (Grant number. 201510161726583); National Science and Technology Major Project, China (Grant number. 2016ZX09101004); Natural Science Foundation of Guangdong Province, China (grant number 2018A030310260) and Medical Scientific Research Foundation of Guangdong Province, China (grant no. 2018102516469945). Declaration of Interests: The authors stated: No potential conflicts of interest were disclosed. Ethics Approval Statement: The animal experiments were approved by Animal Use and Care Committee of Sun Yat-sen University.

FOXP3 and CD25 double staining antibody cocktails identify regulatory T cells in different types of tumor tissues using tissue microarrays

Regulatory T cells (Tregs) are CD4+ T cells that express CD25 and transcription factor Forkhead box P3 (FOXP3), and Tregs play a central role in regulation of tumor immunity. FOXP3 immunohistochemistry has been widely used to study Tregs in paraffin embedded tissue, and flow cytometry using fresh tissue has been used to identify FOXP3+/CD25+ double positive Tregs. In our study, we validated the FOXP3/CD25 double staining antibody cocktails for detecting Tregs in paraffin-embedded tissue. Tissue microarrays (TMA) included 115 malignant tumors, 3 ovarian mucinous borderline tumors and 15 benign tissues. Digital image analysis was performed using ImageJ software. Our results showed that FOXP3/CD25 double positive lymphocytes, a subset of FOXP3+ lymphocytes, accounted for variable percentage of the total FOXP3+ lymphocytes and they were positively correlated with FOXP3+ cell counts. Tumors from different sites had variable FOXP3+/CD25+ and FOXP3+ lymphocyte counts. Tumors from lung, head & neck and colon had more and renal cell carcinoma had minimal FOXP3+/CD25+ and FOXP3+ lymphocytes. In conclusion, FOXP3/CD25 double staining antibody cocktails can be easily applied to paraffin-embedded tissue, and FOXP3+/CD25+ Tregs count was positively correlated with FOPX3+ Tregs count but they were not interchangeable. We recommend using CD25/FOXP3 double staining for studying Tregs in tumor tissue.

Surgical trauma-induced CCL18 promotes recruitment of regulatory T cells and colon cancer progression.

Background: Surgical stress has been suggested to facilitate colon cancer growth and metastasis. However, the precise mechanisms by which surgical trauma promotes colon cancer progression remain poorly understood. Methods: To unravel the mechanisms underlying surgery‐induced colon cancer progression, a syngenic transplantation tumor model was established with CT26 cells, and the effect of laparotomy on tumor progression was investigated. Especially, the expression of several chemokines was assessed, and their roles in recruiting CD4+ CD25+ regulatory T cells (Tregs) after surgery were analyzed. Results: Tregs population was significantly increased in the tumor tissue and peripheral blood of tumor‐bearing mice after laparotomy. C‐C motif chemokine ligand 18 (CCL18) expression was significantly upregulated after laparotomy in tumor tissue and the peritoneal cavity of tumor‐bearing mice, and it was positively correlated with the recruitment of Tregs. Functionally, CCL18 knockdown significantly reduces tumor growth and angiogenesis compared with control. Through analysis of Tregs, we found an upregulated proportion of Tregs in tumor tissue, peritoneal cavity, and peripheral blood after laparotomy, but this enhancement was blocked after CCL18 knockdown. In patients with colon cancer, a higher Tregs proportion is positively correlated to more advanced clinical TNM stages and shorter survival. Furthermore, a positive correlation was found between the serum CCL18 level and the Treg proportion in clinical samples. Conclusion: Surgical trauma contributes to colon cancer progression by increasing CCL18 expression and hence promotes Treg recruitment, which leads to an immunosuppressive environment.

