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Objective: B-cells can contribute to the suppression or progression of the tumor growth through the secretion of various cytokines. In this study, the relevance of B-cell cytokine production to the outcome variables in patients having squamous cell carcinoma (SCC) of the head and neck was studied. Materials and Methods: Thirty-six draining lymph nodes from untreated patients with tongue and larynx SCC were stimulated with CpG, recombinant CD40L and phorbol-12-myristate-13-acetate/ionomycin and analyzed by flow cytometry for the frequency of B-cell subsets based on interleukin (IL)-10 and tumor necrosis factor-alpha (TNF-α) cytokine production. Results: It was indicated that patients with uninvolved lymph nodes and low stage of the disease had an increased percentage of TNF-α+ CD19+ B-cells (p=0.006 and p=0.041, respectively), whereas the frequency of IL-10+ CD19+ B-cells showed an increasing trend in tumor-draining lymph nodes (TDLNs) of patients with grade 2+3 compared to grade 1 (p=0.054). Conclusion: Collectively, TNF-α-producing B-cells in TDLNs has been reported to be associated with good prognosticators in patients with SCC of head and neck that might have a positive role in immunity to SCC of head and neck. [ABSTRACT FROM AUTHOR]

Background: A crucial role for the immune system has been proposed in the establishment and progression of head and neck squamous cell carcinoma (HNSCC). In this study, we investigated the cytokine and regulatory profiles of T cells in tumor draining lymph nodes (TDLNs) of patients with HNSCC. Results: The frequencies of CD4+TNF-α+ and CD4+TNF-αhi negatively were associated with poor prognostic factors such as LN involvement (P = 0.015 and P = 0.019, respectively), stage of the disease (P = 0.032 and P = 0.010, respectively) and tumor size (P = 0.026 and P = 0.032, respectively). Frequencies of CD8+IFN-γ+ and CD8+IFN-γ+ TNF-α+ T cells showed negative relationship with tumor grade (P = 0.035 and P = 0.043, respectively). While, the frequencies of CD4+IL-4+, CD8+IL-10+, CD8+IL-4+T cells were higher in advanced stages of the disease (P = 0.042, P = 0.041 and P = 0.030, respectively) and CD4+IFN-γ+TNF-α−, CD8+IL-4+ and CD8+IFN-γ+TNF-α− T cells were higher in patients with larger tumor size (P = 0.026 and P = 0.032, respectively). Negative associations were found between the frequencies of CD4+CD25+Foxp3+ and CD4+CD25+Foxp3+CD127low/− Treg cells and cancer stage (P = 0.015 and P = 0.059). Conclusion: This study shed more lights on the changes in immune profile of T cells in TDLNs of HNSCC. Larger tumor size and/or LN involvement were associated with lower frequencies of CD4+TNF-α+, CD8+IFN-γ+ and CD8+IFN-γ+TNF-α+ but higher frequency of CD4+IL-4+ T cells. Moreover, Foxp3+Tregs correlated with good prognostic indicators. [ABSTRACT FROM AUTHOR]

