Interleukin (IL)-21 and IL-21 receptor expression in peripheral T and B cells of patients with breast cancer.

IL-21 is a cytokine with versatile antitumor and pro-tumorigenic activities. It is mainly produced by CD4+ T cells and B cells are one of its pivotal targets. In this study, we assessed and compared the expression of IL-21 by CD4+ T cells and the IL-21 receptor (IL-21R) on B cells in the peripheral blood of women with breast cancer and healthy individuals. Blood samples were taken from both patients and controls. Mononuclear cells were seperated using Ficoll-Hypaque density gradient centrifugation. These isolated cells were then stained with either anti-CD19/anti-IL-21R or anti-CD4/anti-IL-21 antibodies and analyzed using flow cytometry. The results showed that there was no significant difference in the percentage of IL-21R+ B cells and IL-21+CD4+ T cells between patients and controls. However, the percentage of CD4+ T cells decreased significantly in patients with breast cancer (P=0.003). This decline was observed from the early stage and before lymph node (LN) involvement. In comparison to the control group, IL-21R+ B cells were relatively lower in patients with stages I+II and those with fewer than 4 involved LNs. The intensity of IL-21 expression in T cells was associated with HER2 expression (P=0.029). Furthermore, we found that the majority of IL-21R+ B cells exhibited a naïve phenotype and most of IL-21+CD4+ T cells did not produce IFN-γ or IL-17. In conclusion, breast cancer from the early stages leads to a significant reduction in the proportion of peripheral CD4+ T cells. However, we did not find a significant change in IL-21 and its receptor expression during disease progression.

Immune Profiling of SARS-CoV-2; What We Know and What We Don't Know.

Coronavirus disease 2019 (COVID-19), described as World War 3, is the current worldwide health challenge and nearly all countries have so far faced this disaster. There is still no cure because of the complicated pathogenesis, however, there are several studies on track investigating different aspects of the immune response to the virus. In this review, we will provide an overview of recent investigations that have analyzed immune cells in patients with COVID-19. We will then discuss the differences in immune profiles between healthy controls and various clinical presentations, including asymptomatic, mild, moderate, and severe cases.

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.

Expression of TNFRs by B and T Lymphocytes in Tumor-Draining Lymph Nodes.

Tumor necrosis factor alpha (TNF-α) has crucial roles in the induction or inhibition of various biological activities in immune and nonimmune cells. This cytokine mainly exerts its effects via two receptors named TNFR1 (CD120a) and TNFR2 (CD120b). Both B and T cells express TNFRs; however, opposing roles have been reported for TNF-α in the adaptive immunity. Lymph nodes (LNs), as the secondary lymphoid organs,  are one of the major places for the formation of immune responses against cancer. In this chapter, we explain the procedure as to how to isolate mononuclear cells from tumor-draining lymph nodes. In addition, we describe the process of surface staining with fluorochrome-conjugated antibodies for the assessment of the TNFRs expression by CD3+, CD3+CD4+, CD3+CD8+, and CD19+ lymphocytes by flow cytometry.

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.

Correlation of 4-1BBL+ B Cells in Tumor Draining Lymph Nodes with Pathological Characteristics of Breast Cancer.

BACKGROUND: B cells can increase the expression of granzyme B in CD8+ T cells through 4-1BBL/4-1BB interaction and promote anti-tumor immunity. OBJECTIVE: To investigate the expression of 4-1BBL on B cells in the breast tumor draining lymph nodes (TDLNs) and its association with disease parameters. METHODS: Using Ficoll-Hypaque gradient centrifugation, mononuclear cells were isolated from axillary lymph nodes of 42 patients. Cells received 4 hours of PMA/Ionomycin stimulation, in vitro. Both unstimulated and stimulated cells were stained with anti‒CD19 and anti‒4-1BBL antibodies and subjected to flow cytometry. RESULTS: 4-1BBL expression was detected on 2.8 ± 1.7% of unstimulated B cells, while 27.4 ± 11.9% of B cells expressed this co-stimulatory molecule following stimulation. In steady state, the percentage of 4-1BBL+ B cells was not associated with cancer characteristics. However, in patients with invasive ductal carcinoma, the percentage of 4-1BBL expressing B cells in stimulated condition had a decreasing trend in grade III, compared to grade II+I. In addition, significantly higher frequency of 4-1BBL+ B cells was seen in the TDLNs of ER+ or PR+ compared with ER‒ or PR‒ patients (p=0.021 and p=0.015, respectively). No significant associations were observed between the frequency of 4-1BBL+ B cells and the number of involved LNs, Her2 expression or disease stage. CONCLUSIONS: The frequency of 4-1BBL+ B cells significantly increased following a short time activation, and showed relative and significant associations with tumor grade and estrogen receptor status, respectively. More investigations are required to evaluate the potential of 4-1BBL+ B cells for use in immunotherapy.

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.