Atypical memory B cells increase in the peripheral blood of patients with breast cancer regardless of lymph node involvement.

BACKGROUND: Breast cancer is the most common cancer in females. The immune system has a crucial role in the fight against cancer. B and T cells, the two main components of the adaptive immunity, are critical players that specifically target tumor cells. However, B cells, in contrast to T cells, and their role in cancer inhibition or progression is less investigated. Accordingly, in this study, we assessed and compared the frequency of naïve and different subsets of memory B cells in the peripheral blood of patients with breast cancer and healthy women. RESULTS: We found no significant differences in the frequencies of peripheral CD19+ B cells between the patients and controls. However, there was a significant decrease in the frequency of CD19+IgM+ B cells in patients compared to the control group (P=0.030). Moreover, the patients exhibited higher percentages of atypical memory B cells (CD19+CD27‒IgM‒, P=0.006) and a non-significant increasing trend in switched memory B cells (CD19+CD27+IgM‒, P=0.074). Further analysis revealed a higher frequency of atypical memory B cells (aMBCs) in the peripheral blood of patients without lymph node involvement as well as those with a tumor size greater than 2cm or with estrogen receptor (ER) negative/progesterone receptor (PR) negative tumors, compared with controls (P=0.030, P=0.040, P=0.031 and P=0.054, respectively). CONCLUSION: Atypical memory B cells (CD19+CD27‒IgM‒) showed a significant increase in the peripheral blood of patients with breast cancer compared to the control group. This increase seems to be associated with tumor characteristics. Nevertheless, additional research is necessary to determine the precise role of these cells during breast cancer progression.

Expression of Interleukin-21 and Interleukin-21 receptor in lymphocytes derived from tumor-draining lymph nodes of breast cancer.

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.

The expression pattern of membranous TNF-α is distinct from its intracellular form in breast cancer-draining lymph nodes.

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.

Regulatory T cells express Tumor Necrosis Factor Receptor 2 with the highest intensity among CD4+ T cells in the draining lymph nodes of breast cancer.

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.

Diversity of KIRs in invasive breast cancer patients and healthy controls along with the clinical significance in ER/PR/HER2+ patients.

Killer cell immunoglobulin-like receptors (KIR) consists of activating and inhibitory genes are essential for natural killer cell education. To determine the association of KIRs with susceptibility to invasive Breast cancer (BC), genotyping of 16 KIRs was performed by sequence-specific primers-polymerase chain reaction in 226 confirmed cases of BC with defined estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER2) status and 226 healthy controls (CNs). We observed a lower frequency of 2DL1 and 2DS4del along with increased frequency of 2DS4fl in cases compared to CNs. Further analysis revealed a higher frequency of KIR2DL2, 2DS1, 2DS2,3DS1 in ER+ cases, 2DL2, 2DL5 in PR+ and 2DL1 in HER2+ cases compared to CNs. The detrimental role of KIR2DS4fl was observed in ER+ and PR+ cases whereas 2DS4del confers protection against ER+, PR+, and HER2+ cases. We noted the predisposing role of Bx genotype, KIR2DS1, 2DS2, 2DS5, 2DL2, 2DL5 for lymphatic invasion in ER+ cases along with a higher rate of lymph node metastasis (LNM) in carriers of Bx genotype and KIR2DS1 in ER+ cases. We suggest a link between B haplotype associated genes with the increased risk of lymphatic invasion and LNM, particularly in ER+ cases of BC.

Significance of TIM-3 expression by CD4+ and CD8+ T lymphocytes in tumor-draining lymph nodes from patients with breast cancer.

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.

The balance of regulatory and stimulatory B cell subsets in breast cancer draining lymph nodes correlates with tumor prognostic factors.

AIMS: 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. MAIN METHODS: 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. KEY FINDINGS: 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. SIGNIFICANCE: 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.

Helios, CD73 and CD39 Induction in Regulatory T Cells Exposed to Adipose Derived Mesenchymal Stem Cells.

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-ß) and IL-17 were assessed using flow cytometry and ELISPOT assays, respectively. RESULT: 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-ß 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-ß 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.

