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Human leukocyte antigen (HLA) class I-specific killer-cell immunoglobulin-like receptors (KIR) regulate natural killer (NK) cell function in eliminating malignancy. Breast cancer (BC) patients exhibit reduced NK-cytotoxicity in peripheral blood. To test the hypothesis that certain KIR-HLA combinations impairing NK-cytotoxicity predispose to BC risk, we analyzed KIR and HLA polymorphisms in 162 women with BC and 278 controls. KIR-Bx genotypes increased significantly in BC than controls (83.3% vs. 71.9%, OR 1.95), and the increase was more pronounced in advanced-cancer (OR 5.3). No difference was observed with inhibitory KIR (iKIR) and HLA-ligand combinations. The activating KIR (aKIR) and HLA-ligand combinations, 2DS1 + C2 (OR 2.98) and 3DS1 + Bw4 (OR 2.6), were significantly increased in advanced-BC. All patients with advanced-cancer carrying 2DS1 + C2 or 3DS1 + Bw4 also have their iKIR counterparts 2DL1 and 3DL1, respectively. Contrarily, the 2DL1 + C2 and 3DL1 + Bw4 pairs without their aKIR counterparts are significantly higher in controls. These data suggest that NK cells expressing iKIR to the cognate HLA-ligands in the absence of putative aKIR counterpart are instrumental in antitumor response. These data provide a new framework for improving the utility of genetic risk scores for individualized surveillance.

Simple Summary: New insights into the foundation of cellular and molecular cancer immunology have revealed that immune cells play crucial roles in the development and growth of breast cancer (BC). T-cells are one of the most important cells in the tumor microenvironment and are divided into several subtypes including helper, cytotoxic, and regulatory subsets according to their transcription factors, markers, and functions. This article provides a comprehensive review of contradictory functions of various T-cell subsets in the prognosis and treatment of patients with BC, and crosstalk between tumor cells and T-cells. The literature shows that the role of T-cells in BC immunity depends on a variety of factors, including the tumor type or subtype, the stage of the disease, the localization of the cells in the tumor tissue and the presence of different cells or cytokines. Breast cancer (BC) is the most common cancer type in women and the second leading cause of death. Despite recent advances, the mortality rate of BC is still high, highlighting a need to develop new treatment strategies including the modulation of the immune system and immunotherapies. In this regard, understanding the complex function of the involved immune cells and their crosstalk with tumor cells is of great importance. T-cells are recognized as the most important cells in the tumor microenvironment and are divided into several subtypes including helper, cytotoxic, and regulatory T-cells according to their transcription factors, markers, and functions. This article attempts to provide a comprehensive review of the role of T-cell subsets in the prognosis and treatment of patients with BC, and crosstalk between tumor cells and T-cells. The literature overwhelmingly contains controversial findings mainly due to the plasticity of T-cell subsets within the inflammatory conditions and the use of different panels for their phenotyping. However, investigating the role of T-cells in BC immunity depends on a variety of factors including tumor types or subtypes, the stage of the disease, the localization of the cells in the tumor tissue and the presence of different cells or cytokines. [ABSTRACT FROM AUTHOR]

BACKGROUND: Interleukin-21 (IL-21) is produced by various cell types inducing positive and negative effects in immunity against tumors. OBJECTIVE: To investigate the expression of IL-21 by CD4+T and IL-21 receptor (IL-21R) by B lymphocytes isolated from breast-tumor draining lymph nodes (TDLNs). METHODS: Fresh lymph node samples were obtained from 45 patients with breast cancer. To assess IL-21 expression, mononuclear cells were briefly stimulated whereas IL-21R expression was assessed in unstimulated B cells. Cells were stained with antibodies for CD4, IL-21, CD19 and IL-21R and acquired by flow cytometry. RESULTS: The frequency of IL-21+CD4+T cells did not show significant association with disease parameters. However, the geometric mean fluorescence intensity (gMFI) of IL-21 in CD4+T cells was significantly lower in patients with grade III tumor than grade I + II (P = 0.042). In non-involved LNs, the intensity of IL-21 was significantly higher in patients with stage II compared with stage III (P = 0.038) and correlated negatively with the number of involved LNs. The frequency of IL-21R+CD19+B cells was significantly higher in grade III than grade I + II (P = 0.037). CONCLUSION: The higher intensity of IL-21 in CD4+T cells showed association with good prognosticators in breast cancer and warrants further investigation of the role played by IL-21 in immunity against breast cancer. [ABSTRACT FROM AUTHOR]

BACKGROUND: Block of programmed cell death protein 1 (PD-1) interaction with its ligand, PD-L1, enhances anti-tumor activity. OBJECTIVES: We aimed to assess the association between PD-L1 expression in tumor cells and CD8+ tumor infiltrating T cells (TILs) as well as soluble (s)PD-L1 serum levels in patients with triple negative breast cancer (TNBC) compared to triple positive (TPBC). METHODS: A total of 113 tumor sections and 133 serum samples were available from 144 patients with breast cancer (72 TNBC and 72 TPBC). Dual immunohistochemistry staining was applied to determine differential PD-L1 expression in tumor cells and CD8+ TILs. Soluble PD-L1 serum levels were also evaluated in patients compared to 40 healthy women by ELISA method. RESULTS: Despite TPBC patients which were mostly grades 1/2, TNBC patients were grade 3 (72% versus 66.7%, P < 0.001). Most of the TNBC patients were stages I/II, whereas most of the TPBC patients were stages III/IV (57.3% versus 68.3%,P = 0.005). There was no difference in tumor size and metastasis between TNBC and TPBC patients, although the number of involved lymph nodes was significantly more in TPBC patients (P = 0.0012). PD-L1 expression was detected in 11.5% of samples mostly in TNBC subtype and was associated with advanced grades (P = 0.039). There was no relationship between PD-L1 expression and tumor stage. PD-L1 expression in CD8+ TILs was nonsignificantly higher than tumor cells. Serum levels of sPD-L1 showed no difference between patients and healthy women. We found no correlation between PD-L1 expression in tumor lesions and serum levels of sPD-L1 in patients. CONCLUSION: PD-L1 expression was more detected in our patients with TNBC. It seems that, these patients who are resistant to standard chemotherapy regimens may get benefit from PD-L1 inhibition therapy and because of its low serum levels, sPD-L1 cannot interfere with this therapy. [ABSTRACT FROM AUTHOR]

