3). CAPRI cell-stimulated cancer cells showed a 40% increase in mean fluorescence intensity (MFI) in HLA class I expression (MFI versus MFI) and a 60% increase

in HLA-DR class II expression (MFI versus MFI) (Fig. 3A). The enhanced MHC class II expression in cancer cells could be pivotal for the Carfilzomib cell line destructive power of CAPRI cells, as CD4 interactions augment cytotoxic T cell responses [34, 35]. Stimulated APC express high levels of MHC class I and class II molecules along with B7 and other costimulatory molecules [36]. We analysed phenotypic markers of CFSE-labelled CD14+ monocytes before activation (day 0) and 1 day (day 1) and 5 days (day 5) after activation (Fig. 4). In CAPRI cells, a considerable number of monocytes lost CD14 expression and matured, as defined by the acquisition of the dendritic cell markers CD1a and GSK3235025 solubility dmso CD83 at day 1 and their marked upregulation at day 5 (Fig. 4B). Upregulation of the costimulatory molecules CD80, CD86 and CD40, and HLA-DR

class II and HLA class I molecules was also observed (Fig. 4B). In only CD3-activated PBMC, the number of CD14+ monocytes and cells expressing CD83 and CD1 remained constant. Upregulation of the costimulatory molecules CD80, CD86, CD40 and HLA class I and of HLA-DR was clearly lower than in CAPRI cell cultures (Fig. 4C). Quantitative analysis of leucocyte subpopulations in CD3-activated PBMC and CAPRI cells from five patients with cancer showed significantly more matured dendritic cells in CAPRI cultures than in CD3-activated PBMC (paired t-test, P = 0.000096) (Table 1) and

a higher percentage of monocytes in CD3-activated PBMC compared to CAPRI cells on day 5 (paired t-test, P = 0.023) (Table 1). Depletion of subpopulations Liothyronine Sodium and the resulting effect on lysis were analysed at the following time points: 1) in unstimulated PBMC before CD3 activation; 2) in unstimulated PBMC to be added to CD3-activated PBMC; and 3) from CAPRI cells before coculture with cancer cells (Fig. 5). Depletion of CD3+CD8+ T lymphocytes at each time point prevented CAPRI cells from developing any lytic capacity (Fig. 5D), and depletion of CD3+CD4+ T cells had the same effect at each time point (Fig. 5C). Depletion of CD14+ monocytes at time point 1) or 2) completely abrogated the lytic activity of CAPRI cells (Fig. 5A), whereas depletion of monocytes at time point 3) did not significantly influence the lysis of cancer cells. Depletion of CD83+ dendritic cells reduced the development of CAPRI cell lytic efficiency by 50% (Fig. 5B). This ‘medium’ contribution to the lytic capacity of CAPRI cells may indicate a continuous supply of contact information and/or of cytokines to T effector cells during cancer cell destruction. The failure of immune responses as a consequence of rudimentary immunogenic information from cancer cells has been previously demonstrated [32, 33].