Figure 3: NCL downregulation activates GAr-limited antigen presentation and recognition by specific T lymphocytes.

(a) Effect of NCL knockdown on antigen presentation. H1299 were transfected with 235GAr-OVA and murine MHC class I K^b plasmids and with control siRNA or siRNA targeting NCL, as indicated, and the levels of Kb/OVA-derived antigenic peptide complexes determined using FACS analysis (a.u.: arbitrary units). These experiments were performed three times and the mean quantification of the complex level obtained in cells treated by si-NCL or by si-control are shown and compared using the Student’s t-test (***P<0.001). (b) Same experiment as in a except that H1299 cells were transfected with OVA plasmid (*P<0.05). (c) Effect of NCL knockdown on T-cell proliferation. H1299 cells were transfected with mouse K^b and 235GAr-OVA plasmids and control siRNA (left) or siRNA targeting NCL (right). Afterwards cells were mixed with mouse naive OVA_257–264 (SIINFEKL) specific CD8⁺ T-cells isolated from peripheral and mesenteric lymph-nodes and stained with CellTrace Violet. The proliferation of these SIINFEKL-specific T cells was determined by FACS analysis. Quantification of proliferating T lymphocytes when incubated in presence of cells treated by siRNA targeting NCL (si-NCL) or with control siRNA-treated cells (si-control) are shown on the right. The results were compared using the Student’s t-test (*P<0.05). (d) Same experiment as in c except that H1299 were transfected with OVA plasmid (NS: not significant).