Induction of enhanced immunogenic cell death through ultrasound-controlled release of doxorubicin by liposome-microbubble complexes

ABSTRACTImmunogenic cell death (ICD) is a specific kind of cell death that stimulates the immune system to combat cancer cells. Ultrasound (US)-controlled targeted release of drugs by liposome-microbubble complexes is a promising approach due to its non-invasive nature and visibility through ultrasound imaging. However, it is not known whether this approach can enhance ICD induced by drugs, such as doxorubicin. Herein, we prepared a doxorubicin-liposome-microbubble complex (MbDox), and the resultant MbDox was then characterized and tested for US-controlled release of Dox (MbDox+US treatment) to enhance the induction of ICD in LL/2 and CT26 cancer cells and in syngeneic murine models. We found that MbDox+US treatment caused more cellular uptake and nuclear accumulation of Dox in tumor cells, and more accumulation of Dox in tumor tissues. Enhanced induction of ICD occurred both in vitro and in vivo. MbDox+US treatment induced more apoptosis, stronger membrane exposure and the release of ER stress proteins and DAMPs in tumor cells, and increased DC maturation in vitro. In addition, MbDox+US treatment also resulted in stronger therapeutic effects in immunocompetent mice than in immunodeficient mice. Moreover, MbDox+US enhancement of ICD was also evidenced by a higher proportion of activated CD8+ T-lymphocytes but lower Treg in tumor tissues. Taken together, our results demonstrate that US-controlled release of ICD inducers into nuclei using liposome-microbubble complexes may be an effective approach to enhance the induction of ICD for tumor treatment.

CCL22/CCR4 signaling induces immune escape in hepatocellular carcinoma

Objective To investigate the regulation of CCL22/CCR4 signaling on CD4+ CD25+ regulatory T cells (Tregs) and immune escape in hepatocellular carcinoma (HCC). Methods CCL22, interleukin-2 (IL-2), transforming growth factor-β (TGF-β), and interleukin-10 (IL-10) levels in tumor tissue of 30 HCC patients were determined by ELISA. Tumor infiltrating lymphocytes were isolated and assayed by flow cytometry to evaluate the change of CD4+ CD25+ Tregs in tumor tissue, and CCR4 in CD4+ CD25+ Tregs were detected. Results The CCL22 level in tumor tissue was obviously increased. The level of CCL22 in tumor tissue was (920.1±180.1)ng/L, which was significantly higher than that in non-tumor tissue[(227.2±108.6) ng/L; P<0.05]. The tumor infiltrating CD4+ CD25+ Tregs was obviously increased, reaching approximately to (13.3±4.0)%, and the CCR4 expression in CD4+ CD25+ Tregs increased to (8.8±3.0)%. Along with progression in clinical TNM staging, the levels of CD4+ CD25+ Tregs and CD4+ CD25+ CCR4+ Tregs in tumor tissue increased, and were correlated with the CCL22 level. IL-2 level in tumor tissue was decreased, but TGF-β and IL-10 levels were increased. HCC tissue can secrete a large amount of CCL22 that could recruit CD4+ CD25+ Tregs to tumor tissue by activating CCL22/CCR4 signaling. CD4+ CD25+ Tregs played an important role in the immune escape of HCC by releasing plenty of TGF-β and IL-10 and inhibiting IL-2 secretion. Conclusion This study validates CCL22/CCR4 as therapeutic targets in immunotherapy for HCC. Key words: Hepatocellular carcinoma; Regulatory T cells; Immune escape

CD4+ regulatory T cells in gastric cancer mucosa are proliferating and express high levels of IL-10 but little TGF-β.

An increase of regulatory T cells, defined as CD25high- and/or FOXP3+-expressing CD4+ T cells, within tumors has been reported in several studies. Tregs promote tumor growth by modulating the antitumor immune response, mainly through inhibition of T-cell-mediated tumor cell killing: this has been suggested to be dependent on IL-10 and/or TGF-β. In stomach cancer, the mechanisms behind the accumulation of Tregs in tumor tissue has not been fully elucidated, and neither has Treg gene expression in situ. Stomach tissue from gastric cancer patients undergoing gastric resection was analyzed using flow cytometry and cell sorting, followed by RT-PCR. We observed that stomach CD4+ FOXP3+ T cells proliferated to a higher degree than CD4+ FOXP3- T cells, which may contribute to Treg accumulation in the mucosa. By analyzing DNA methylation, we demonstrated that both proliferating and nonproliferating FOXP3+ T cells exhibited complete demethylation of the FOXP3 gene, indicating a stable FOXP3 expression in both cell populations. Furthermore, analysis of T-cell populations isolated directly from the tumor and tumor-free mucosa demonstrated that CD4+ CD25high T cells have a higher IL-10/IFN-γ gene expression ratio but express lower levels of TGF-β than CD4+ CD25low/- T cells. We demonstrate strong proliferation among regulatory CD4+ FOXP3+ CD25high T cells in the gastric cancer mucosa. These local Treg express a suppressive cytokine profile characterized by high IL-10 and low TGF-β and IFN-γ production.