Context: Through the expression of different immunomodulatory molecules, mesenchymal stem cells (MSCs) play a significant role in the regulation of immune responses against tumor cells. Herein, the expression of major histocompatibility complex class I polypeptide-related sequence B (MIC B) as an immunomodulatory molecule was investigated on adipose-derived stem cells (ASCs) isolated from breast cancer patients (Stage II and III) and healthy individuals.Materials and Methods: ASCs were isolated enzymatically, and the expression of MIC B was measured using quantitative real-time polymerase chain reaction method before and after treatment with interferon γ (IFN-γ). The concentration of MIC B in the supernatant of ASCs and also sera of breast cancer and normal individuals were determined using ELISA method.Results: The expression of MIC B in normal ASCs and Stage II ASCs was higher than Stage III ASCs. However, after treatment with IFN-γ expression of MIC B in ASCs was conversely changed as cancer ASCs showed approximately 3.5 fold higher expression of MIC B compared to normal ASCs. The mRNA expression of MIC B in Stage III, Stage II, and normal ASCs showed 61 (P = 0.02), 13 (P = 0.01) and 3 (P > 0.05) fold higher expression after stimulation with IFN-γ compared to cells with no stimulation.Conclusion: Expression of MIC B and upregulation of this molecule in response to IFN-γ in cancer ASCs draw attention to the effective role of MSCs in the tumor microenvironment. However, more studies will be needed to further elucidate Natural-killer Group 2, member D (NKG2D) ligands-dependent immunomodulatory roles of ASCs in the tumor progression. [ABSTRACT FROM AUTHOR]

• TNFR2 was expressed by B or T lymphocytes in breast tumor draining lymph nodes. • Lymphocytes of breast tumor draining LNs expressed negligible amounts of TNFR1. • TNFR1 expression was not induced by polyclonal activators in lymphocytes. • TNFR2 expression in B cells was associated with good prognosticators. Tumor necrosis factor-α (TNF-α) is a key cytokine in inflammation and a driving force for leukocyte migration and recruitment. However, it may exert contrasting effects on the immune responses depend on its differential binding to its receptors (TNFR1 or TNFR2). The expression of TNF receptors by lymphocytes in the tumor draining lymph nodes (TDLNs) has not been thoroughly investigated. Herein, the expression of TNFRs on lymphocytes in the breast TDLNs was assessed. 40 axillary LN samples were obtained from breast cancer patients. Mononuclear cells were isolated using Ficoll-Hypaque gradient centrifugation and stained with anti-CD3, CD4, CD8, CD19, TNFR1 and TNFR2 and subjected to flow cytometry. Results showed that TNFR2 was detected on unstimulated B or T cells in the breast TDLNs while TNFR1 was nearly absent on these cells. Short or long term activation did not increase the expression of TNFR1. The percentage of TNFR2+ cells was significantly higher in CD4+ compared to CD19+ or CD8+ cells. No significant association was observed between the percentage of TNFR2 expressing T cells and prognostic indicators. However, the percentage of TNFR2+ B cells in the metastatic LNs had negative associations with tumor grade and the number of involved LNs (P = 0.009 and P = 0.04, respectively). Collectively, TNFR2 was the main TNFR expressed by unstimulated B or T lymphocytes in the breast TDLNs and the frequency of TNFR2+ B cells was connected to good prognosticators. The effects of TNFR2 expression by lymphocytes of breast TDLNs on their functions requires more functional studies. [ABSTRACT FROM AUTHOR]

The immune‐modulatory effect of adipose‐derived stem cells (ASCs) on B cells in cancer has not been well elucidated. Herein, the interaction between B cells and ASCs isolated from the breast fat of either normal (nASCs) or breast cancer women (cASCs) was investigated. B cells derived from breast tumor draining lymph nodes were co‐cultured with nASCs or cASCs and B cells proliferation was assessed in direct and transwell assays. Moreover, B cells were co‐cultured with cASCs, nASCs or mesenchymal stromal cells of the tumor tissue (TSCs) and B cell cytokine production was assessed using flow cytometery. cASCs or TSCs were co‐cultured with either intact or B cell depleted lymphocytes and frequencies of CD25+FoxP3+ Tregs, IL‐10+ or IFN‐γ+CD4+ T cells were assessed. Results showed that co‐culture of B cells with ASCs in transwell chambers did not affect B cell proliferation. nASCs, however, was able to significantly reduce B cell proliferation in direct co‐culture experiments (P = 0.004). The frequencies of IL‐10+, TNF‐α+, IL‐2+, and IFN‐γ+ B cells were not significantly different in the co‐cultures of B cells with ASCs or TSCs. But the TNF‐α+/ IL‐10+ B cells ratio decreased in all co‐cultures, a reduction merely significant in B cell‐cASCs co‐culture (P = 0.01). The frequencies of CD4+ T cells subsets in either intact or B cell depleted lymphocytes did not undergo significant changes following co‐culture with ASCs or TSCs. Therefore, ASCs is capable of inhibiting B cell proliferation in a contact dependent manner and shifting the cytokine profile of B cells toward an anti‐inflammatory profile. [ABSTRACT FROM AUTHOR]

