Autocrine and paracrine loops between cancer cells and macrophages promote lymph node metastasis via CCR4/CCL22 in head and neck squamous cell carcinoma

Lymph node metastasis is a poor prognostic factor for patients with head and neck squamous cell carcinoma (HNSCC). However, its molecular mechanism has not yet been fully understood. In our study, we investigated the expression of CCR4 and its ligand CCL22 in the HNSCC tumor microenvironment and found that the CCR4/CCL22 axis was involved in lymph node metastasis of HNSCC. CCR4 was expressed in 20 of 31 (64.5%) human tongue cancer tissues, and its expression was significantly correlated with lymph node metastasis (p < 0.01) and lymphatic invasion (p < 0.05). CCR4 was expressed in three of five human HNSCC cell lines tested. CCR4⁺ HNSCC cells, but not CCR4^? cells, showed enhanced migration toward CCL22, indicating that functional CCR4 was expressed in HNSCC cell lines. CCL22 was also expressed in cancer cells (48.4% of tongue cancer tissues) or CD206⁺ M2‐like macrophages infiltrated in tumors and draining lymph nodes. CCL22 produced by cancer cells or CD206^high M2‐like macrophages increased the cell motility of CCR4⁺ HNSCC cells in vitro in an autocrine or paracrine manner. In the mouse SCCVII in vivo model, CCR4⁺ cancer cells, but not CCR4^? cells, metastasized to lymph nodes which contained CCL22 producing M2‐like macrophages. These results demonstrate that lymph node metastasis of CCR4⁺ HNSCC is promoted by CCL22 in an autocrine or M2‐like macrophage‐dependent paracrine manner. Therefore, the CCR4/CCL22 axis may be an attractive target for the development of diagnostic and therapeutic strategies for patients with HNSCC.     

Improved therapeutic efficacy of unmodified anti-tumor antibodies by immune checkpoint blockade and kinase targeted therapy in mouse models of melanoma

The use of specific anti-tumor antibodies has transformed the solid cancer therapeutics landscape with the relative successes of therapies such as anti-HER2 in breast cancer, and anti-EGFR in HNSCC and colorectal cancer. However, these therapies result in toxicity and the emergence of resistant tumors. Here, we showed that removing immune suppression and enhancing stimulatory signals increased the anti-tumor activity of unmodified TA99 antibodies (anti-TYRP1) with a significant reduction of growth of solid tumors and lung metastases in mouse models of melanoma. Immune checkpoint blockade enhanced the efficacy of TA99, which was associated with greater CD8⁺/Foxp3⁺, NK1.1⁺ and dendritic cell infiltrates, suggestive of an increased anti-tumor innate and adaptive immune responses. Further, MEK inhibition in melanoma cell lines increased the expression of melanosomal antigens in vitro, and combining TA99 and MEKi in vivo resulted in enhanced tumor control. Moreover, we found an improved therapeutic effect when YUMM tumor-bearing mice were treated with TA99 combined with MEKi and immune checkpoint blockade (anti-PD1 and anti-CTLA4). Our findings suggest that MEKi induced an increased expression of tumor-associated antigens, which in combination with anti-tumor antibodies, generated a robust adaptive anti-tumor response that was sustained by immune checkpoint inhibition therapy. We postulate that combining anti-tumor antibodies with standard-of-care strategies such as immune checkpoint blockade or targeted therapy, will improve therapeutic outcomes in cancer.     

Immune‐checkpoint molecules on regulatory T‐cells as a potential therapeutic target in head and neck squamous cell cancers

