Tumor-draining Lymph Nodes Of Patients Research Articles

Natural Killer Cell Subsets in Tumor Draining Lymph Nodes of Patients with Bladder Cancer and Their Clinical Implications.

Natural killer (NK) cells are crucial innate components in anti-tumor immunity. However, the clinical impacts and their phenotypes in bladder cancer (BC) remain unclear. To assess the clinical significance of NK cell subsets in tumor-draining lymph nodes of patients with BC. In a cross-sectional study, pelvic lymph nodes were obtained from 49 untreated patients with BC. Mononuclear cells were isolated and immunophenotyped using CD3, CD56, CD16, CD27, and CD11b markers. NK cells were then classified based on their expression patterns of CD56/CD16 (conventional) and CD27/CD11b (new). On average, NK cells constituted 2.99±1.44% of the total lymphocytes in the draining lymph node of patients with BC. The CD56dim and regulatory NK subsets (CD27+CD11b+/-) were the predominant old and new NK, respectively. The NK cells significantly increased in patients with at least one involved node (LN+) compared with those with free nodes (LN-; p=0.022). Conversely, CD56dimCD16- subset significantly decreased in higher stages (p=0.032) and in tumors with muscle invasion (p=0.038). Significant variations were also observed in different T-stages (p<0.05). Regarding new classification, the frequency of CD11b+ regulatory NK cells was significantly lower in node-positive patients (p=0.025). These findings emphasize the dynamic nature of NK cell subsets in bladder cancer and their potential relevance in disease progression and management, suggesting potential implications for therapeutic strategies targeting these specific subsets.

Diversity of Memory CD8+ T Cells in Tumor-Draining Lymph Nodes from Patients with Bladder Cancer.

The role of memory T cells in orchestrating memory responses to previously known tumor antigens is well documented. The aim of this study was to assess the frequency of different memory T cell subsets in tumor-draining lymph nodes of patients with bladder cancer (BC) and their prognostic significance. Mononuclear cells were isolated from 50 tumor-draining lymph nodes of untreated patients with BC and stained with antibodies against the markers CD8, CD95, CD45RO and CCR7. Data were collected using the FACSCalibur flow cytometer and analyzed using FlowJo software. Among the CD8+ cytotoxic lymphocytes, the frequency of different subsets was determined including total memory cells (CD8+CD45RO+CD95+), T central memory (TCM: CD8+CCR7+CD45RO+CD95+), T effector memory (TEM: CD8+CCR7-CD45RO+CD95+), T stem cell memory (TSCM: CD8+CCR7+CD45RO-CD95+) and naïve T cells (CD8+CCR7+CD45RO-CD95-). The analysis revealed that on average 49.32±20.15 (between 1.62% and 87.20%) percent of CD8+ lymphocytes in draining lymph nodes of BC had a memory phenotype. TCM cells showed the highest frequency (34.71±17.04), while TSCM cells (7.51±8.53) demonstrated the lowest. The total frequency of memory cells tended to be higher in patients with tumor invasion to muscle layer (P=0.052) and stage III (P=0.042) than in patients without invasion and stage I. The TCM subset was more frequent in patients with necrotic tumors than in patients without necrosis (P=0.048). TSCM significantly increased in patients with N2 compared to N0 (P=0.042). Conversely, the ratio of TSCM cells to total memory cells was higher in lower tumor stages (P=0.059), tumors without muscle invasion (P=0.026) and low T grouping (P=0.043). Overall the data indicated an increase in the frequency of memory T cells and their TSCM and TCM cells with tumor progression. In contrast, the ratio of TSCM to total memory cells was higher in less advanced tumors. These results suggest that the immune system is frequently exposed to tumor antigens and strives to create a memory T cell reservoir, but this is suppressed by inhibitory factors provided by the tumor. These findings emphasize the importance of understanding the dynamic interplay between memory T cell subsets and BC progression.

Regulatory and effector T cell subsets in tumor-draining lymph nodes of patients with squamous cell carcinoma of head and neck

BackgroundA 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.ResultsThe 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).ConclusionThis 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.

IFNγ-IL-17-IL-22+CD4+ subset and IL-22-producing cells in tumor draining lymph nodes of patients with breast cancer.

