Figure 4. VLCs suppress CD8⁺ and CD4⁺ T cell responses.

(A) 5×10⁵ C57Bl/6 CFSE-labeled splenocytes were incubated for 72h in the absence (Negative) or presence of either αCD3 antibody (αCD3) or PMA and ionomycin (PMA). After incubation the cells were stained with αCD8 antibody, and CFSE dilution, indicative of T cell division, was assessed by FACS. Data gated on CD8⁺ cells. (B) As in A, shown as histogram of stimulated (black line) and unstimulated (grey line) CD8⁺ cells. (C) Cells were treated as described for A and B and the percentages of CD8⁺ T cells having undergone cell division, as assessed by CFSE fluorescence, were analyzed. Where indicated, 1×10⁵ CD11c⁺ VLCs from tumor bearing mice or 1×10⁵ CD11c⁺ splenocytes from naïve mice were added. (D) Cells were prepared as in C, and CD4⁺ division was assessed by FACS. The standard deviation of three independent experiments is shown (C, D). The statistical significance (*p<0.05, **p<0.01, ***p<0.005) of stimulated cells with CD11c⁺ VLCs in comparison to stimulated controls with media alone was determined with the Student's t test.