Functional and molecular analysis of follicular helper T lymphocytes in breast cancer patients

T follicular helper (Tfh) cells are involved in the maturation of antigen-specific antibody-producing B cells in the lymph node (LN). The molecular and functional characterization of Tfh cells found in the LN and peripheral blood of cancer patients has not been clearly defined. In this study, lymphocytes isolated from the breast cancer patients' LN and peripheral blood were used in phenotypical characterization; CD4, CD8, PD-1, CXCR5, CCR7, CD45RO, CD127, CD25, TIM-3, LAG-3, CTLA-4 and ICOS expression were analyzed with flow cytometer and evaluated in the light of clinical data. Immunofluorescence staining was conducted for CD4, CD19 and BCL-6 on LN sections. In functional analysis, CD4+ T cells were isolated from samples, stimulated with anti-CD3/CD28 microbeads and time dependent expression of PD-1 and CXCR5 related early activation and inhibitory markers and their proliferation were examined. After stimulation, CD4+ T cells were isolated as PD-1 and CXCR5 dependent and in these cells BCL-6, ICOS and TIM-3 expressions were investigated by immunofluorescence staining and the mRNA level of molecules which are associated with other T cell subgroups were analysed with RT-PZR technique. As a result, higher Tfh related CD4+PD-1+CXCR5+ cells were detected in the LN compared to the circulation, these cells were found to have CCR7+CD45RO+ central memory phenotypic features and have high levels of CD127, TIM-3 and ICOS expression. In addition, it was determined that CD4+PD-1+CXCR5+ cells have a higher proliferation capacity and increase early activation and inhibitory receptor expressions with stimulation. High levels of BCL-6 mRNA, ICOS and TIM-3 expression were also observed in CD4+PD-1+CXCR5+ cells after stimulation. Thus, our study highlights that Tfh cells can be characterized mainly as CD4+PD-1+CXCR5+BCL-6+ICOS+TIM-3+ cells with high proliferative capacity in LN, and it is suggested that targeting the activation of these cells can induce anti-cancer humoral immune response.