Figure 6. HIVBr27 immunization elicits polyfunctional CD4⁺ and CD8⁺ T cells.

Two weeks after the last immunization with HIVBr27 or empty pVAX1, pooled spleen cells from 6 BALB/c mice were collected, labeled with CFSE (1.25 µM) and cultured for 4 days in the presence of pooled HIV-1 peptides (5 µM) or medium only. On day 4, cells were pulsed for 12 hours with pooled peptides in the presence of Brefeldin A and costimulatory antibody (anti-CD28). A) Multiparameter flow cytometry strategy used to determine the frequency of IFN-γ, IL-2 or TNF-α producing CFSE^low CD4⁺ and CD8⁺ T cells. B) Frequency of IFN-γ, IL-2 or TNF-α producing CFSE^low CD4⁺ (left) and CD8⁺ (right) T-cells. C) Boolean combinations of IFN-γ, IL-2 and TNF-α producing CFSE^low CD4⁺ and CD8⁺ T cells from HIVBr27 immunized mice. Background responses detected in negative control tubes (cells stimulated with medium and cells from pVAX1 immunized mice stimulated with pooled peptides) were subtracted from those detected in stimulated samples. Data are representative of three independent experiments.