Cytokine profile of CD4+CD25-FoxP3+ T cells in tumor-draining lymph nodes from patients with breast cancer.

BACKGROUND: 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). MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: We suggest that CD4+CD25-FoxP3+ cells in patients with BC are exhausted cells with an intermediate phenotype between effector and regulatory cells.

Expression of major histocompatibility complex class I polypeptide-related sequence B in adipose-derived stem cells from breast cancer patients and normal individuals.

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.

The significance of cytokine-producing B cells in breast tumor-draining lymph nodes.

PURPOSE: The role of cytokine-producing B cells in antitumor immunity is mostly overlooked. In the present study, we investigated changes in B cell cytokine profiles in breast tumor-draining lymph nodes (TDLNs) during disease progression, and associations of these changes with prognostic indicators. METHODS: Flow cytometry was used to measure the expression of TNF-α, IL-10, TGF-ß, IL-2 and IFN-γ in B cells from 42 axillary lymph nodes. The frequencies of IL-10+ and FoxP3+ T regulatory cells (Tregs) were also determined. RESULTS: No significant changes in B cell cytokine profiles were observed during breast cancer progression from stage I to III, but the percentage of B cells with high TNF-α expression (TNFhi) showed a negative relationship with lymph node involvement and Her2 expression (p < 0.05). The percentage of IL-10+ B cells was found to be significantly higher in non-metastatic lymph nodes in node-negative compared to node-positive patients (p = 0.001). The frequencies of IL-10+ and TNFhi B cells were found to be negatively correlated with the number of involved lymph nodes. The frequency of TNFhi B cells showed an inverse correlation with the frequency of FoxP3+ Tregs, which in turn was associated with indicators of a poor prognosis. CONCLUSIONS: Our data indicate that the cytokine profiles of B cells in TDLNs of patients with breast cancer show associations with various disease parameters. TNFhi and IL-10+ B cells correlated positively with indicators of a good prognosis. Further functional studies are required to elucidate the role of cytokine production by B cells in immunity against breast cancer.

TNFR2 but not TNFR1 is the main TNFR expressed by B and T lymphocytes in breast cancer draining lymph nodes.

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.

Importance of CD45RO+ tumor-infiltrating lymphocytes in post-operative survival of breast cancer patients.

PURPOSE: In recent years, the prognostic/predictive significance of tumor infiltrating lymphocytes (TILs) has become a topic of interest. Here, we aimed to evaluate the prognostic significance of CD3+, CD8+, CD45RO+ and Foxp3+ TILs in breast cancer, as well as the relation of these markers to other clinicopathological features of this disease. METHODS: FFPE tumor samples from 94 females with invasive ductal carcinoma of the breast were retrospectively selected and immunohistochemically assessed for CD3, CD8, CD45RO and Foxp3 expression. Digital photos were acquired from the center (CT) and invasive margins (IM) of the tumors, after which positive cells were counted using ImageJ software. RESULTS: We found that greater infiltrations of target lymphocyte subpopulations were associated with TNM stage III, lymph node metastasis, high histological grade, ER negativity and HER2 positivity. The ratios of CD8+ cytotoxic T cells to CD3+, CD45RO+ and Foxp3+ TILs were found to be relatively higher in tumors exhibiting the aforementioned characteristics. In univariate survival analyses, CD8+ TILs in the IM and total CD45RO+ TILs were found to be significantly associated with overall survival (OS). Infiltration of CD45RO+ TILs in the CT and lymph node status were variables that significantly correlated with disease-free survival (DFS). Multiple Cox regression analyses revealed independent significant prognostic effects of total CD45RO+ TILs and lymph node status (HR of 3.24 and 3.19, respectively) in predicting OS. Infiltration of CD45RO+ TILs in the CT (HR 3.12) and lymph node status (HR 3.15) also exhibited significant prognostic effects on DFS. CONCLUSION: From our data we conclude that CD45RO+ TILs serve as prognostic factors for predicting OS and DFS of breast cancer patients.