A recently introduced CD4+ T subset that mainly secretes interleukin (IL-) 22 has been reported to be associated with a variety of tumors, including colon, gastric, hepatocellular, and small- and large-cell lung carcinoma. Both tumor-promoting and - suppressing roles have been suggested for these cells. In the present study, we aimed to investigate the frequency of IL-22-producing subsets in tumor-draining lymph nodes (TDLNs) of the patients with breast cancer and determine their association with the clinicopathological characterizations of the disease. Thirty untreated women diagnosed with breast cancer were enrolled and their axillary lymph nodes were dissected during surgery. Mononuclear cells were isolated using Ficoll density gradient, activated, permeabilized, and stained by fluorochrome-conjugated antibodies against CD4, IL-22, IL-17, and IFNγ. The cells were then acquired on the FACSCalibur flow cytometer, and raw data was analyzed by the FlowJo software package (V10). Our results demonstrated that 2.39% ± 0.39 of CD4+ lymphocytes in TDLNs of patients with breast cancer produced IL-22. Among them, 0.64% ± 0.8 just produced IL-22 but were negative for IFNγ and IL-17. Statistical analysis indicated that the frequency of CD4+IL-22+ cells was significantly higher in the patients with stageIII and the ones with 3-9 tumor involved lymph nodes (N2) compared to those with stageII and those having 1-3 tumor involved lymph nodes (N1) (P =0.008 and P =0.004, respectively). The higher frequency of IL-22-producing cells in draining lymph nodes of patients with more advanced tumors (higher stage (stageIII) and more involved lymph nodes) suggests a role for IL-22-producing cells in the tumor progression and invasion. However, further studies with larger sample size and more functional studies are needed to clarify the role of IL-22-producing cells in breast cancer pathogenesis.

CD86+ Antigen-Presenting B Cells Are Increased in Cancer, Localize in Tertiary Lymphoid Structures, and Induce Specific T-cell Responses

The role of B cells in antitumor immunity and their impact on emerging immunotherapies is increasingly gaining attention. B-cell effector functions include not only secretion of antibodies, but also presentation of antigens to T cells. A physiologic B-cell subset with immunostimulatory properties was described in humans, defined by a high expression of CD86 and downregulation of CD21. We used multicolor flow cytometry and IHC to elucidate abundance and spatial distribution of these antigen-presenting B cells (BAPC) in blood (peripheral blood mononuclear cells, PBMC) and tumor samples of 237 patients with cancer. Antigen-specific T-cell responses to cancer testis antigens were determined using tetramer staining and sorted BAPCs in FluoroSpot assays for selected patients. We found that BAPCs were increased in the tumor microenvironment of 9 of 10 analyzed cancer types with site-specific variation. BAPCs were not increased in renal cell carcinoma, whereas we found a systemic increase with elevated fractions in tumor-infiltrating lymphocytes (TIL) and PBMCs of patients with colorectal cancer and gastroesophageal adenocarcinoma. BAPCs were localized in lymphoid follicles of tertiary lymphoid structures (TLS) and were enriched in tumors with increased numbers of TLSs. BAPCs isolated from tumor-draining lymph nodes of patients with cancer showed increased percentages of tumor antigen-specific B cells and induced responses of autologous T cells in vitro. Our results highlight the relevance of BAPCs as professional antigen-presenting cells in tumor immunity and provide a mechanistic rationale for the observed correlation of B-cell abundance and response to immune checkpoint inhibition.

GM-CSF-producing lymphocytes in tumor-draining lymph nodes of patients with bladder cancer.

Bladder cancer (BC) is the tenth common cancer worldwide. Despite progress in treatment and the use of chemotherapoeutic drugs, the survival rate of BC patients is still low. Manipulation of the immune system was recently introduced as an interesting alternative treatment for this immunogenic cancer with fewer side effects. Accordingly, in the present study, we assessed the frequency of GM-CSF-producing lymphocytes in tumor-draining lymph nodes (TDLNs) of BC patients and evaluated their relationship with clinicopathological factors and survival rate. Fifty-four patients with BC who had received no treatment were recruited. Mononuclear cells were isolated from fresh homogenized lymph nodes by centrifugation over Ficoll-Hypaque, activated and subsequently analyzed by flow cytometry for the cell surface expression of CD4 and CD8 and the intracellular production of GM-CSF. Flow cytometric analysis revealed that 4.97 ± 2.7% of lymphocytes in TDLNs of patients with BC produced GM-CSF. The mean frequency of GM-CSF-producing cells was 5.5% among CD4+ lymphocytes and 11.7% in the CD8+ population. Elevated frequencies of GM-CSF-producing lymphocytes, as well as a higher production of GM-CSF by CD4+ lymphocytes was observed in the patients with tumor-free lymph nodes, as compared to those with at least one tumor-infiltrated lymph node (p <0.05). On the other hand, the lower frequency of GM-CSF-producing CD4+ lymphocytes (ThGM) was associated with improved overall, but not one-year, survival. No other significant relationship was observed between clinicopathological parameters and the frequency of GM-CSF-producing subsets. Collectively, our findings suggest a protective role for GM-CSF in the early stages of BC; however, the unfavorable association of ThGM frequency with survival rate may imply a more complex role for this cytokine in BC.