To find out if the T cell repertoire is efficiently and specifically provoked in patients with breast cancer, we have investigated the clonotypes of main T cell subsets (based on V β -Chain) in tumor draining lymph nodes. CD4+ helper, CD8+ cytotoxic and (CD4+CD127 dim CD25+) regulatory T cells, were negatively selected, and isolated, from lymph node mononuclear cells of 14 untreated patients with breast cancer. Four non-malignant patients, who underwent surgical operation, were also recruited as the control group. Based on sequences and new nomenclature of the T cell Receptor β Variables (TRBVs) available in the international ImMunoGeneTics (IMGT) database, 28 TRBV specific forward primers and two TRB Constant region (TRBC) specific reverse primers were developed to amplify all functional alleles. Fluorescent-labeled PCR products were then run on an ABI PRISM 310 Genetic-Analyzer. The data was analyzed by GeneMapper software version 3.1. Clonotype analysis suggested that activated T cells are present in breast cancer. More TRBV usage were detected among CD4+ helper and regulatory subsets, with Gaussian-like pattern in the majority of functional TRBV families; whereas CD8+ cytotoxic T cells showed oligoclonality in almost all TRBV families with one or two dominant peaks in each family. Similar pattern in some of these TRBVs were also observed among controls. Having no expression or polyclonality in the controls, the oligoclonal pattern observed in the TRBV18, however, appears to be specific to breast cancer patients. This phenomenon may reflect the existence of new antigenic stimulation(s) in BC patients, preferentially activating those clones of T cells that express TRBV18. [ABSTRACT FROM AUTHOR]

Abstract: Adipose‐derived mesenchymal stem cells (ASCs) are known to have immunomodulatory properties through soluble factors or by direct cell‐to‐cell contact. This study aimed to assess the expression of HLA‐G and IDO activity in breast cancer and normal ASCs and to see whether ASC is capable of modulating both tumor cells and immune system cells in vitro. ASCs were enzymatically isolated from 15 breast cancer patients and 10 normal individuals. Then they were cultured, and the impact of their conditioned media on the movement of the MDA‐MB‐231 breast cancer cell line was studied in wound healing scratch assay. Next, PBLs from the peripheral blood of normal individuals were separated and co‐cultured with breast cancer and normal ASCs. PBLs proliferation and apoptosis were assessed using CFSE labeling dye and annexin V/7AAD staining, respectively. IDO activity and HLA‐G protein expression in ASCs were examined using kynurenine assay and Western blotting, respectively. Tumor‐derived ASCs, especially those from higher stages of breast cancer, have stronger effects on the proliferation and movement of MDA‐MB‐231 cells than normal ASCs (P‐value < 0.05). Apoptosis in PBLs increased in the presence of ASCs compared to PBLs cultured alone (P‐value < 0.05). In contrast, necrosis of PBLs decreased in the presence of ASCs compared to apoptosis in these cells (P‐value < 0.001). Collectively, ASCs may have strategic effects on both tumor cells and cells of the immune system in the tumor microenvironment, resulting in tumor development, growth, and metastasis. [ABSTRACT FROM AUTHOR]

Background: The compartments of memory T cells play a fundamental role in the immune system by substantiating specific and acquired immunity. A new subset of memory cells, T stem cell memory (T) cells, with stem cell-like properties, a high capacity to proliferate, a long survival, and an ability to differentiate into all effector and memory cells has recently been introduced. In the present study, we aimed to determine the frequency of CD4 T and other T memory cell subsets in tumor draining lymph nodes of breast cancer patients. Materials and methods: Mononuclear cells were obtained from axillary lymph nodes of 52 untreated patients with breast cancer (BC) and stained with fluorochrome conjugated anti-CD4, −CCR7, −CD45RO and -CD95 antibodies to detect different subtypes of memory cells in CD4 lymphocyte populations. Data were acquired using a four-color FACSCalibur flow cytometer and analyzed using CellQuest Pro software. Results: We found that >70% of CD4 lymphocytes in draining lymph nodes of BC patients exhibited a memory phenotype of which 7.04 ± 1.04% had a T phenotype (CD4CCR7CD45ROCD95). The frequency of T cells was significantly higher in tumor positive lymph nodes compared to tumor negative lymph nodes ( p = 0.026) as well as among those patients who had at least one affected lymph node ( p = 0.012). Moreover, we found that the total frequency of central memory T cells (T) with a low expression of CD45RO was significantly higher among these patients. The percentage of CD45RO T cells was also found to increase with tumor progression from stage I to stage III ( p = 0.020). On the other hand, we found that the percentage of CD95 effector memory T cells (T) was significantly decreased in involved lymph nodes ( p = 0.009). Conclusion: Our data suggest that following long-term exposure to putative tumor antigens, T cells proliferate to generate a pool of committed memory and effector T cells. As the tumor progresses, the immunosuppressive milieu induced by tumor cells may slow down the differentiation of CD45RO T cells to more functional sub-populations. [ABSTRACT FROM AUTHOR]