Immune‐checkpoint inhibitors improve the survival of head and neck squamous cell carcinoma (HNSCC) patients. Although recent studies have demonstrated that the tumor immune microenvironment (TIME) has critical roles in immunotherapy, the precise mechanisms involved are unclear. Therefore, further investigations of TIME are required for the improvement of immunotherapy. The frequency of effector regulatory T‐cells (eTregs) and the expression of immune‐checkpoint molecules (ICM) on eTregs and conventional T‐cells (Tconvs) both in peripheral blood lymphocytes (PBL) and tumor‐infiltrating lymphocytes (TIL) from HNSCC patients were analyzed by flow cytometry and their distributions were evaluated by multi‐color immunofluorescence microscopy. High frequency eTreg infiltration into HNSCC tissues was observed and high expressions of CD25, FOXP3, stimulatory‐ICM (4‐1BB, ICOS, OX40 and GITR) and inhibitory‐ICM (programmed cell death‐1 [PD‐1] and cytotoxic T‐lymphocyte‐associated protein‐4 [CTLA‐4]) were found on invasive eTregs. In contrast, the expression of stimulatory‐ICM on Tconvs was low and the expression of inhibitory‐ICM was high. In addition, ICM‐ligands (programmed cell death‐1 [PD‐L1], galectin‐9 and CEACAM‐1) were frequently expressed on cancer cells. PD‐L1 and galectin‐9 were also expressed on macrophages. PD‐1⁺ T‐cells interacted with PD‐L1⁺ cancer cells or PD‐L1⁺ macrophages. This suggested that in TIL, eTregs are highly activated, but Tconvs are exhausted or inactivated by eTregs and immune‐checkpoint systems, and ICM and eTregs are strongly involved in the creation of an immunosuppressive environment in HNSCC tissues. These suggested eTreg targeting drugs are expected to be a combination partner with immune‐checkpoint inhibitors that will improve immunotherapy of HNSCC.     

Intratumoral IL22-producing cells define immunoevasive subtype muscle-invasive bladder cancer with poor prognosis and superior nivolumab responses

Our previous researches have identified immunoevasive subtype muscle-invasive bladder cancer (MIBC) characterized with immune cells infiltration patterns. Our study explored the clinical significance, immunoregulatory role and therapeutic value of intratumoral IL22-producing cells in MIBC. Two hundred and fifty-nine formalin-fixed paraffin-embedded MIBC samples and 83 freshly resected MIBC tissues and 391 TCGA MIBC samples were retrospectively evaluated. Immunohistochemistry and flow cytometry were applied to identify immune cell infiltration and functional status. In vitro intervention studies were to test the therapeutic and predictive potential of IL22⁺ cells. Our data revealed patients with high IL22⁺ cells infiltration suffered poor overall survival and recurrence-free survival in both training and validation cohorts. Only pT2 patients of combined cohort with low IL22⁺ cells infiltration gained survival benefits from adjuvant chemotherapy (ACT) significantly. Besides, immune contexture featured with increased pro-tumor cells and immunosuppressive cytokines was identified in patients with high IL22⁺ cells density. The expression pattern of exhausted and effector markers in CD8⁺ T cells from high IL22⁺ cells subgroup indicated their dysfunctional status. Importantly, nivolumab showed tumor-killing efficacy in tumors with high IL22⁺ cells infiltration, and immunosuppressive contexture with CD8⁺ T cells exhaustion was abrogated in tumors treated with anti-IL22 antibody. In summary, IL22⁺ cells infiltration determined immunosuppressive contexture with CD8⁺ T cell dysfunction. Tumor-infiltrating IL22⁺ cells could be used as an independent marker to predict prognosis and ACT responses. IL22⁺ cells infiltration possessed the potential to be a favorable predictor for nivolumab application and IL22 blockade could be a novel therapeutic strategy in MIBC.     

CD4+ T cell exhaustion leads to adoptive transfer therapy failure which can be prevented by immune checkpoint blockade