CD8-positive memory T cells in tumor-draining lymph nodes of patients with breast cancer

BackgroundHuman immunological memory is a hallmark of the adaptive immune system and plays an important role in the development of effective immune responses against tumors. In the present study, we aimed to determine the frequencies of CD8+ memory T cell subsets including T stem cell memory (TSCM) in tumor-draining lymph nodes of patients with breast cancer (BC).MethodsMononuclear cells were obtained from axillary lymph nodes of 52 untreated patients with BC and stained for CD8, CCR7, CD45RO, CD95 markers to detect different subtypes of memory cells in the CD8+ lymphocyte population. Data were acquired on four-color flow cytometer and analyzed with CellQuest Pro software.ResultsWe observed that 47.65 ± 2.66% of CD8+ lymphocytes expressed the CD45RO, a marker for memory T cells. Statistical analysis showed that the total frequency of central memory T cells (TCM) and their subset with low CD45RO expression was significantly higher in tumor-involved nodes compared to tumor-free ones (P = 0.024 and P = 0.017, respectively). The level of CD95 expression (based on mean fluorescence intensity) on the surface of TCM, their CD45ROhi and CD45ROlow subsets, and TSCM was higher in patients with stage II compared to those in stage I (P < 0.05). In addition, the percentage of naive CD8+ T cells was significantly lower in tumor-involved lymph nodes compared to tumor-free ones (P = 0.025).ConclusionsOur data collectively indicate no significant differences in the frequencies of CD8+ lymphocytes or their memory subsets in tumor-draining lymph nodes of patients with BC. However, the frequency of CD45low TCM was higher in tumor-involved nodes. Along with a decrease in the frequency of naive T cells, the higher frequency of CD45low TCM suggests that despite the immune reaction to provide a pool of effective memory cells, it is blocked in early-stage of memory cells’ differentiation (CD45ROlow), probably by tumor-derived suppressive factors. Identifying the molecular and cellular mechanisms behind this suppression can provide invaluable tools for adoptive T cell therapies in cancer.

NK, NKT and Invariant-NKT Cells in Tumor Draining Lymph Nodes of Patients with Breast Cancer.

NK (natural killer) and NKT (natural killer T) cells, as components of innate immune system, play a crucial role in tumor progression and dissemination. To investigate the percentages of NK cells, NKT cells, iNKT (invariant natural killer T) cells, total T lymphocytes as well as activated T lymphocytes, in tumor draining lymph nodes (TDLNs) of patients with breast cancer (BC) and their association with different clinic-pathological features of the patients. Axillary lymph nodes were obtained from 30 Iranian women with breast cancer. After routine pathological evaluations, mononuclear cells were separated from their lymph nodes and incubated with appropriate fluorochrome conjugated monoclonal antibodies specific for CD3, HLA-DR, CD16/56, and Vα24Jα18-TCR. Data were collected on a four-color flow cytometer and analyzed by CellQuest software. The mean percentages of NK (CD3-CD16/56+), NKT (CD3+CD16/56+) and iNKT (Vα24Jα18-TCR+) cells in TDLNs mononuclear cells of BC patients were 2.04%, 2.44% and 0.1%, respectively. A significant decrease in the percentages of NK and iNKT subsets in patients with grade I was observed compared to grade III (p=0.03 and p=0.01, respectively). Moreover, NK cells were increased in patients with grade III of BC compared to grade II (p= 0.003). The increase in the percentage of NK and iNKT cells in TDLNs of patients with higher grade of BC might suggest a suppressive phenotype for these cells in breast cancer, which merit more functional investigation.

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

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. Flow cytometry was used to measure the expression of TNF-α, IL-10, TGF-β, 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. 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. 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.

