Figure 3.

Nab2_E51K affects CD8⁺ T-cell expansion after restimulation mediated through TRAIL. Sixty naive OTI T cells expressing Nab2_E51K or the EV were transferred into C57BL/6J mice before immunization with OVA-loaded splenocytes in a helped or helpless setting. Seven days later, lymphocytes were harvested and the number of OVA_257-264–specific T cells was determined by K^b-OVA_257-264 tetramer staining. Cells were restimulated with MEC.B7.SigOVA cells for 7 days, and the number of OVA_257-264–specific T cells was reassessed. The -fold expansion was determined for (A) exogenous Nab2_E51K-expressing or EV-expressing OT-I T cells (A) and for the corresponding endogenous OVA_257-264-specific CD8⁺ T-cell responses (B). Each data point represents an individual mouse or 2 pooled mice (n = 3). Graph shows data compiled from 3 individual experiments (GFP WT, n = 11; all other groups, n = 14). (C-D) CD8⁺ T cells were isolated from helped and helpless mice that had received OT-I-Nab2_E51K cells (C) or OT-I-EV cells (D) before immunization with OVA-coated splenocytes. CD8⁺ T cells were restimulated with MEC.B7.SigOVA cells for 7 days in the presence of 5 μg/mL of TRAL-FC, 5 μg/mL of Fas-FC, or 20 CU/mL of recombinant human IL-2 or medium alone (ctrl). The secondary proliferation of GFP⁺ OTI T cells was assessed with Kb-OVA_257-264 tetramer staining, as described previously.²³ Secondary expansion of helped OT1-EV cells with Ag alone was significantly higher than that of Nab2_E51K–expressing T cells (P < .05; supplemental Figure 2). Each data point represents T cells pooled from 2 mice; the figure depicts 3 biologic replicates. **P < .02; ***P < .002.