Cytotoxic CD8⁺ T cell exhaustion is one of the mechanisms underlying the tumor immune escape. The paradigm-shifting immune checkpoint therapy can mitigate CD8⁺ T lymphocyte exhaustion, reinvigorate the anticancer immunity, and achieve durable tumor regression for some patients. Emerging evidence indicates that CD4⁺ T lymphocytes also have a critical role in anticancer immunity, either by directly applying cytotoxicity toward cancer cells or as a helper to augment CD8⁺ T cell cytotoxicity. Whether anticancer CD4⁺ T lymphocytes undergo exhaustion during immunotherapy of solid tumors remains unknown. Here we report that melanoma antigen TRP-1/gp75-specific CD4⁺ T lymphocytes exhibit an exhaustion phenotype after being adoptively transferred into mice bearing large subcutaneous melanoma. Exhaustion of these CD4⁺ T lymphocytes is accompanied with reduced cytokine release and increased expression of inhibitory receptors, resulting in loss of tumor control. Importantly, we demonstrate that PD-L1 immune checkpoint blockade can prevent exhaustion, induce proliferation of the CD4⁺ T lymphocytes, and consequently prevent tumor recurrence. Therefore, when encountering an excessive amount of tumor antigens, tumor-reactive CD4⁺ T lymphocytes also enter the exhaustion state, which can be prevented by immune checkpoint blockade. Our results highlight the importance of tumor-specific CD4⁺ T lymphocytes in antitumor immunity and suggest that the current immune checkpoint blockade therapy may achieve durable anticancer efficacy by rejuvenating both tumor antigen-specific CD8⁺ T lymphocytes and CD4⁺ T lymphocytes.     

乌司他丁抑制CCL17/CCL22-CCR4介导的小鼠乳腺癌肝转移

背景与目的：越来越多的研究表明趋化因子及其受体在癌症的发生、发展和转移中起关键作用。本文研究了乌司他丁（ulinastatin）对胸腺和活化调节趋化因子（thymus and activation-regulated chemokine,TARC）即CCL17/巨噬细胞来源的趋化因子（macrophage-derived chemokine,MDC）即CCL22-CC族趋化因子受体4（CC chemokine receptor 4,CCR4）信号通路介导的乳腺癌肝转移的影响及其作用机制。方法：通过小鼠乳腺脂肪垫（mfp）接种4T1乳腺癌细胞的方式构建小鼠乳腺癌模型,15 d后取小鼠乳腺肿瘤,并记录乳腺肿瘤的质量;采用免疫组织化学法检测乳腺肿瘤组织中CCR4及肝脏转移瘤中CCL22、CCL17蛋白的表达;采用慢病毒转染的方式抑制4T1乳腺癌细胞CCR4基因,采用蛋白质印迹法（Western blot）检测抑制效果,并以同样方式进行小鼠乳腺成瘤并观察肿瘤生长情况;用三种不同浓度乌司他丁处理4T1细胞荷瘤小鼠,15 d后采用免疫组织化学法检测乳腺癌组织中CCR4及肝脏组织中CCL22及CCL17的表达,采用Westernblot和实时荧光定量聚合酶链反应（real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR）分别检测肝脏组织中TGF-β的表达以及microRNA-34a和microRNA-31的含量,并进行TGF-β和microRNA-34a、microRNA-31、CCL22以及CCL17的相关性分析。结果：小鼠乳腺癌组织高表达CCR4,肝脏转移瘤高表达CCL22和CCL17;沉默4T1乳腺癌细胞CCR4的表达,可抑制乳腺癌成瘤性;乌司他丁抑制CCR4、TGF-β、CCL22、microRNA-31和CCL17的表达,促进microRNA-34a的表达。结论：乌司他丁具有抑制乳腺癌肝转移的作用,其具体机制可能与乌司他丁通过TGF-β-microRNA-34a-CCL22轴及microRNA-31-TGF-β-CCL17轴抑制肝乳腺癌;肝转移;乌司他丁;CCR4;CCL22;CCL17脏组织中与乳腺癌肝转移密切相关的CCL17/CCL22-CCR4信号通路有关。     

Complement receptor C5aR1 blockade reprograms tumor-associated macrophages and synergizes with anti-PD-1 therapy in gastric cancer