High and interrelated rates of PD-L1+CD14+ antigen-presenting cells and regulatory T cells mark the microenvironment of metastatic lymph nodes from patients with cervical cancer.

A better understanding of the microenvironment in relation to lymph node metastasis is essential for the development of effective immunotherapeutic strategies against cervical cancer. In the present study, we investigated the microenvironment of tumor-draining lymph nodes of patients with cervical cancer by comprehensive flow cytometry-based phenotyping and enumeration of immune-cell subsets in tumor-negative (LN(-), n = 20) versus tumor-positive lymph nodes (LN(+), n = 8), and by the study of cytokine release profiles (n = 4 for both LN(-) and LN(+)). We found significantly lower CD4(+) and higher CD8(+) T-cell frequencies in LN(+) samples, accompanied by increased surface levels of activation markers (HLA-DR; ICOS; PD-1; CTLA-4) and the memory marker CD45RO. Furthermore, in LN(+), we found increased rates of a potentially regulatory antigen-presenting cell (APC) subset (CD11c(hi)CD14(+)PD-L1(+)) and of myeloid-derived suppressor cell subsets; the LN(+) APC subset correlated with significantly elevated frequencies of FoxP3(+) regulatory T cells (Treg). After in vitro stimulation with different Toll-like receptor (TLR) ligands (PGN; Poly-IC; R848), we observed higher production levels of IL6, IL10, and TNFα but lower levels of IFNγ in LN(+) samples. We conclude that, despite increased T-cell differentiation and activation, a switch to a profound immune-suppressive microenvironment in LN(+) of patients with cervical cancer will enable immune escape. Our data indicate that the CD14(+)PD-L1(+) APC/Treg axis is a particularly attractive and relevant therapeutic target to specifically tackle microenvironmental immune suppression and thus enhances the efficacy of immunotherapy in patients with metastasized cervical cancer.

IL-17 and IL-4 producing CD8+ T cells in tumor draining lymph nodes of breast cancer patients: positive association with tumor progression.

CD8+ cytotoxic T lymphocytes have been recently divided based on their cytokine expression profile. To evaluate the percentages of CD8+ lymphocytes and their effector subsets including Tc1, Tc2 and Tc17 in the tumor draining lymph nodes (TDLNs) of patients with breast cancer. Single cell suspensions were obtained from TDLNs of 42 patients with breast cancer. Staining of the cell surface markers and intracellular cytokines was performed using appropriate fluorochrome-conjugated antibodies. The data was acquired on a four-color flow cytometer and was analyzed by CellQuestPro software package. The percentages of different CD8+ cell subtypes (Tc1, Tc2 and Tc17) were quantified in CD8+ T lymphocytes. The comparison was made between LN+ versus LN- patients, as well as patients in different clinico-pathological status. The percentage of Tc1, Tc2 and Tc17 subsets were not significantly different between LN+ and LN- patients. Despite no difference in the percentages of Tc1 cells in LN+ patients with infiltrative ductal carcinoma (IDC), the mean expression of IFN-γ by Tc1 cells decreased significantly in comparison to LN- patients. On the other hand, the percentages of Tc2 and Tc17 effector subsets were increased in advanced stages (p=0.018 and p=0.009, respectively). As the first study to investigate various effector subtypes of CD8+ lymphocytes in TDLNs of patients with breast cancer, our data collectively suggests a positive association between IL-17- and IL-4-producing CD8+ T cell percentages (Tc2 and Tc17) in TDLNs with breast cancer progression. Although the number of Tc1 cells seems not to be affected by cancer progression, down-regulation of IFN-γ by these cells seems to be associated with tumor metastasis to TDLNs. These findings may have implications in cancer immunotherapy based on CD8+ effector subsets.