Objective(s): IL-12 as an anti-tumor cytokine and IL-33 a novel identified cytokine with both pro- or anti-tumor activities, play important roles in response against tumor cells. Our aim was to evaluate the IL-12 and IL-33 levels and single nucleotide polymorphisms (SNP) in their genes in patients with breast cancer. Materials and Methods: Blood samples were collected from 100 patients with breast cancer, and 100 healthy women were controls. The serum IL-12 and IL-33 levels were measured by ELISA. The SNP rs3212227 (in IL-12 gene) and rs 1929992 (in IL-33 gene) were determined using PCR-RFLP. Results: The IL-12 levels similarly expressed in patients and controls. IL-12 levels in patients at stage1 were significantly lower than in the healthy group (P<0.05). IL-33 levels and the IL-33/IL-33/IL-12 ratio in stage IV patients were significantly higher than other stages and controls (P<0.0001 and P<0.001, respectively). There were no significant differences in the frequencies of genotypes in rs3212227 and res1929992 between patients and the control group. No sigficant differences were observed between subjects with various genotypes at rs3212227 and res1929992 with respect to related cytokine levels. Conclusion: These results indicate that the diminished IL-12 production may contribute to the tumor establishment. The higher IL-33 levels and IL-33/IL-12 ratio in patients also indicate an imbalance in Th1/Th2 responses that may contribute to tumor development. Thus, correcting the imbalance of Th1/Th2 could be an important strategy for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Background: Interleukin-19, amemberoftheinterleukin-10 family of cytokines, contributes to breast cancer pathogenesis. High interleukin-19 expression in breast tumor tissues is associated with poor clinical outcome. This study aimed to assess the changes in serum level of interleukin-19 in breast cancer patients in comparison with normal women and its association with the clinicopathological parameters of this disease. Methods: Enzyme-linked immunosorbent assay was used to analyze serum levels of interleukin-19 in 116 women with breast cancer before chemotherapy or radiotherapy, and in 60 healthy age-matched women without any acute or chronic diseases or family history of cancer. Results: There were significantly lower serum interleukin-19 levels in breast cancer patients (median: 27.3 pg/ml; range: 10.5-2443.6 pg/ml) compared to healthy controls (median: 35.1 pg/ml; range: 10.9-13676.6 pg/ml; P<0.01). Compared to the healthy control group, the decrease in serum interleukin-19 concentration was seen in all breast cancer stages. However the decrease was only significant for stage III (P=0.02). We found no significant association between serum interleukin-19 levels and stage, grade, lymph node involvement or other clinicopathological variables of the disease. However, when compared to the healthy control group, we found significantly decreased serum interleukin-19 levels in patients with involved lymph nodes (P<0.01) or tumor size greater than 2 cm (P=0.01). Conclusion: There were significantly decreased interleukin-19 levels in breast cancer patients compared to the healthy control group. We observed no association between serum interleukin-19 levels and clinicopathological parameters in breast cancer patients. [ABSTRACT FROM AUTHOR]

Background: Cytokines produced by different subsets of T cells are the key mediators to adjust the quality of immune responses. Despite evidence of induction of an immune reaction against tumors such as breast cancer, not all responses are protective. In this study we attempt to evaluate the expression profile of several cytokines from the T helper 1 and 2 subsets in blood cells from patients with breast cancer. Methods:We recruited 100 recently diagnosed patients with confirmed pathological reports. Peripheral blood samples were taken and the expression of the transcripts for IL-2, IL-12A, IL-12B, IFN-γ, IL-4, IL-10, IL-13 and IL-13Rα2 were measured by quantitative real time PCR. We correlated the results to clinical findings and compared the results to those from 64 healthy individuals. Results: Among the studied cytokines, we observed a significant increase in the expression of T helper 1 pro-inflammatory cytokines IL-12B, IL-2 and IFN-γ compared to healthy controls. Elevated expression of those cytokines was associated with high stage and/or high grade tumors but there was no association with other clinical determinants. Simultaneously, blood cells showed high expression of IL-10, but not the other studied T helper 2 cytokines. Conclusion: The mixed and complex cytokine profile of T helper 1 and 2 immunity in blood cells may show an immunological imbalance which makes the overall system favor cancer. This may be a potential target for immunotherapy approaches, however more comprehensive results are needed. [ABSTRACT FROM AUTHOR]

Objective(s): Breast cancer is the leading cause of cancer-related death in women worldwide. It has been revealed that elevated risk for malignancy may be associated with certain HLA alleles. This study was performed to assess the association of HLA class I alleles with breast cancer in women in Southern Iran. Materials and Method: Eighty nine patients included for analyzing the HLA class I alleles frequency using complement dependent cytotoxicity microassay and results were compared to 86 gender-matched healthy volunteers. Results: There were significantly more patients with A24(9) allele than those of healthy individuals (38.2% versus 16.3%) (P-value=0.002). In contrast, HLA-A1 had significantly much less expression in the patient group compared to the controls (P- value=0.04). Conclusion: A24(9) allele appears to be one of the factors increasing an individual's the susceptibility to breast cancer in our population but further investigation might be required. [ABSTRACT FROM AUTHOR]

Background: MDM2, E-cadherin, survivin and Her2 genes are involved in the regulation of normal cell growth. However, any crucial change in their expression levels can convert a normal cell into a cancer cell. Numerous studies have identified alterations of these gene expression levels in various cancers, particularly in breast cancer. Thus, they may be used as diagnostic biomarkers. In this experiment, we aim to evaluate these gene transcripts in patients' peripheral blood and compare the results with healthy individuals. Methods: RNA was extracted from peripheral blood cells of 52 breast cancer patients and 52 healthy volunteers. Then, cDNA was synthesized and assessed for MDM2, E-cadherin, and survivin and Her2 gene transcriptions in peripheral blood samples by quantitative real-time polymerase chain reaction. Results: There were no considerable differences in the expression of MDM2, Ecadherin, survivin and Her2 in cancer patients compared to healthy individuals. However, there were significantly correlation between E-cadherin, survivin and Her2 expression and some of the clinicopathological characteristics of patients studied. Also, survivin transcripts expression was positively correlated with Her2, E-cadherin, and MDM2 gene expressions. Conclusion: These results indicated no variations in MDM2, E-cadherin, Her2 and survivin gene expressions in patients compared to controls. We might not consider the examined biomarkers as valuable prognosis factors in primary breast cancer. However, additional research should be undertaken to assess these four genes in a larger sample size. [ABSTRACT FROM AUTHOR]