In gastric cancer (GC), the therapeutic response of immune checkpoint blockade (ICB) remains suboptimal. Targeting myeloid cell checkpoints might be feasible as adjuvant to current ICB regimens. We sought to evaluate the crucial role of C5aR1⁺ TAMs in regulating antitumor immunity and the efficacy of combinatorial treatment with antiprogrammed cell death protein-1 (PD-1) and C5aR1 blockade. Here, we found that C5aR1 was predominantly expressed on macrophages and high level of C5aR1⁺ TAMs infiltration could predict poor prognosis and inferior chemotherapeutic response. The flow cytometry (FCM) and single-cell RNA-seq (scRNA-seq) data revealed that C5aR1⁺ TAMs exhibited immunosuppressive property which might contribute to CD8⁺ T cell dysfunction. Blockade of C5aR1 could diminish the immunosuppressive function of TAMs and led to reinvigorated CD8⁺ T cells mediated antitumor immunity. Moreover, using in vitro intervention experiment based on fresh GC surgical specimens, we discovered that C5aR1 blockade exert a synergistic effect when combined with PD-1 inhibitor for tumor clearance. Our study demonstrated that C5aR1 is a critical myeloid checkpoint and plays a crucial role in regulating the immunosuppressive property of TAMs and CD8⁺ T cell immune tolerance. C5aR1 blockade reprograms TAMs and reinvigorated the cytotoxicity of CD8⁺ T cells, thus improving the efficacy of anti-PD-1 therapy for tumor eradication in GC.     

NK depletion results in increased CCL22 secretion and Treg levels in Lewis Lung Carcinoma via the accumulation of CCL22‐secreting CD11b+CD11c+ cells

Tumor‐induced immune suppression involves the accumulation of suppressive infiltrates in the tumor microenvironment such as regulatory T‐cells (Tregs). Previous studies demonstrated that NK‐dependant increases in CCL22 secretion selectively recruit Tregs toward murine lungs bearing Lewis Lung Carcinoma (LLC). To extend the in vitro studies, the present studies utilized in vivo depletion of NK cells to ascertain the contribution of NK‐derived CCL22 toward total CCL22 and subsequent Treg levels in both normal and LLC‐bearing lungs. However, NK depletion had the unexpected effect of increasing both CCL22 secretion and Treg levels in the lungs of NK‐depleted LLC‐bearing mice. This was concurrent with an increase in tumor burden. Flow cytometry and a series of both immunomagnetic and FACS isolations were used to identify the CCL22‐producing cellular fractions in LLC‐bearing lungs. A novel CD11b⁺CD11c⁺ cell population was identified that accumulates in large numbers in NK‐depleted LLC‐bearing lung tissue. These CD11b⁺CD11c⁺ cells secreted large amounts of CCL22 that may overcompensate for the loss of NK‐derived CCL22 in the lungs of NK‐depleted LLC‐bearing mice. Taken together, these data suggest that NK cells play both a positive and negative role in the regulation of CCL22 secretion and, in turn, the recruitment of Tregs toward LLC‐bearing lungs.     

Chemokine C-C motif ligand 21 synergized with programmed death-ligand 1 blockade restrains tumor growth

Programmed death-ligand 1 (PD-L1) blockade has revolutionized the prognosis of several cancers, but shows a weak effect on pancreatic cancer (PC) due to poor effective immune infiltration. Chemokine C-C motif ligand 21 (CCL21), a chemokine promoting T cell immunity by recruiting and colocalizing dendritic cells (DCs) and T cells, serves as a potential antitumor agent in many cancers. However, its antitumor response and mechanism combined with PD-L1 blockade in PC remain unclear. In our study, we found CCL21 played an important role in leukocyte chemotaxis, inflammatory response, and positive regulation of PI3K-AKT signaling in PC using Metascape and gene set enrichment analysis. The CCL21 level was verified to be positively correlated with infiltration of CD8⁺ T cells by the CIBERSORT algorithm, but no significant difference in survival was observed in either The Cancer Genome Atlas or the International Cancer Genome Consortium cohort when stratified by CCL21 expression. Additionally, we found the growth rate of allograft tumors was reduced and T cell infiltration was increased, but tumor PD-L1 abundance elevated simultaneously in the CCL21-overexpressed tumors. Then, CCL21 was further verified to increase tumor PD-L1 level through the AKT-glycogen synthase kinase-3β axis in human PC cells, which partly impaired the antitumor T cell immunity. Finally, the combination of CCL21 and PD-L1 blockade showed superior synergistic tumor suppression in vitro and in vivo. Together, our findings suggested that CCL21 in combination with PD-L1 blockade might be an efficient and promising option for the treatment of PC.     

