Abstract
B-cell malignancy-derived immunoglobulin (idiotype) and survivin, a member of the inhibitor of apoptosis gene family and a shared tumor-associated antigen, are expressed by B-CLL cells. Idiotype- and survivin-specific cytotoxic T cells (CTLs), capable of lysing primary autologous B-CLL cells, can be induced in patients with B-CLL. However, the leukemia cell microenvironment was shown to protect B-CLL cells from apoptosis. The protective effects of stromal cells can be reversed by CXCR4 antagonists in vitro and resensitize CLL cells to spontaneous and chemotherapy-induced apoptosis. The aim of the present study is to investigate whether stromal cell contact impairs CLL killing by CTLs raised against immunoglobulin- or survivin-derived peptides and whether the addition of CXCR4 inhibitors enhances T cell mediated cytotoxicity. To analyze the T cell response, we isolated CD8+ T cells and PBMCs from HLA-A2+ healthy donors. PBMCs were differentiated into dendritic cells (DCs) and CD40-activated B cells. CD8+ T cells were primarily stimulated with peptide-pulsed DCs and then restimulated weekly with peptide-pulsed CD40-activated B cells. Heteroclitic framework region (FR−), heteroclitic complementarity-determining region (CD−) derived peptides, and native and heteroclitic survivin-derived peptides were used for CTL induction. As expected, heteroclitic peptide modifications increased the binding affinity to HLA-A*0201 compared to the native peptide as predicted by the Parker Score (Median change of predicted half-time of dissociation to HLA class I molecules 1429 minutes) and measured by the T2 binding assay (Fluorescence Index (FI) native 0.2; FI heteroclitic 0.9). Cytotoxicity of T cells was assessed by chromium release assay and by flow cytometry against CFSE-labelled CLL cells alone and in co-culture with unlabelled stromal cells in the absence or presence of CXCR4 blocking agents. The induced CTLs efficiently lysed allogenic HLA-A2+ CLL cells (mean cytotoxicity at 30:1, 10:1, 3:1 effector-to-target (E:T) ratio: 15,5%+/−2,8; 7,5%+/−2,8; and 1,9%+/− 0,6), but not HLA-A2 negative CLL cells. Co-culture of CLL cells with the murine stromal cell line M2-10B4 resulted in protection of CLL cells from lysis by antigen-specific cytotoxic T cells in vitro, indeed suggesting a protective role of the microenvironment (mean cytotoxicity at 30:1, 10:1, 3:1 E:T ratio: 5,2%+/−4,1; 0,4%+/−1,6; 1,2%+/−2,0). In contrast to apoptosis induced by fludarabine, CXCR4 blocking agents did not reverse the protective effects of the stromal cell line on T cell mediated cytotoxicity (mean cytotoxicity 30:1, 10:1, 3:1 E:T ratio: 3,1%+/−2,4; 0,8%+/−2,5; 2,3%+/−1,6). These data indicate that the microenvironment may exert protective effects against immunotherapeutic strategies in CLL. However, the protective interaction is not entirely mediated by the CXCR4 - CXCL12 axis. Additional cell-cell interactions appear to play a role and need to be identified as therapeutic targets in order to effectively interrupt the protective effect of the microenvironment on T cell mediated cytotoxicity of B-CLL cells.
Maintenance of high levels of pluripotent hematopoietic stem cells in vitro: effect of stromal cells and c-kit