The tumor microenvironment consists of a dynamic interaction with several cell types including infiltrating cells from the immune system, fibroblasts, adipose derived stem cells (ASCs), and an appropriate milieu for tumor cell growth, expansion and spreading. The aim of this study was to focus on ASCs function by isolating and characterizing them from both breast cancer and normal breast adipose tissues. Therefore, the expressions of Bcl-2 and Fas mRNAs in these cells were determined using real-time quantitative PCR (Q-PCR). Also the proliferative properties of ASCs were compared among different pathological states and normal ASCs utilizing the MTT assay. It was observed that, Bcl-2 mRNA had 5-fold more expression in ASCs of patients than in controls. In contrast, Fas had a lower expression of mRNA in ASCs of patients compared to the controls. ASCs isolated from patients with stage 3 breast cancer had a statistically significant higher rate of proliferation compared to stage 2 and normal ASCs (P-value < 0.001). Based on these results, ASCs of the tumor microenvironment may contribute to tumor growth and progression and consequently protect tumor cells from the host immune response. Therefore these cells may be considered as therapeutic targets of cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Background: Several recent studies demonstrated that transforming growth factor beta (TGF-β), by stimulating T regulatory cells, and interleukins 6 and 17 (IL-6, IL- 17), by inducing inflammatory reactions, may be critical factors in cancer pathogenesis. Methods: We used quantitative real-time polymerase chain reaction assays to quantify the expression of IL-17, IL-6 and TGF-β mRNA in peripheral blood mononuclear cells and lymphocytes from draining lymph nodes of 60 women with breast cancer. The results were compared according to the patients' clinical or pathological status. Results: Higher amounts of IL-17 and IL-6 mRNA, but not TGF-β transcripts, were found in patients compared to controls. There were no significant differences between patients with negative or positive nodes or with different histological grades or stages of disease. Conclusion: Most women in this analysis had stage I or II disease. We thus conclude that IL-17, a prominent proinflammatory cytokine, may play an important role in recruiting and infiltrating antitumor immune responses in early stages of breast cancer. [ABSTRACT FROM AUTHOR]

Soluble CTLA4 (sCTLA4) was assessed in sera of 43 new cases with breast cancer and 44 age- and sex-matched healthy individuals by enzyme-linked immunosorbent assay method. Associations of sCTLA4 levels with the six important CTLA4 Single Nucleotide Polymorphisms (SNPs) and with the resulted haplotypes were investigated. Results revealed significant concentration of sCTLA4 in patients with breast cancer (23.5 ± 14.96 versus 7.69 ± 4.04 ng/ml, p < .001). Patients’ sCTLA4 concentration observed to be affected by the SNPs at positions +49 and +1822 and haplotype combination TACACA/TACGTG. These findings suggest that increased sCTLA4 may contribute in CTLA4-induced suppression of tumor immunity and provide explanations for the widely reported association of CTLA4 gene with cancer. [ABSTRACT FROM AUTHOR]

Objective: Mesenchymal stem cells (MSCs) have prominent immunomodulatory roles in the tumor microenvironment. The current study intended to elucidate Treg subsets and their cytokines after exposing naïve T lymphocytes to adiposederived MSCs (ASCs). Materials and Methods: In this experimental study, to obtain ASCs, breast adipose tissues of a breast cancer patient and a normal individual were used. Magnetic cell sorting (MACS) was employed for purifying naïve CD4+ T cells from peripheral blood of five healthy donors. Naïve CD4+ T cells were then co-cultured with ASCs for five days. The phenotype of regulatory T cells (Tregs) and production of interleukine-10 (IL-10), transforming growth factor beta (TGF-β) and IL-17 were assessed using flow cytometry and ELISPOT assays, respectively. Results: CD4+CD25-FOXP3+CD45RA+ Tregs were expanded in the presence of cancer ASCs but CD4+CD25+Foxp3+CD45RA+ regulatory T cells were up-regulated in the presence of both cancer- and normal-ASCs. This up-regulation was statistically significant in breast cancer-ASCs compared to the cells cultured without ASCs (P=0.002). CD4+CD25+ FOXP3+Helios+, CD4+CD25- FOXP3+Helios+ and CD25+ FOXP3+CD73+CD39+ Tregs were expanded after co-culturing of T cells with both cancer-ASCs and normal-ASCs, while they were statistically significant only in the presence of cancer-ASCs (P<0.05). Production of IL-10, IL-17 and TGF-β by T cells was increased in the presence of either normal- or cancer-ASCs; however, significant effect was only observed in the IL-10 and TGF-β of cancer-ASCs (P<0.05). Conclusion: The results further confirm the immunosuppressive impacts of ASCs on T lymphocytes and direct them to specific regulatory phenotypes which may support immune evasion and tumor growth. [ABSTRACT FROM AUTHOR]