MiR-21 controls in situ expansion of CCR6⁺ regulatory T cells through PTEN/AKT pathway in breast cancer.

Our recent evidence showed that prior expansion of CCR6(+) Foxp3(+) regulatory T cells (Tregs) was important for their dominant enrichment in tumor tissue, which was closely related to poor prognosis of breast cancer patients. However, the underlying regulation mechanism of expansion of CCR6(+) Tregs in situ remains largely unknown. In this study, we reported that miR-21 was highly expressed in CCR6(+) Tregs in tumor tissues from a murine breast cancer model. And silencing of miR-21 could significantly reduce the proliferation of CCR6(+) Tregs in vitro. Adoptive cell-transfer assay further showed that silencing of miR-21 could alter the enrichment of CCR6(+) Tregs in the tumor mass and endow effectively antitumor effect of CD8(+) T cells using a murine breast cancer model. Mechanistic evidence showed that silencing of miR-21 enhanced the expression of its target phosphatase and tensin homolog deleted on chromosome ten (PTEN) and subsequently altered the activation of Akt pathway, which was ultimately responsible for reduced proliferation activity of CCR6(+) Tregs. Finally, we further revealed that miR-21 was also highly expressed on CCR6(+) Tregs in clinical breast cancer patients. Therefore, miR-21 can act as a fine tuner in the regulation of PTEN/Akt pathway transduction in the expansion of CCR6(+) Tregs in tumor sites and provided a novel insight into the development of therapeutic strategies for promoting T-cell immunity by regulating distinct subset of Tregs through targeting specific miRNAs.

Human hepatocellular carcinoma-infiltrating CD4+CD69+Foxp3− regulatory T cell suppresses T cell response via membrane-bound TGF-β1

Tumors can recruit, induce, and expand regulatory T cells (Tregs) to suppress antitumor immune responses for survival and progression. The complicated tumor-related Treg subsets and their functional mechanisms are not fully addressed yet. We have previously identified a novel CD4⁺CD69⁺Foxp3⁻ Treg subset in tumor-bearing mice, which suppresses CD4 T cell response via membrane-bound transforming growth factor beta 1 (mTGF-β1) and then promotes tumor progression. In hepatocellular carcinoma patients, here, we identified tumor-infiltrating human CD4⁺CD69⁺ Tregs which represent ~67.2 % of tumor-infiltrating CD4 T cells that is significantly higher than conventional CD4⁺CD25⁺Foxp3⁺ Tregs. They expressed mTGF-β1, PD-1, and CTLA-4, but not CD25 or Foxp3, and only produced a little interleukin (IL)-10 and TGF-β1. More importantly, they significantly suppressed CD4 T cell response via mTGF-β1 in vitro. Furthermore, the percentage of these CD4⁺CD69⁺ Tregs in tumor tissue was significantly correlated with tumor progression, which is more pronounced at the late stage of cancer patients. Thus, we have identified a tumor-induced new population of human CD4⁺CD69⁺ Tregs in cancer patients with phenotype of CD25⁻Foxp3⁻mTGF-β1⁺CTLA-4⁺PD-1⁺, and these Tregs can suppress antitumor immune response via mTGF-β1. Our results not only enrich the family of Treg subsets, providing new mechanistic insight to tumor-induced immune suppression in human, but also suggest a potential target for cancer immunotherapy. CD4⁺CD69⁺Foxp3⁻ regulatory T cells were identified in hepatocellular carcinoma patients. These Treg cells inhibit T cell response via membrane-bound TGF-β. The percentage of these cells was significantly correlated with tumor progression. The percentage of these cells was higher than conventional CD4⁺CD25⁺Foxp3⁺ Tregs. These Treg cells not only exist in tumor-bearing mice, but also in cancer patients.