Intratumoral IL17-producing cells infiltration correlate with antitumor immune contexture and improved response to adjuvant chemotherapy in gastric cancer

BackgroundTumor IL17-producing (IL17A⁺) cells infiltration has different prognostic values among various cancers. The objective of this study was to assess the effect of IL17A⁺ cells in gastric cancer.Patients and methodsThe study included two patient cohorts, the Cancer Genome Atlas cohort (TCGA, n = 351) and the Zhongshan Hospital cohort (ZSHC, n = 458). The TCGA and ZSHC were used for mRNA-related and cells infiltration-related analyses, respectively. The roles of IL17A mRNA and IL17A⁺ cells in overall survival (OS), response to adjuvant chemotherapy (ACT), and immune contexture were evaluated. Another independent cohort was included to identify the correlation between mRNA of IL17A and IL17A⁺ cells infiltration (the preliminary Zhongshan Hospital cohort, PZSHC, n = 21).ResultsThe infiltration of IL17A⁺ cells was positively correlated with the expression of IL17A mRNA (Spearman’s ρ = 0.811; P < 0.001). High IL17A mRNA expression and intratumoral IL17A⁺ cells were correlated with improved OS and remained to be significant after adjusted for confounders. Patients with TNM II/III disease whose tumor present higher intratumoral IL17A⁺ cells or lower peritumoral IL17A⁺ cells can benefit more from ACT. Elevated IL17A mRNA expression and increased intratumoral IL17A⁺ cells infiltration was associated with more antitumor mast cells and nature killer cells infiltration and less pro-tumor M2 macrophages infiltration. High IL17A mRNA expression represented a Th17 cells signature and immune response process and was correlated with increased cytotoxic GZMA, GZMB, IFNG, PRF1, and TNFSF11 expression.ConclusionsIL17A mRNA expression and intratumoral IL17A⁺ cells infiltration were correlated with antitumor immune contexture. IL17A⁺ cells infiltration could be used as an independent prognostic biomarker for OS and predictive biomarker for superior response to ACT, and further prospective validation needs to be conducted.     

Specific blockade CD73 alters the “exhausted” phenotype of T cells in head and neck squamous cell carcinoma

The adenosine‐induced immunosuppression hampers the immune response toward tumor cells and facilitates the tumor cells to evade immunosurveillance. CD73, an ecto‐5‐nucleotidase, is the ectoenzyme dephosphorylating extracellular AMP to adenosine. Here, using immunocompetent transgenic head and neck squamous cell carcinoma (HNSCC) mouse model, immune profiling showed high expression of CD73 on CD4⁺ and CD8⁺ T cells was associated with an “exhausted” phenotype. Further, treatment with anti‐CD73 monoclonal antibody (mAb) significantly blunted the tumor growth in the mouse model, and the blockade of CD73 reversed the “exhausted” phenotype of CD4⁺ and CD8⁺ T cells through downregulation of total expression of PD‐1 and CTLA‐4 on T cells. Whereas the population of CD4⁺CD73^hi/CD8⁺CD73^hi T cells expressed higher CTLA‐4 and PD‐1 as compared to untreated controls. In addition, the human tissue microarrays showed the expression of CD73 is upregulated on tumor infiltrating immune cells in patients with primary HNSCC. Moreover, CD73 expression is an independent prognostic factor for poor outcome in our cohort of HNSCC patients. Altogether, these findings highlight the immunoregulatory role of CD73 in the development of HNSCC and we propose that CD73 may prove to be a promising immunotherapeutic target for the treatment of HNSCC.     