• Most TNFR2-expressing CD4+ T lymphocytes do not show regulatory phenotype. • Most CD4+Foxp3+CD25+ regulatory T cells express TNFR2 with the highest intensity. • The intensity of Foxp3 expression is higher in TNFR2+ Tregs than TNFR2– ones. Tumor Necrosis Factor Receptor 2 (TNFR2) is one of the receptors of TNF-α, which is expressed on various cell types. TNFR2 signaling has a balancing role between regulatory and effector functions of T cells. Herein, we investigated the expression of TNFR2 on regulatory T cells (Tregs) and non-Tregs in breast tumor-draining lymph nodes. Mononuclear cells were isolated from 16 axillary lymph nodes, and the expressions of TNFR2, Foxp3 and CD25 were assessed in CD4+ T cells by flow cytometry. Our results showed that the majority of TNFR2+CD4+ T cells were Foxp3−CD25−. However, the percentage of TNFR2+ cells was significantly higher in Foxp3+CD25+CD4+ Tregs compared to Foxp3−CD25−CD4+, Foxp3+CD25−CD4+, and Foxp3−CD25+CD4+ T cell subsets. Among these subsets, Foxp3+CD25+TNFR2+CD4+ T cells were found to have the highest intensity of TNFR2 expression. The intensity of Foxp3 expression in Foxp3+CD25+TNFR2+CD4+ Treg cells was significantly higher than in their TNFR2− counterpart. Collectively, we showed that most of TNFR2+CD4+ T lymphocytes were Foxp3−CD25−, while the majority of Foxp3+CD25+CD4+ Tregs were TNFR2+, and they expressed TNFR2 with the highest intensity. This report highlights the importance of TNFR2 expression on Tregs and paves the way for further investigation of the effects of TNF-α on the suppressive activity of Tregs in the tumor microenvironment. [ABSTRACT FROM AUTHOR]

• The expression of TIM-3 was assessed on T cells in breast tumor draining lymph nodes. • TIM-3 expression on T cells showed association with the number of involved LNs. • The majority of CD4+TIM3+ T cells didn't express Foxp3. T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), which is expressed by immune and nonimmune cells, has been shown to play immunoregulatory roles in the tumor microenvironment. In this study we assessed the expression of TIM-3 by T cells from tumor draining lymph nodes (TDLNs) of patients with breast cancer and its association with disease progression. Lymphocytes were isolated from 41 TDLNs, and flow cytometry was used to determine the expression of TIM-3 on CD4+ and CD8+ T cells, along with the simultaneous expression of CD25, Foxp3 and TIM-3 in CD4+ T cells. The results showed that the frequency of TIM-3+CD8+ T cells was associated with higher tumor grade, and the geometric mean fluorescence intensity (gMFI) of TIM-3 in CD4+ and CD8+ T cells was significantly higher in patients with more than 9 involved lymph nodes than those with fewer involved nodes. The gMFI of TIM3 in CD4+ T cells also showed a direct correlation with the number of metastatic lymph nodes. Phenotypic characterization of TIM-3+CD4+ T cells showed that the majority of CD4+TIM3+ lymphocytes were Foxp3 ̶ CD25 ̶, and the majority of Foxp3+CD25+ regulatory T cells were TIM-3−. Our findings showed that TIM-3 was expressed by CD4+, CD8+ and regulatory T cells in breast TDLNs, and that expression on CD4+ and CD8+ T cells was mostly associated with poor prognosticators such as a higher number of involved lymph nodes or higher tumor grade. More studies are required to confirm TIM-3 as a prognostic marker and a target for immunotherapy in breast cancer. [ABSTRACT FROM AUTHOR]

• About 4–5% of CD4+ lymphocytes in TDLNs of BC had CD4+ CD25− FoxP3+CD127low/− phenotype. • A very small fraction of CD25− cells produce cytokines, lower than regulatory and effector subsets. • CD25− cells display an exhausted phenotype intermediate between effector and regulatory cells. • They have higher expression of IFNγ and IL-2 than Tregs but lower than effector subset. • Expression of IL-10 in this subset is significantly higher than effector but lower than Tregs. A T cell subtype with the CD4+ CD25− FoxP3+ phenotype was recently described. We aimed to investigate the frequency of these cells and their ability to produce cytokines in tumor-draining lymph nodes from patients with breast cancer (BC). Mononuclear cells from lymph nodes of 20 patients with BC were activated and stained for appropriate markers. The cells were assayed with four-color flow cytometry. A very small fraction of CD4+ CD25− FoxP3+ cells produced cytokines at levels that were significantly lower than in the regulatory (CD4+ CD25+ FoxP3+) and effector cell (CD4+ CD25+ FoxP3 −) subpopulations. The expression of IFNγ and IL-2 in the CD4+ CD25− FoxP3+ subset was significantly higher than in Treg cells, but lower than in the effector subset. Conversely, IL-22 expression in Treg cells was significantly higher than in the CD4+ CD25− FoxP3+ subpopulation. The expression of IL-10 in the CD4+ CD25− FoxP3+ subset was also significantly higher than in effector cells. We suggest that CD4+ CD25− FoxP3+ cells in patients with BC are exhausted cells with an intermediate phenotype between effector and regulatory cells. [ABSTRACT FROM AUTHOR]

• B cells derived from breast tumor draining lymph nodes expressed granzyme B (GZMB). • Stimulation with Both IL-21 and anti-BCR were necessary for B cell production of GZMB. • A minimal percentage of GZMB+ B cells expressed perforin. • Metastatic lymph nodes contained less GZMB+ B cells compared to tumor free nodes. B lymphocytes with regulatory or effector functions synthesize granzyme B (GZMB). We investigated the frequency and phenotype of GZMB-producing B cells in breast tumor-draining lymph nodes (TDLNs). Mononuclear cells were isolated from 48 axillary lymph nodes and were stimulated with anti-BCR (B cell receptor), recombinant interleukin (IL)-21 and CD40 L alone or in combination. Flow cytometry was used to evaluate the expression of GZMB in B cells, and in 4 samples the phenotype of GZMB+ B cells was determined. B cells produced GZMB only when stimulated with a combination of IL-21 and anti-BCR for at least 16 h. Adding CD40 L to IL-21 and anti-BCR stimuli resulted in lower GZMB production in B cells. A small fraction of B cells was able to produce perforin in all stimulation conditions, and the majority of GZMB+ B cells were perforin-negative. Both naïve (CD24lowCD27−) and active/memory (CD24hiCD27+) B cells expressed GZMB. In patients with invasive ductal carcinoma, the frequency of GZMB+ B cells was significantly lower in metastatic compared to non-metastatic lymph nodes. The frequency of GZMB+ B cells did not significantly correlate with prognostic factors such as stage, tumor size or Her2 expression. In summary, a subpopulation of both naïve and memory B cells expressed GZMB in breast TDLNs. Our findings underscore the need to investigate the function of GZMB+ B cells in breast tumor immunity. [ABSTRACT FROM AUTHOR]