Mesenchymal stem cells induced anti-inflammatory features in B cells from breast tumor draining lymph nodes.

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.

Cancer and normal adipose-derived mesenchymal stem cells (ASCs): Do they have differential effects on tumor and immune cells?

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.

Memory CD4+ T cell subsets in tumor draining lymph nodes of breast cancer patients: A focus on T stem cell memory cells.

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 (TSCM) 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+ TSCM 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 TSCM phenotype (CD4+CCR7+CD45RO-CD95+). The frequency of TSCM 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 (TCM) with a low expression of CD45RO was significantly higher among these patients. The percentage of CD45ROLow TCM 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 CD95Hi effector memory T cells (TEM) was significantly decreased in involved lymph nodes (p = 0.009). CONCLUSION: Our data suggest that following long-term exposure to putative tumor antigens, TSCM 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 CD45ROLow TCM cells to more functional sub-populations.

Adipose Derived Stem Cells Exert Immunomodulatory Effects on Natural Killer Cells in Breast Cancer.

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.

Induction of T regulatory subsets from naïve CD4+ T cells after exposure to breast cancer adipose derived stem cells.

BACKGROUND: Adipose derived stem cells (ASCs) provoke the accumulation and expansion of regulatory T cells, leading to the modulation of immune responses in tumor microenvironment. OBJECTIVE: To assess the effect of tumoral ASCs on the trend of regulatory T cells differentiation. METHODS: Peripheral blood naïve CD4+ T cells were co-cultured with ASCs derived from breast cancer or normal breast tissues. In separate cultures peripheral blood naïve CD4+ T cells were exposed to the culture supernatants of ASCs. RESULTS: Generation of CD4+CD25+Foxp3+ and CD4+CD25-Foxp3+ Treg subsets was observed after coculture of naïve CD4+ T cell with either ASCs or the related supernatant. The percentage of CD4+CD25+Foxp3+ cells increased after exposing naïve CD4+ T cells to both ASCs and their supernatants while augmentation of CD4+CD25-Foxp3+ subset mostly depended on the presence of ASCs. Similarly, upregulation of FoxP3 molecule was more significant in condition of cell to cell contact. IL-4 and IL-10 were up-regulated in the cocultured naïve CD4+ T cells after exposure to ASCs/supernatant while IFN-γ was down-regulated in the presence of ASCs. CONCLUSION: ASC may act as one of the major players in tumor site with immunomodulatory effects, which may mostly be carried out through direct cell-cell interaction.

Immune profiles of CD4+ lymphocyte subsets in breast cancer tumor draining lymph nodes.

Tumor draining lymph nodes form the first line of defense against tumor dissemination. Lymphocyte subpopulations activated during anti-tumor response determine the outcome of host-tumor interaction. In the present study we explored the percentages of different subtypes of CD4+ lymphocytes, including regulatory cells (TFR, CD25-, and CD25+ Treg cells), helper subsets (Th1, Th2, Th17, and Tfh cells), and the expression level of their cognate cytokines (IFNγ, IL4, and IL17) in tumor draining lymph nodes of patients with breast cancer, and compared the results between node negative (LN-) and node positive (LN+) patients. Forty seven sentinel and non-sentinel auxiliary lymph nodes with or without tumor involvement were collected from untreated breast cancer patients undergoing surgical resection. Mononuclear cells obtained from fresh homogenized lymph nodes were subjected to surface and intracellular staining by flow cytometry. The results revealed the presence of a newly identified subtype of regulatory T cells, TFR, as well as CD25- Treg cells in TDLNs of the breast cancer patients. In addition, evaluation of different helper and regulatory subgroups of CD4+ T lymphocytes showed that upon metastasis of tumor cells to lymph nodes together with the progression of the disease stage, the immune responses changed from an inflammatory to an inhibitory state, as evidenced by a reduction in pro-inflammatory and anti-tumor cytokines, IL17 and IFNγ, as well as an increase in pro-tumorigenic phenotypes, Th2 and Treg cells. This situation may provide a favorable condition for tumor growth and spread.