PD-L1 is highly expressed in Enzalutamide resistant prostate cancer

Efficacy of Enzalutamide (ENZ) in castration resistant prostate cancer (CRPC) patients is short-lived. Immunotherapy like T cell checkpoint blockade may improve patient survival. However, when and where checkpoint molecules are expressed in CRPC and whether immune evasion is a mechanism of ENZ resistance remains unclear. Thus, we investigated whether clinically relevant immunotherapy targets, specifically PD-L1/2, PD-1 and CTLA-4, are upregulated in ENZ resistant (ENZR) patients and in a pre-clinical model of ENZ resistance. We show for the first time that patients progressing on ENZ had significantly increased PD-L1/2⁺ dendritic cells (DC) in blood compared to those naïve or responding to treatment, and a high frequency of PD-1⁺T cells. These data supported our pre-clinical results, in which we found significantly increased circulating PD-L1/2⁺ DCs in mice bearing ENZR tumors compared to CRPC, and ENZR tumors expressed significantly increased levels of tumor-intrinsic PD-L1. Importantly, the expression of PD-L1 on ENZR cells, or the ability to modulate PD-L1/2⁺ DC frequency, was unique to ENZR cell lines and xenografts that did not show classical activation of the androgen receptor. Overall, our results suggest that ENZ resistance is associated with the strong expression of anti-PD-1 therapy targets in circulating immune cells both in patients and in a pre-clinical model that is non-AR driven. Further evaluation of the contribution of tumor vs. immune cell PD-L1 expression in progression of CRPC to anti-androgen resistance and the utility of monitoring circulating cell PD-L1 pathway activity in CRPC patients to predict responsiveness to checkpoint immunotherapy, is warranted.     

Breast Cancer: Coordinated Regulation of CCL2 Secretion by Intracellular Glycosaminoglycans and Chemokine Motifs

The chemokine CCL2 (MCP-1) has been identified as a prominent tumor-promoting factor in breast cancer. The major source for CCL2 is in the tumor cells; thus, identifying the mechanisms regulating CCL2 release by these cells may enable the future design of modalities inhibiting CCL2 secretion and consequently reduce tumorigenicity. Using cells deficient in expression of glycosaminoglycans (GAGs) and short hairpin RNAs reducing heparan sulfate (HS) and chondroitin sulfate (CS) expression, we found that intracellular HS and CS (= GAGs) partly controlled the trafficking of CCL2 from the Golgi toward secretion. Next, we determined the secretion levels of GFP-CCL2-WT and GFP-CCL2-variants mutated in GAG-binding domains and/or in the 40s loop of CCL2 (⁴⁵TIVA⁴⁸). We have identified partial roles for R18+K19, H66, and the ⁴⁵TIVA⁴⁸ motif in regulating CCL2 secretion. We have also demonstrated that in the absence of R24 or R18+K19 +⁴⁵TIVA⁴⁸, the secretion of CCL2 by breast tumor cells was almost abolished. Analyses of the intracellular localization of GFP-CCL2-mutants in the Golgi or the endoplasmic reticulum revealed particular intracellular processes in which these CCL2 sequences controlled its intracellular trafficking and secretion. The R24, ⁴⁵TIVA⁴⁸ and R18+K19 +⁴⁵TIVA⁴⁸ domains controlled CCL2 secretion also in other cell types. We propose that targeting these chemokine regions may lead to reduced secretion of CCL2 by breast cancer cells (and potentially also by other malignant cells). Such a modality may limit tumor growth and metastasis, presumably without affecting general immune activities (as discussed below).     

Expression of CCL17 and CCL22 by latent membrane protein 1-positive tumor cells in age-related Epstein-Barr virus-associated B-cell lymphoproliferative disorder