Cervical cancer remains a public health concern despite all the efforts to implement vaccination and screening programs. Conventional treatment for locally advanced cervical cancer consists of surgery, radiotherapy (with concurrent brachytherapy), combined with chemotherapy, or hyperthermia. The response rate to combination approaches involving immunomodulatory agents and conventional treatment modalities have been explored but remain dismal in patients with locally advanced disease. Studies exploring the immunological effects exerted by combination treatment modalities at the different levels of the immune system (peripheral blood (PB), tumor-draining lymph nodes (TDLN), and the local tumor microenvironment (TME)) are scarce. In this systemic review, we aim to define immunomodulatory and immunosuppressive effects induced by conventional treatment in cervical cancer patients to identify the optimal time point for immunotherapy administration. Radiotherapy (RT) and chemoradiation (CRT) induce an immunosuppressive state characterized by a long-lasting reduction in peripheral CD3, CD4, CD8 T cells and NK cells. At the TDLN level, CRT induced a reduction in Nrp1+Treg stability and number, naïve CD4 and CD8 T cell numbers, and an accompanying increase in IFNγ-producing CD4 helper T cells, CD8 T cells, and NK cells. Potentiation of the T-cell anti-tumor response was particularly observed in patients receiving low irradiation dosage. At the level of the TME, CRT induced a rebound effect characterized by a reduction of the T-cell anti-tumor response followed by stable radioresistant OX40 and FoxP3 Treg cell numbers. However, the effects induced by CRT were very heterogeneous across studies. Neoadjuvant chemotherapy (NACT) containing both paclitaxel and cisplatin induced a reduction in stromal FoxP3 Treg numbers and an increase in stromal and intratumoral CD8 T cells. Both CRT and NACT induced an increase in PD-L1 expression. Although there was no association between pre-treatment PD-L1 expression and treatment outcome, the data hint at an association with pro-inflammatory immune signatures, overall and disease-specific survival (OS, DSS). When considering NACT, we propose that posterior immunotherapy might further reduce immunosuppression and chemoresistance. This review points at differential effects induced by conventional treatment modalities at different immune compartments, thus, the compartmentalization of the immune responses as well as individual patient's treatment plans should be carefully considered when designing immunotherapy treatment regimens. [ABSTRACT FROM AUTHOR]

Innate lymphoid cells (ILCs) - which include cytotoxic Natural Killer (NK) cells and helper-type ILC - are important regulators of tissue immune homeostasis, with possible roles in tumor surveillance. We analyzed ILC and their functionality in human lymph nodes (LN). In LN, NK cells and ILC3 were the prominent subpopulations. Among the ILC3s, we identified a CD56+/ILC3 subset with a phenotype close to ILC3 but also expressing cytotoxicity genes shared with NK. In tumor-draining LNs (TD-LNs) and tumor samples from breast cancer (BC) patients, NK cells were prominent, and proportions of ILC3 subsets were low. In tumors and TD-LN, NK cells display reduced levels of NCR (Natural cytotoxicity receptors), despite high transcript levels and included a small subset CD127- CD56- NK cells with reduced function. Activated by cytokines CD56+/ILC3 cells from donor and patients LN acquired cytotoxic capacity and produced IFNg. In TD-LN, all cytokine activated ILC populations produced TNFα in response to BC cell line. Analyses of cytotoxic and helper ILC indicate a switch toward NK cells in TD-LN. The local tumor microenvironment inhibited NK cell functions through downregulation of NCR, but cytokine stimulation restored their functionality. [ABSTRACT FROM AUTHOR]

