4). To ensure the transfer of MHC information, resting/naïve T cells expressing high levels of the αβ TCR were added because CD3 activation downmodulates the αβ TCR [19, 20]. The highly efficient lysis of autologous cancer cells by these CAPRI immune cells (Fig. 1G) confirmed our notion that stimulated APC of patients with cancer harbour/present sufficient tumour-immunogenic information to generate T effector cells. The nearly complete blocking of lysis with antibodies
against HLA class I and class II molecules demonstrated the MHC restriction selleck inhibitor of the lysis (Fig. 2B, C). Furthermore, lysis of allogeneic cancer cells was more efficient when CAPRI cells and cancer cells shared HLA class II antigens (Fig. 2A). To assess the expression levels of costimulatory and MHC molecules of activated APC,
we labelled CD14+ monocytes with LY2109761 datasheet CFSE (Fig. 4). In CAPRI cultures, but not in CD3-activated PBMC, labelled monocytes showed an increased expression of CD40, CD80, CD86 and HLA molecules (Fig. 4). Particularly interesting was the numerical decrease in CD14+ monocytes and the numerical increase in CFSE-labelled cells with the CD1a+CD83+ mature dendritic cell phenotype, which was not seen in CD3-activated PBMC (P = 0.000096, Fig. 4A–C, Table 1). To determine the contribution of CAPRI cell subpopulations during priming and lysis, we depleted subpopulations from Branched chain aminotransferase PBMC before CD3 activation, from unstimulated PBMC before their addition to previously activated PBMC or from CAPRI cells before cancer cell lysis (Fig. 5). Depleting either CD8+ T cells or CD4+ T cells at any time point prevented cancer lysis (Fig. 5). Supernatants from undepleted CAPRI cell cultures did not rescue the effect of CD4+ T cell depletion, indicating a significant cytotoxic activity of CD4+ T cells (not shown). The ‘unrealized potential’ of CD4+
T cells for cancer ACT has been proposed and evaluated [48, 49]. Depletion of APC populations revealed that CD14+ monocytes but not dendritic cells were absolutely required for priming. Monocytes could not be removed from PBMC cultures before CD3 activation or from unstimulated PBMC before their coculture with CD3-activated PBMC. One might speculate that capture of tumour material may silence monocytes in vivo and prevent their differentiation to dendritic cells. Until now, failing immune responses have been explained mainly by the inactivation of T cells at the tumour site rather than by mute monocytes. We do not know whether activated monocytes, activated monocytes in transition of differentiation or rather de novo matured dendritic cells are the crucial cells required to prime naïve T cells. Differentiation of monocytes here may have been induced by activated monocytes priming naïve T cells, and primed T cells could drive monocyte differentiation to dendritic cells.