Abstract Breast cancer is the most prevalent malignancy among women around the world such that more than 1,400,000 new cases are being diagnosed each year. Despite immense studies over many years on diagnosis and treatment of breast cancer, about 30% of treated patients will relapse and require subsequent therapy. By development of hybridoma technology, murine monoclonal antibodies (MAbs) against several human tumor-associated antigens have been produced and characterized in many laboratories. The purpose of these studies is to generate effective monoclonal antibodies that could be useful in tumor diagnosis and therapy. In this study, splenic lymphocytes of immunized BALB/c mouse with a new established breast cancer cell line (Pari-ICR cell line, established in Shiraz Institute for Cancer Research) were fused with the mouse myeloma cell line SP2/0 in the presence of polyethylene glycol. We generated a panel of monoclonal antibodies against the newly established cell line. The hybrid cultures were screened by flow cytometry. Hybridomas that produced antibody to surface antigens of immunizing cell line but not to Human Gingival Fibroblasts, adipose stem cells, and leucocytes isolated from peripheral blood were selected and cloned by limiting dilution method. The 1E3 clone (IgG2a type) that displayed clonal stability was further analyzed for specificity by flow cytometry. MAb 1E3 showed weak to strong reactivity to other cell lines compared with Pari-ICR cell line. Antigen identification was performed by a workflow consisting of immunoaffinity purification, SDS-PAGE, Western blotting, and mass spectrometry analysis. The target of 1E3 mAb was identified as NCAM1. In conclusion, using the antibody-based strategy we identified NCAM1 as a potential therapeutic target and biomarker for breast cancer. Highlights • Discovery of potential biomarkers that can predict tumor behavior is essential in breast cancer. • A panel of monoclonal antibodies was produced by hybridoma technology. • MAbs that reacted with surface antigens of immunizing cell line but not normal cells were selected. • Target was identified as NCAM1 by immunoprecipitation and mass spectrometry. • The anti-NCAM1 antibody has potential application in cancer diagnosis. [ABSTRACT FROM AUTHOR]

Objective: Adipose derived stem cells (ASCs), as one of the important stromal cells in the tumor microenvironment, are determined with immunomodulatory effects. The principle aim of this study was to evaluate the immunosuppressive effects of ASCs on natural killer (NK) cells. Materials and Methods: In this experimental study, we assessed the expressions of indolamine 2, 3-dioxygenase (IDO1), IDO2 and human leukocyte antigen-G5 (HLA-G5) in ASCs isolated from breast cancer patients with different stages as well as normal individuals, using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Immunomodulatory effects of ASCs on the expression of CD16, CD56, CD69, NKG2D, NKp30, NKG2A and NKp44 was also assessed in peripheral blood lymphocytes (PBLs) by flow-cytometry. Results: Our result showed that IDO1, IDO2 and HLA-G5 had higher mRNA expressions in ASCs isolated from breast cancer patients than those from normal individuals (P>0.05). mRNA expression of these molecules were higher in ASCs isolated from breast cancer patients with stage III tumors than those with stage II. The indirect culture of ASCs isolated from breast cancer patients and normal individuals with activated PBLs significantly reduced NKG2D+ and CD69+ NK cells (P<0.05). Conclusion: Results of the present study suggest more evidences for the immunosuppression of ASCs on NK cells, providing conditions in favor of tumor immune evasion. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor-alpha (TNF-α) is mostly known as a soluble cytokine. This study, however, focused on its membranous form whose significance is rarely investigated in antitumor immunity. Herein, we assessed the expression of both membranous and intracellular forms of TNF-α (m/icTNF-α) in the lymphocytes derived from breast cancer-draining lymph nodes. CD4+T cells were the main subset expressing mTNF-α with the highest intensity, whereas icTNF-α expression was most intense in CD8+T cells. An inverse correlation was seen between the frequency of mTNF-α and the expression intensity of this cytokine in B cells. In the clinical context, the higher intensity of mTNF-α expression in CD19+ cells correlated with poor prognosticators, while the frequency of mTNF-α+CD19+ cells showed a reverse correlation with the number of involved lymph nodes. The two forms of TNF-α did not show similar associations with cancer parameters, which highlights the complex role of this cytokine in breast cancer immunity. • B and T cells express membranous TNF-α (mTNF-α) in breast tumor-draining lymph nodes. • The frequency of mTNF-α-expressing cells and its intensity are higher in CD4+ T cells. • More intense expression of mTNF-α in B cells correlates with poor prognosticators. • The higher frequency of mTNF-α+ B cells is associated with a good prognosticator. [ABSTRACT FROM AUTHOR]

Highlights: [•] Accumulation of Tfh, TFR and CD25− Treg cells in tumor draining lymph nodes (TDLNs). [•] In TDLNs, IL17 and IFNγ expression are decreased upon tumor progression. [•] In TDLNs, Treg and Th2 cells are increased in advance stages of disease. [Copyright &y& Elsevier]