• Accumulation of NK cells in tumor tissues of the patients with breast cancer may play roles in breast tumor regression. • The expression of activating NKG2D receptor by tumor infiltrating NK cells (TINKs) may play roles in breast tumor regression especially in the patients without tumor involved lymph nodes. • As the tumor growths and the size of tumor increases the accumulation of regulatory NK cells may facilitate the tumor improvement. NK cells have been introduced as the main innate arm of immunity against malignancies. Recent advances introduced new subsets of, and new effector molecules on NK cells suggesting new paradigms for NK cell functions in tumor immunity. Considering these new paradigms, in the current research we investigated the frequency of tumor infiltrating NK cell (TINK) subsets and their functional molecules in breast tumor tissues by flowcytometry method. Breast tumor tissues were obtained from 32 untreated patients with breast cancer. The tissues were then minced mechanically to acquire a single cell suspension and surface-stained with monoclonal antibodies against CD3, CD56, CD11b, CD27, NKG2A, NKG2D and CXCR3. For intracellular staining (ICS), the surface-stained cells were then fixed, permeabilized and stained with anti-Perforin and anti-Granzyme B antibodies. The samples were run and the data were acquired on a four-color flowcytometer. The cell suspension derived from tumor tissue encompassed 3.10 ± 0.52 % CD3−CD56+(bright/dim) total NK cells. Based on the conventional classification the percentages of cytotoxic (CD3− CD56dim) and regulatory (CD3− CD56bright) NK cells were respectively 1.74 ± 0.24 % and 1.36 ± 0.48 %. According to the new classification the percentages of cytotoxic (CD3- CD56+ CD11b+ CD27−), regulatory (CD3−CD56+ CD11b+/- CD27+) and tolerant (CD3−CD56+ CD27- CD11b−) NK cells were respectively 0.48 ± 0.07, 1.55 ± 0.34 and 1.15 ± 0.51. A significant higher frequency of total NK cells (CD3−CD56+ (bright/dim)) in the breast tumor tissues of the patients whose tumor draining lymph nodes (TDLNs) has not been yet involved by tumor cells (LN− patients) compared with the ones with lymph nodes involvement (LN+) (5.91 ± 1.79 % Vs. 2.20 ± 0.20 %, P < 0.004). Furthermore, NK cells with overexpressed activating receptor; NKGD2 (CD3- CD56+(bright/dim) NKG2D+ NK cells) was observed to be elevated in LN− patients compared with the LN+ ones (70.01 ± 7.96 Vs. 42.5 ± 4.81, P < 0.011). Correlation analysis revealed the percentages of conventional regulatory NK cells (CD3- CD56bright) in breast tumor tissue to be in positive correlation with the tumor size (R = 0.380, P < 0.04). The mean percentage of this cell subset was also observed to be higher in patients with T3 tumor size compared with smaller T1 tumor size (1.61 ± 0.20 % vs. 0.75 ± 0.15 %, P < 0.023. Our observations suggest that accumulation of NK cells as well as the expression of activating NKG2D receptor by TINKs may play roles in breast tumor regression especially in the LN− patients. As the tumor growths and the size of tumor increases the accumulation of regulatory NK cells may facilitate the tumor improvement. These observations may have implications in cancer NK cell-based immunotherapy. [ABSTRACT FROM AUTHOR]

Certain chemo-drugs could induce immunogenic cell death (ICD) activating T cell antitumor immunity while trigger indoleamine-2,3-dioxygenase (IDO) upregulation suppressing immune responses. Moreover, to achieve therapeutic efficacies on both primary tumors and tumor draining lymph nodes (TDLNs) in the meantime is still a big challenge. In this study, transfersomes functionalized with a tumor targeting, cell penetrating peptide tLyp1 (CGNKRTR) was developed to co-encapsulate doxorubicin (DOX, ICD inducer) and 1MT (IDO inhibitor). The functionalized transfersomes were complexed with microneedles (MNs) to realize co-delivery towards primary tumors and TDLNs via transdermal administration. The transfersomes were concentrated in the needles of MNs and released with needle dissolution after insertion into skin. After being internalized by cells, DOX induced tumor ICD effect to promote DCs maturation and dramatically activated cytotoxic T lymphocytes (CD8+ T), while 1MT inhibited IDO activity in DCs and reduced the immunosuppressive Tregs, thus mitigating tumor suppressive microenvironment. The nano-complexed microneedles exhibited 2.2-fold suppression in tumor growth compared with the I.V. group, which significantly enhanced anti-tumor effect. [Display omitted] [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]

Abstract CD4+ CD25+ regulatory T (Treg) cells express the transcription factor FOXP3 and play an essential role in preventing autoimmunity. Abundant Treg cell accumulation in tumors and tumor draining lymph nodes (TDLNs) has been reported to correlate with both poor and favorable prognosis in various cancers, which suggests that Tregs may have multiple effects on antitumor immunity. However, the heterogeneity of tumor- and TDLN-infiltrating Treg cells remains unclear. Here we provide heterogeneity analysis of tumor infiltrating human CD4+ Treg cells and their matched adjacent tissues and TDLNs. We defined three different subpopulations of tumor- and TDLN-infiltrating Treg cells by Helios and CCR8 expression in pancreatic ductal adenocarcinoma (PDAC) and confirmed their functional heterogeneity. Helios+ CCR8+ Treg cells with potent suppressor function and limited IL-2 and IFN-γ secretion were identified in tumors and TDLNs. On the contrary, Helios− CCR8− Treg cells have impaired suppressive activity, and elevated expression of pro-inflammatory cytokines. More advanced grades of PDAC have predominantly Helios+ CCR8+ Treg cells and few Helios− CCR8− Treg cells both in tumors and TDLNs that suggests poor prognosis. These data could help further define the role of Treg cells and their functional role in tumors and TDLNs. [ABSTRACT FROM AUTHOR]