An Increased Abundance of Tumor-Infiltrating Regulatory T Cells Is Correlated with the Progression and Prognosis of Pancreatic Ductal Adenocarcinoma

CD4+CD25+Foxp3+ regulatory T cells (Tregs) can inhibit cytotoxic responses. Though several studies have analyzed Treg frequency in the peripheral blood mononuclear cells (PBMCs) of pancreatic ductal adenocarcinoma (PDA) patients using flow cytometry (FCM), few studies have examined how intratumoral Tregs might contribute to immunosuppression in the tumor microenvironment. Thus, the potential role of intratumoral Tregs in PDA patients remains to be elucidated. In this study, we found that the percentages of Tregs, CD4+ T cells and CD8+ T cells were all increased significantly in tumor tissue compared to control pancreatic tissue, as assessed via FCM, whereas the percentages of these cell types in PBMCs did not differ between PDA patients and healthy volunteers. The percentages of CD8+ T cells in tumors were significantly lower than in PDA patient PBMCs. In addition, the relative numbers of CD4+CD25+Foxp3+ Tregs and CD8+ T cells were negatively correlated in the tissue of PDA patients, and the abundance of Tregs was significantly correlated with tumor differentiation. Additionally, Foxp3+ T cells were observed more frequently in juxtatumoral stroma (immediately adjacent to the tumor epithelial cells). Patients showing an increased prevalence of Foxp3+ T cells had a poorer prognosis, which was an independent factor for patient survival. These results suggest that Tregs may promote PDA progression by inhibiting the antitumor immunity of CD8+ T cells at local intratumoral sites. Moreover, a high proportion of Tregs in tumor tissues may reflect suppressed antitumor immunity.

Expression and significance of regulatory T cells in patients with breast cancer

Objective To investigate the expression and clinical signifycance of CD4 + CD25 + CD127low/-regulatory T cells (Tregs) in peripheral blood and tumor tissue of breast cancer patients.Methods The percentage of Tregs in tumor tissues,and adjacent tissues from 10 patients with breast cancer,peripheral blood from 48 patients with breast cancer and 12 healthy volunteers was detected by using flow cytometry.Results The percentage of Tregs in 10 breast cancer patients was (39.74 ± 9.02) ％ in tumor tissues,significantly higher than that in the adjacent tissues [(29.81 ± 8.52)％,P ＜0.05].The percentage of Tregs in peripheral blood of 48 breast cancer patients was (7.04 ± 1.56) ％,significantly higher than in 12 healthy volunteers [(4.01 ± 1.05) ％,P ＜ 0.05].With the progress of the disease,the proportion of Tregs in peripheral blood of breast cancer patients had a higher level.The levels of Tregs in peripheral blood of breast cancer patients with stage (Ⅱ + Ⅲ) were signifycantly higher than those of patients with stage Ⅰ [(6.39 ± 0.81) ％,P ＜ 0.05] and heathy volunteers [(4.01 ± 1.05) ％,P ＜ 0.05].Conclusion There is high expression of Tregs in peripheral blood and tumor tissues of patients with breast cancer,which might be closely related to long-term immunoescape of tumor.Besides,the populations of Tregs in peripheral blood may contributed to the progression of breast cancer. Key words: Breast cancer; Peripheral blood; T cells; Flow cytometry

CD4 helper T cells, CD8 cytotoxic T cells, and FOXP3+ regulatory T cells with respect to lethal prostate cancer

Prostate cancer represents a major contributor to cancer mortality, but the majority of men with prostate cancer will die of other causes. Thus, a challenge is identifying potentially lethal disease at diagnosis. Conflicting results have been reported when investigating the relationship between infiltration of lymphocytes and survival in prostate cancer. One of the mechanisms suggested is the recruitment of regulatory T cells (Tregs), a subpopulation of T cells that have a role in promoting tumor growth. Tregs counteract tumor rejection through suppressive functions on the anti-immune response but their prognostic significance is still unknown. We report here the results of a conducted case–control study nested in a cohort of men treated with transurethral resection of the prostate and diagnosed incidentally with prostate cancer. Cases are men who died of prostate cancer (n=261) and controls are men who survived >10 years after their diagnosis (n=474). Infiltration of both Thelper and Tcytotoxic cells was frequently observed and the majority of the Tregs were CD4+. Thelper or Tcytotoxic cells were not associated with lethal prostate cancer. However, we found a nearly twofold increased risk of lethal prostate cancer when comparing the highest with the lowest quartile of CD4+ Treg cells (95% confidence interval: 1.3–2.9). Our conclusion is that men with greater numbers of CD4+ Tregs in their prostate tumor environment have an increased risk of dying of prostate cancer. Identification of CD4+ Tregs in tumor tissue may predict clinically relevant disease at time of diagnosis independently of other clinical factors.

