Purpose: Cellular immunotherapy frequently fails to induce sustained remissions in patients with multiple myeloma, indicating the ability of multiple myeloma cells to evade cellular immunity. Toward a better understanding and effective therapeutic modulation of multiple myeloma immune evasion mechanisms, we here investigated the role of the tumor microenvironment in rendering multiple myeloma cells resistant to the cytotoxic machinery of T cells.Experimental Design: Using a compartment-specific, bioluminescence imaging-based assay system, we measured the lysis of luciferase-transduced multiple myeloma cells by CD4 þ or CD8 þ CTLs in the presence versus absence of adherent accessory cells of the bone marrow microenvironment. We simultaneously determined the level of CTL activation by measuring the granzyme B release in culture supernatants. Conclusion: These results reveal the cell adhesion-mediated induction of apoptosis resistance as a novel immune escape mechanism and provide a rationale to improve the efficacy of cellular therapies by pharmacologic modulation of CAM-IR.

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<p>Supplementary Figure 1 - PDF file 53K, Antigen specific, HLA-restricted and dose-dependent recognition of the MM cell lines U266 and UM9, but not of accessory cells by CD8+ and CD4+ mHag-specific CTLs. The CD4+ (A) and CD8+ (B) CTLs were co-cultured with the luc+ MM cell lines UM9 and U266 in different effector to target ratios. The HLA- and mHag typing of these MM cell lines are indicated. MM cell survival was determined 24 h after addition of T cells by CS-BLI. Results represent the mean values of triplicate cultures (+/- SEM). Results are representative of 3 independent assays. In (C and D) CD4+ (C) and CD8+ (D) CTLs were co-cultured with accessory cells. Accessory cell survival was determined 24h after addition of T cells by FACS annexine V/ propidium iodide staining of CD4 and CD8 negative cells in the co-cultures. Results are depicted as % surviving cells, measured as relative number annexin V/propidium iodide negative cells, compared to no T cells control</p>

<p>Supplementary Figure 6 - PDF file 74K, Synergistic interaction of YM155 and CTLs. In A, the effect of YM155 on survivin and MCL-1 protein levels of MM cells is shown. The cells were cultured with YM155 during 24h and the western blot assays were carried out as indicated in material methods. Band intensities were quantified using image J software, calculated as relative values to the control protein tubuline and plotted as relative to the expression levels of cells cultured without YM155. In B, the type of interaction between YM155 and CTLs (see figure 5C-D) in the presence of accessory cells was analyzed using Compusyn software (version 1.0, 2004 �). In the plots (A) the CI values corresponding to the doses in figure 5 are shown. A CI value of <1, 1 or >1 indicates synergy, additive effects or antagonism, respectively. In C the supernatants of assays as described in Fig 5C-D granzyme B release was measured using ELISA (B). Results are expressed as the mean values of the triplicate cultures. Error bars represent the SEM. Results are representative of 3 independent assays</p>

<p>Supplementary Figure 3 - PDF file 55K, RGDw reduces adhesion of MM cells to accessory cells. Adherent HS-5 en HUVEC accessory cells were incubated with RGDw or an irrelevant peptide for 1hour. After washing away the unbound peptide, UM9 (A) or U266 (B) cells were added and incubated overnight. Plates were reversely centrifuged to determine adhesion to accessory cells. Results represent the mean values of triplicate cultures (+/- SEM). Differences in adhesion were tested by unpaired two tailed student's t test. *= p<0.05; **= p<0.01, ***= p<0.001</p>

<p>Supplementary Figure 5 - PDF file 41K, Bortezomib does not improve the CTL mediated lysis of Fas negative L363 cellsL3L3 reactive CD8+CTLs were incubated at the indicated effector to target ratios with luc-transduced L3L3 cells alone or in the presence of various dilutions of bortezomib. The CTL mediated and bortezomib mediated lysis of L3L3 cells was determined after 48 hours by BLI. Results are depicted as % surviving cells using the triplicate cultures without T cells and bortezomib as the 100% survival control</p>