Abstract: FOXP3+ regulatory T cells (Tregs) play an important role in the maintenance of tumor immunity tolerance. Compared with conventional myeloid dentritic cells (mDCs), plasmacytoid dendritic cells (pDCs) exhibit poor immunostimulatory ability, and their interaction with T cells often promotes the development of Tregs. The aim of this study was to determine FOXP3+ Tregs and CD123+pDCs infiltration in colorectal cancer and tumor draining lymph node (TDLN), and to evaluate the clinical significance and relationship between pDCs infiltration and Tregs development in the CRC tolerogenic milieu. An immunohistochemical assay was conducted to assess FOXP3+Tregs and CD123+pDCs infiltration in tumor tissue and in metastatic-free TDLN (mfTDLN) and metastatic TDLN (mTDLN). The results showed that FOXP3+ Tregs infiltration was more frequent in tumor tissue than in adjacent normal mucosa (P <0.001). FOXP3+Tregs infiltration was associated with advanced TNM stage and lymph node metastasis (P <0.01 and P <0.01 for TNM stage and lymph node metastasis, respectively). Different from FOXP3+Tregs, CD123+pDCs frequencies were lower in most CRC tumor tissues, whereas the positive rate of CD123 expression in CRC was significantly higher than in adjacent normal mucosa tissue (P <0.01). Compared to mfTDLN, mTDLN was significantly enriched in FOXP3+ Tregs (P <0.01) and increased in pDC/mDC ratio (P <0.01). The statistical analysis demonstrated a significant correlation in both Tregs and pDC/mDC ratio in mTDLN. These results suggest that there are more FOXP3+ Tregs with a stronger prognostic significance which might promote tumor tolerance, and that CD123+pDCs might contribute to Tregs development in the CRC tolerogenic milieu. [Copyright &y& Elsevier]

Objective. In the present study we compared the lymphocyte composition of tumor draining lymph nodes (TDLN) in 13 cervical (CC) and 26 endometrial cancer (EC) patients with special emphasis on the suppressive CD4+ T cell subset constitutively expressing CD25 (T regulatory, Treg, cells).Methods. Microscopically uninvolved TDLNs (n = 122) collected at different distances from the primary tumor site were used. Proximal TDLN included obturator, and internal and external iliac nodes. Distal TDLN included common iliac, presacral, and aortic nodes. Sixteen relevant lymphocyte subsets were assessed. The proliferative response to PHA and TCR crosslinking, the suppressive activity of Treg cells, and IFN-γ and IL-4 production were also assessed.Results. Compared to EC patients, TDLN from CC patients contained a higher proportion of naive CD4+ and CD8+ cells (P < 0.01), a lower proportion of CD4+ pre-effector cells (P < 0.05), and a higher proportion of the most potent Treg cell subset identified by coexpression of a high level of CD25 and CD152 (P < 0.05). Functionally, Treg cells were anergic and efficiently inhibited other T cell proliferation, thereby fulfilling the requirements of genuine Treg cells. The proliferative response of TDLN to both PHA and TCR crosslinking tended to be lower in CC than EC patients and was inversely related to the Treg cell content in both type of tumors. No differences were noticed between CC and EC patients in terms of IFN-γ and IL-4 production. Using pairs of TDLN from individual patients, we showed that proximal TDLN in CC and EC patients displayed a higher CD4/CD8 ratio (P < 0.05 and 0.001, respectively) compared to distal TDLN of the same individual. By contrast, only in EC patients did proximal TDLN contained a proportion of Treg cells higher than distal TDLN (P < 0.02).Conclusions. The present data highlight differences in immune competency of TDLN in CC and EC patients, suggesting that the former is in a relative immunosuppressive status, and also underlines the importance of the proximity to the primary tumor site. [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]