Prognostic impact of FOXP3 expression in triple-negative breast cancer

Background. Forkhead Box Protein 3 (FOXP3) is a marker for immunosuppressive CD4+CD25+ regulatory T cells (Tregs). We investigated whether there were significant numbers of FOXP3-positive Tregs in triple-negative breast cancer (TNBC) using immunohistochemistry, and whether the presence of FOXP3-positive Tregs was associated with other prognostic factors, such as stage or histologic grade. We investigated the number of tumor-infiltrating FOXP3-positive Tregs in formalin-fixed TNBC specimens obtained from patients who received palliative treatment between 1999 and 2007. Material and methods. Immunohistochemistry was used to assess the number of CD4+, CD25+, and FOXP3+ Tregs in tumor tissue and normal breast tissue from 86 TNBC patients. Univariate and multivariate analyses evaluated outcomes according to the number of FOXP3-positive Tregs. Results. Of the 86 tumor specimens, 22 (25.6%) expressed more than 15 FOXP3-positive Tregs per 10 high power fields in the peritumoral area. On multivariate analysis, staining showing ≥ 15 FOXP3-positive Tregs was an independent prognostic factor for overall survival and progression free survival with hazard ratios of 2.4 (95% CI 1.0–5.6; p = 0.049) and 2.0 (95% CI 1.1–3.6; p = 0.032), respectively. In TNBC, FOXP3-positive Tregs had stronger prognostic significance than did FOXP3-negative Tregs. The finding of improved survival associated with highly infiltrating FOXP3-positive Tregs in TNBC contrasted with several other types of solid cancer. Conclusion. TNBC may be differently driven by FOXP3 via an immune mechanism. The inclusion of FOXP3+ Tregs may help to improve prognostication for TNBC.

New immune therapy targets tumor-associated environment: from bone marrow to tumor site

New immune therapy targets tumor-associated environment: from bone marrow to tumor site

Tumor-Infiltrating FOXP3+ T Regulatory Cells Show Strong Prognostic Significance in Colorectal Cancer

To determine the prognostic significance of FOXP3(+) lymphocyte (Treg) density in colorectal cancer compared with conventional histopathologic features and with CD8(+) and CD45RO(+) lymphocyte densities. Tissue microarrays and immunohistochemistry were used to assess the densities of CD8(+), CD45RO(+), and FOXP3(+) lymphocytes in tumor tissue and normal colonic mucosa from 967 stage II and stage III colorectal cancers. These were evaluated for associations with histopathologic features and patient survival. FOXP3(+) Treg density was higher in tumor tissue compared with normal colonic mucosa, whereas CD8(+) and CD45RO(+) cell densities were lower. FOXP3(+) Tregs were not associated with any histopathologic features, with the exception of tumor stage. Multivariate analysis showed that stage, vascular invasion, and FOXP3(+) Treg density in normal and tumor tissue were independent prognostic indicators, but not CD8(+) and CD45RO(+). High FOXP3(+) Treg density in normal mucosa was associated with worse prognosis (hazard ratio [HR] = 1.51; 95% CI, 1.07 to 2.13; P = .019). In contrast, a high density of FOXP3(+) Tregs in tumor tissue was associated with improved survival (HR = 0.54; 95% CI, 0.38 to 0.77; P = .001). FOXP3(+) Treg density in normal and tumor tissue had stronger prognostic significance in colorectal cancer compared with CD8(+) and CD45RO(+) lymphocytes. The finding of improved survival associated with a high density of tumor-infiltrating FOXP3(+) Tregs in colorectal cancer contrasts with several other solid cancer types. The inclusion of FOXP3(+) Treg density may help to improve the prognostication of early-stage colorectal cancer.