Age-related Epstein–Barr virus-positive (EBV⁺) B-cell lymphoproliferative disorder (ALPD) is a disease entity identified from a large-scale re-survey of cases diagnosed as diffuse large B-cell lymphoma. ALPD is a group of EBV⁺ polymorphic B-cell lymphoma typically seen in elderly patients. An age-associated decline in host immunity against EBV might be partly responsible for the pathogenesis of ALPD. Histologically, ALPD is often characterized by a minor proportion of EBV-encoded RNA-positive tumor cells in a background of extensive cellular infiltration, similar to that of classical Hodgkin's lymphoma. In contrast to Hodgkin and Reed–Sternberg cells, ALPD tumor cells are clearly positive for B cell markers CD20 and/or CD79a. Hodgkin and Reed–Sternberg cells produce various chemokines, including CCL17 and CCL22, that attract chemokine receptor CCR4-expressing Th2 cells and regulatory T cells. Previously, we have shown that EBV-immortalized B cells also produce CCL17 and CCL22 through latent membrane protein 1 (LMP1)-mediated activation of nuclear factor κB. Here we examined expression of CCL17 and CCL22 in ALPD. ALPD tumor cells were often heterogeneous in size in accordance with the differential expression of EBV latent genes at the single cell level. LMP1-expressing tumor cells were typically large in size and selectively positive for CCL17 and CCL22. CCR4⁺ cells and forkhead box protein 3⁺ regulatory T cells were abundantly present, and the majority of forkhead box protein 3⁺ cells were CCR4⁺. Collectively, our data show production of CCL17 and CCL22 by LMP1⁺ large-sized tumor cells and accumulation of CCR4-expressing cells including regulatory T cells in ALPD. ( Cancer Sci 2008; 99: 296–302)     

Dynamics of T‐cell infiltration during the course of ovarian cancer: The gradual shift from a Th17 effector cell response to a predominant infiltration by regulatory T‐cells

The type of immune cells that are present within the tumor microenvironment can play a crucial role in the survival of patients. However, little is known about the dynamics of the tumor‐infiltrating immune cells during disease progression. We studied the immune cells that infiltrated the tumor tissues of ovarian cancer patients at different stages of disease. The early stages of development of ovarian carcinomas were characterized by a strong Th17 immune response, whereas in stage II patients, recruitment of high numbers of Th1 cells was observed. In disseminated tumors (Stages III–IV), we detected a dominant population of Helios⁺ activated regulatory T cells (Tregs) along with high numbers of monocytes/macrophages and myeloid dendritic cells (mDCs). Tumor‐infiltrating Tregs had markedly lower expression of CCR4 than circulating Tregs, and the numbers of tumor‐infiltrating Tregs significantly correlated with the levels of CCL22 in ovarian tumor cell culture supernatants, suggesting their recruitment via a CCR4/CCL22 interaction. CCL22 was mainly produced by tumor cells, monocytes/macrophages and mDCs in the primary ovarian tumors, and its expression markedly increased in response to IFNγ. Taken together, the specific recruitment of Tregs, probably triggered by inflammatory stimuli, leads to a significant immune suppression in the advanced stages of ovarian cancer.     

Involvement of local renin‐angiotensin system in immunosuppression of tumor microenvironment

To improve current cancer immunotherapies, strategies to modulate various immunosuppressive cells including myeloid derived suppressor cells (MDSC) which were shown to be negative factors in immune‐checkpoint blockade therapy, need to be developed. In the present study, we evaluated the role of the local renin‐angiotensin system (RAS) in the tumor immune‐microenvironment using murine models bearing tumor cell lines in which RAS was not involved in their proliferation and angiogenetic ability. Giving angiotensin II receptor blockers (ARB) to C57BL/6 mice bearing murine colon cancer cell line MC38 resulted in significant enhancement of tumor antigen gp70 specific T cells. ARB administration did not change the numbers of CD11b⁺ myeloid cells in tumors, but significantly reduced their T‐cell inhibitory ability along with decreased production of various immunosuppressive factors including interleukin (IL)‐6, IL‐10, vascular endothelial growth factor (VEGF), and arginase by CD11b⁺ cells in tumors. ARB also decreased expression of immunosuppressive factors such as chemokine ligand 12 and nitric oxide synthase 2 in cancer‐associated fibroblasts (CAF). Last, combination of ARB and anti‐programmed death‐ligand 1 (PD‐L1) antibodies resulted in significant augmentation of anti‐tumor effects in a CD8⁺ T cell‐dependent way. These results showed that RAS is involved in the generation of an immunosuppressive tumor microenvironment caused by myeloid cells and fibroblasts, other than the previously shown proliferative and angiogenetic properties of cancer cells and macrophages, and that ARB can transform the immunosuppressive properties of MDSC and CAF and could be used in combination with PD‐1/PD‐L1 immune‐checkpoint blockade therapy.