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.

Glucosamine-Modified Mesoporous Silica-Coated Magnetic Nanoparticles: A "Raisin-Cake"-like Structure as an Efficient Theranostic Platform for Targeted Methotrexate Delivery.

This study presents the synthesis of glucosamine-modified mesoporous silica-coated magnetic nanoparticles (MNPs) as a therapeutic platform for the delivery of an anticancer drug, methotrexate (MTX). The MNPs were coated with mesoporous silica in a templated sol-gel process to form MNP@MSN, and then chloropropyl groups were added to the structure in a post-modification reaction. Glucosamine was then reacted with the chloro-modified structure, and methotrexate was conjugated to the hydroxyl group of the glucose. The prepared structure was characterized using techniques such as Fourier transform infrared (FT-IR) spectroscopy, elemental analysis (CHN), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), a vibrating sample magnetometer (VSM), and X-ray diffraction (XRD). Good formation of nano-sized MNPs and MNP@MSN was observed via particle size monitoring. The modified glucosamine structure showed a controlled release profile of methotrexate in simulated tumor fluid. In vitro evaluation using the 4T1 breast cancer cell line showed the cytotoxicity, apoptosis, and cell cycle effects of methotrexate. The MTT assay showed comparable toxicity between MTX-loaded nanoparticles and free MTX. The structure could act as a glucose transporter-targeting agent and showed increased uptake in cancer cells. An in vivo breast cancer model was established in BALB/C mice, and the distribution of MTX-conjugated MNP@MSN particles was visualized using MRI. The MTX-conjugated particles showed significant anti-tumor potential together with MRI contrast enhancement.

Disease Endotypes Predict the Severity of Mucous Membrane Pemphigoid.

Mucous membrane pemphigoid is an autoimmune disease with variable clinical presentation and multiple autoantigens. To determine whether disease endotypes could be identified on the basis of the pattern of serum reactivity, the clinical and diagnostic information of 70 patients with mucous membrane pemphigoid was collected, and reactivity to dermal or epidermal antigens, using indirect immunofluorescence, and specific reactivity to bullous pemphigoid (BP) autoantigens BP180 and BP230, collagen VII, and laminin 332 were evaluated. Most patients had lesions at multiple mucosae, with the most prevalent being oropharyngeal (mouth, gingiva, pharynx; 98.6%), followed by ocular (38.6%), nasal (32.9%), genital or anal (31.4%), laryngeal (20%), and esophageal (2.9%) sites and skin (45.7%). Autoantigen profiling identified BP180 (71%) as the most common autoantigen, followed by laminin 332 (21.7%), collagen VII (13%), and BP230 IgG (11.6%). Reactivity to dermal antigens predicted a more severe disease characterized by a higher number of total sites involved, especially high-risk sites, and a decreased response to rituximab. In most cases, identification of dermal indirect immunofluorescence reactivity is an accurate predictor of disease course; however, confirmation of laminin 332 reactivity is important, with dermal indirect immunofluorescence positivity because of an increased risk of solid tumors. In addition, the ocular mucosae should be monitored in patients with IgA on direct immunofluorescence.

Prognostic significance of CD4-positive regulatory T cells in tumor draining lymph nodes from patients with bladder cancer.

INTRODUCTION AND METHODS: To clarify the role of CD4+ regulatory T cells in bladder cancer, we investigated the frequency of these cells in tumor draining lymph nodes of 50 patients with bladder cancer who underwent radical cystectomy using flow cytometry method. We also assessed their association with prognosis and survival. RESULTS: On average, 30.13 ± 2.17% of lymphocytes in draining lymph nodes from patients with bladder cancer were positive for both CD4 and FOXP3 molecules. Analyses also showed that 9.92 ± 0.8% of CD4+ lymphocytes had a regulatory phenotype (CD4+CD25+FOXP3+CD127low/neg). The frequency of total CD4+FOXP3+ lymphocytes as well as regulatory T cells was significantly greater in patients with at least one tumor-involved lymph node compared to those with tumor-free nodes (P = 0.026 and P = 0.036, respectively). Mean FOXP3 expression in CD4+ lymphocytes was greater in patients with stage IV compared with those in stage III (P = 0.046). No other significant associations were found between the frequency of regulatory T cells and other clinicopathological characteristics or patient survival. CONCLUSIONS: The increased frequency of regulatory T cells in patients with involved lymph nodes suggests that these cells may negatively regulate antitumor immune responses in draining lymph nodes. Our findings may have implications for immunotherapy-based treatments for bladder cancer.

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.