Abstract: Immunomodulatory function of bone marrow derived mesenchymal stem cells in cancer has recently been investigated. But the resident mesenchymal stem cells as whole in cancer and in the breast cancer tissue have not been studied well. In the present work we isolated adipose derived stem cells (ASCs) from breast cancer and normal breast tissues to investigate the expressions of IL-4, IL-10 and transforming growth factor (TGF)-β1 in ASCs and to see if ASCs isolated from patients can modulate the regulatory molecules on peripheral blood lymphocytes. Our results showed that IL-10 and TGF-β1 have significantly higher mRNA expressions in ASCs isolated from breast cancer patients than those from normal individuals (P value <0.05). The culture supernatant of ASCs isolated from breast cancer patients with pathological stage III induced upregulation of the mRNA expression levels of IL-4, TGF-β1, IL-10, CCR4 and CD25 in PBLs. In addition, the percentage of CD4+CD25highFoxp3+ T regulatory cells was increased in vitro. When the same culture supernatant was added to ASCs isolated from normal subjects augmentation of the mRNA expressions of IL-4, IL-10, IL-8, MMP2, VEGF and SDF-1 in normal ASCs was also observed. These data collectively conclude that resident ASCs in breast cancer tissue may have crucial roles in breast tumor growth and progression by inducing regulatory molecules and promoting anti-inflammatory reaction within the tumor microenvironment. Further investigation is required to see if the immune suppression induced by ASCs is an independent property from tumor cells or ASCs gain their immunosuppressive potential from malignant cells. [Copyright &y& Elsevier]

Abstract: Adipose-derived stem cells (ASCs) are regarded as a major player of breast cancer microenvironment. By production of various growth factors and expression of regulatory molecules, it is postulated that ASCs protect breast cancer cells from the host immune responses. In this study, the expressions of insulin-like growth factor-1 (IGF-1), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), CXCL8 (IL-8) in breast cancer cells and adipose-derived stem cells isolated from breast tissue of women with breast cancer were investigated. The results were analyzed comparatively in normal ASCs isolated from healthy normal women. In case of breast cancer tissues, results were analyzed between high stage and low stage patients. The expressions of extracted mRNAs were determined using real-time quantitative RT-PCR. As a result, in breast cancer tissues, IGF-1 and IL-8 mRNAs had 28.6 and 56-fold more expressions in high stage compared to low stage patients. In ASCs, relative quantifications (RQ) of VEGF, IL-8, HGF and IGF-1 was about 2-fold higher in patients than controls. Data of this study conclude that presence of resident ASCs within the scaffold of breast tissue may support breast tumor growth and progression through the expressions of tumor promoting factors. [Copyright &y& Elsevier]

Abstract: Breast cancer is the most common female malignancy worldwide. Despite the high incidence of sporadic cases, the rate of familial breast cancer is low. The tumor suppressor gene TP53 (alias p53), located on chromosome 17, has been involved in various malignancies. Mutations in codon 72 of TP53 have been studied in breast cancer and most solid tumors. For study of polymorphisms and allele frequency, 221 female patients with sporadic breast cancer and 205 healthy blood donors as control group were recruited. DNA from peripheral blood mononuclear cells was extracted and amplified using allele-specific polymerase chain reaction. Frequency of homozygotic arginine at codon 72 was 37.6% in patients and 36.6% in controls, for homozygotic proline it was 13.1 and 19.5%, and for heterozygotic Arg/Pro it was 49.3 and 43.9%, respectively. No significant difference was found between patients and controls regarding allele frequencies. Mutation in codon 72 of TP53 gene was not associated with breast cancer in Iranian patients. [Copyright &y& Elsevier]

Abstract: Stromal cell-Derived Factor-1 (SDF-1, CXCL12) is one the ELR− CXC angiogenic chemokines. It contributes to hematopoiesis and lymphocyte trafficking. SDF-1 and its exclusive receptor, CXCR4, are reported to play important roles in tumor growth, angiogenesis and metastasis of different types of tumors such as breast, lung, prostate and pancreatic cancers. SDF-1 gene polymorphism, known as SDF1-3′A, has been investigated in HIV-1 infection and the incidence of breast cancer. This investigation was aimed to study the frequency of SDF1-3′A mutation in Iranian women with breast cancer. Results showed that the frequency of AA and AG genotypes was higher among patients, while the frequency of GG genotype was lower compared to the controls. Thus AA and AG genotypes of SDF-1 may be considered as factors increasing the susceptibility of Iranian women to breast cancer. [Copyright &y& Elsevier]

B cells can promote or inhibit immune responses against breast cancer. We investigated changes in the frequency of B cells with stimulatory or regulatory capacity in breast tumor draining lymph nodes during cancer progression. We isolated mononuclear cells from fresh axillary lymph nodes (LNs) of 44 patients with breast cancer and stained lymphocytes with antibodies against CD19, CD80, CD86, CD39 and CD73. To assess programmed death-1 (PD-1) and PD-ligand 1 (PD-L1) expression, lymphocytes were briefly stimulated, stained for CD19, PD-1 and PD-L1, and examined with flow cytometry. The frequency of CD80+ B cells was higher in nonmetastatic lymph nodes, while the percentage of CD86+ B cells showed a positive relationship with higher tumor grade and higher numbers of involved LNs. A small proportion of unstimulated B cells expressed PD-1 or PD-L1 but these molecules were rapidly upregulated on B cells following activation. The frequency of stimulated PD-L1+ B cells showed an inverse association with estrogen and progesterone receptor expression and a nonsignificant positive association with tumor grade. In addition, the percentage of unstimulated PD-1+ B cells was higher in patients with higher-grade tumors. CD73 expression on B cells was associated with lower numbers of involved LNs, and the frequency of CD39+ B cells was higher in patients with larger tumors. CD86+, CD39+, PD-1+ and PD-L1+ B cells showed associations with poor prognostic factors, therefore their potential role in the suppression of the immune responses against breast cancer should be evaluated in greater detail. [ABSTRACT FROM AUTHOR]