Immunologic Monitoring of Cellular Immune Responses in Cancer Vaccine Therapy

Immunologic Monitoring of Cellular Immune Responses in Cancer Vaccine Therapy

Selective Changes in the Immune Profile of Tumor-Draining Lymph Nodes After Different Neoadjuvant Chemoradiation Regimens for Locally Advanced Cervical Cancer

To assess how neoadjuvant chemoradiation regimens modulate the immune system state in tumor-draining lymph nodes (TDLN), in the setting of advanced cervical cancer. Tumor-draining lymph nodes of patients undergoing chemotherapy only (nonirradiated, NI-TDLN) and chemoradiation with lower-dose (39.6 Gy, LD-TDLN) and higher-dose radiation (50 Gy, HD-TDLN) were analyzed by multicolor flow cytometry. Enlarging our previous data, LD-TDLN showed features overall indicative of an enhanced antitumor response as compared with NI-TDLN, namely a significant Th1 and Tc1 polarization and a lower amount of the potent CD4(+)Foxp3(+)CD25(high) regulatory T cell (Treg) subset identified by neuropilin-1 expression. Conversely, compared with NI-TDLN, HD-TDLN showed features overall indicative of an impaired antitumor response, namely a significantly inverted CD4/CD8 cell ratio, a higher Nrp1(+)Treg frequency, and a higher frequency of CCR4(+)Treg, a Treg subset facilitated in migrating out from TDLN to suppress the immune response against distant cancer cells. Moreover, the Th1 and Tc1 polarization induced by LD radiation was lost, and there was an unfavorable tolerogenic/immunogenic dendritic cell ratio compared with LD-TDLN. Even minor differences in radiation dose in neoadjuvant regimens for locally advanced cervical cancer are crucial for determining the balance between a tolerogenic and an efficacious antitumor immune response in TDLN. Because most of the anticancer immune response takes place in TDLN, the present findings also emphasize the importance of chemoradiation protocols in the context of immunotherapeutic trials.

Detection of Wilms' tumor antigen--specific CTL in tumor-draining lymph nodes of patients with early breast cancer.

The Wilms' tumor antigen (WT1) is overexpressed in approximately 90% of breast tumors and, thus, is a potential target antigen for the immunotherapy of breast cancer. We have tested the working hypotheses that WT1 can be immunogenic in patients with breast cancer and can stimulate CTL of sufficient avidity to kill tumor cells. Paired tumor-draining lymph node and peripheral blood samples were analyzed from five HLA-A2-positive patients with stage I/II breast cancer. Fluorescent HLA-A*0201/WT1 tetramers were used to quantify WT1-specific CTL and the functional capacity of the CTL was assessed using cytotoxicity assays and intracellular cytokine staining. WT1 tetramer-binding T cells expanded from all lymph node samples but none of the corresponding peripheral blood samples. Functional assays were carried out on T cells from the patient who had yielded the highest frequency of HLA-A*0201/WT1 tetramer-positive cells. The cytotoxicity assays showed WT1 peptide--specific killing activity of the CTL, whereas intracellular cytokine staining confirmed that the tetramer--positive T cells produced IFN-gamma after stimulation with WT1 peptide. These WT1-specific T cells killed HLA-A2-positive breast cancer cell lines treated with IFN-gamma but no killing was observed with untreated tumor cells. These results show that WT1-specific CTL can be expanded from the tumor-draining lymph nodes of breast cancer patients and that they can display peptide-specific effector function. However, the CTL only killed IFN-gamma-treated tumor targets expressing high levels of HLA-A2 and not tumor cells with low HLA expression. This suggests that induction of autologous WT1-specific CTL may offer only limited tumor protection and that strategies that allow a high level of peptide/MHC complex presentation and/or improve CTL avidity may be required.

Characterization of Intracellular Cytokine Profile of CD4(+) T Cells in Peripheral Blood and Tumor-draining Lymph Nodes of Patients with Gastrointestinal Cancer

Analysis of serum cytokine levels has shown that cancer-bearing hosts have lower levels of IL-2 and IFN-gamma, suggesting that Th1-type immunity is impaired by cancer. However, the mechanisms of the Th1 dysfunction are not clearly understood. The frequencies of Th1 cells in CD4(+) helper T cells were evaluated with an intracytoplasmic cytokine staining method in peripheral blood lymphocytes (PBL) and lymph node lymphocytes (LNL) of patients with gastrointestinal cancer. Activation of lymphocytes with PMA + Ionomycin induced the expression of IL-2 and IFN-gamma in each lymphocyte population. Compared with PBL of non-malignant donors, PBL in cancer patients contained significantly lower frequencies of CD4(+) T cells that produced IL-2 and IFN-gamma. LNL in cancer patients also contained lower levels of IL-2- and IFN-gamma-producing CD4(+) T cells, although the percentages did not show significant differences from those of PBL in the same patients. Our data suggest that suppression of Th1 cytokine in cancer patients is, at least in part, due to the decreased frequency of Th1 cells with CD4(+) phenotype.