Figure 3. Immunization with HIVBr18 induces proliferating T cells with a polyfunctional type 1 cytokine profile.

Two weeks after the last immunization with HIVBr18 or the empty vector pVAX1, 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 or medium only. On day 4, cells were pulsed for 12 hours with pooled peptides in the presence of costimulatory antibody and Brefeldin A. Cells were then surface stained with antibodies to CD4 and CD8, permeabilized and stained for intracellular cytokines (IFNγ, TNFα and IL-2) and CD3. (A) Multiparameter flow cytometry was used to determine the frequency of IFNγ, IL-2 or TNFα CD4⁺ and CD8⁺ T cells. (B) Total frequencies of proliferating (CFSE^low) and cytokine-producing CD4⁺ and CD8⁺ T cells; (C) After gating on proliferating (CFSE^low) and cytokine-producing cells, Boolean combinations were then created using FlowJo software to determine the frequency of each response based on all possible combinations of cytokine expression. Background responses detected in negative control tubes were subtracted from those detected in stimulated samples for every specific functional combination. Negative control tubes include cells stimulated with medium and cells from pVAX1 immunized mice stimulated with pooled peptides. For each sample 500,000 events were collected in the live lymphocyte gate. Results are representative of two to three independent experiments.