Flow cytometric analysis of circulating dendritic cell subsets and intracellular cytokine production in advanced breast cancer patients

Patients with advanced cancer are known to have dysfunctions of the immune system. Dendritic cells (DCs) are potent antigen-presenting cells that play a crucial role in antitumor immune response. At least two peripheral blood DC subsets have been described: myeloid-derived CD11c+CD123- DCs (DC1) and lymphoid-derived CD11c-CD123+ DCs (DC2). Upon interaction with T cells, DC2 seemed to support the generation of a Th2 response, while DC1 predominantly prime a Th1 response. Our study was aimed at investigating the number of circulating DCs, and their subsets and functions in 32 patients with advanced breast cancer that achieved an objective response after a standard-dose sequential chemotherapy (CT), compared to 40 healthy controls. Circulating DC subsets and intracellular cytokine production in CD4+ and CD8+ subsets were analyzed using a tri-color flow cytometry assay. DC subsets were identified in peripheral blood, calculating their percentage gated as lin- HLA-DR+ and using BDCA-1, BDCA-2 and BDCA-3 specific markers, as DC1 and DC2 according to expression of CD11c and CD123, respectively. Intracellular cytokines were evaluated in CD4+(Th1 and Th2) and CD8+ (Tc1 and Tc2) T lymphocytes. The mean percentage of BDCA-1 + BDCA-2 + BDCA-3 was similar to that of DC1 + DC2 (p=ns). The mean percentage of DCs and DC1/DC2 ratio were slightly decreased before CT in cancer patients compared with healthy controls (p=ns). After CT, the percent-age of DC1 further decreased (p=0.02). The production of IFN-γ (Th1 and Tc1) significantly decreased (p<0.03) while that of IL-4 (Th2 and Tc2) increased (p=0.04), thus confirming a shift toward a Th2 CD4 and Tc2 CD8 phenotype and the predominance of type 2 DCs. Our results could help clarify the mechanisms of the immune response or immune status of patients with advanced breast cancer that undergo cytotoxic CT and contribute to improve the selection of potential candidates for active immunotherapy trials.