Peritoneal murine macrophages stimulated with recombinant TSA-1-C4 plus Tc24-C4 antigen combination activates CD8+ T cells
With the purpose to evaluate whether TSA-1-C4 plus Tc24-C4 stimulated macrophages activates CD8+ T cells, peritoneal murine macrophages stimulated with 100, 50, 25 and 12.5 µg/mL of TSA-1-C4 plus Tc24-C4 recombinant antigen combination were co-cultured with CD8+ T cells from naïve mice. IFN-γ, TNF-α, IL-4 and IL-10 were measured from supernatants by ELISA kit-test as we mentioned previously.
As we describe in Table 2 , supernatants from co-cultures of CD8+ T cells and peritoneal macrophages stimulated with either 100, 50, 25 and 12.5 µg/mL of TSA-1-C4 plus Tc24-C4 recombinant antigen combination exhibited significant IFN-γ, TNF-α, IL-4 and IL-10 levels compared to CD8+ T cells co-cultured with non-stimulated macrophages. As we expected, LPS and MTX controls induced the highest production of IFN-γ and TNF-α, or, IL-4 and IL-10 (Table 2 ). Furthermore, we observed a proportional dose-dependent effect when we evaluated pro-inflammatory cytokines (IFN-γ and TNF-α), while higher is the antigen-stimulation, higher is the cytokines production, meanwhile, we observed an inverse dose-dependent effect to evaluate anti-inflammatory cytokines (IL-4 and IL-10), while higher is the antigen-stimulation, lower is the cytokine-production (Fig. 3,A-D ). Our data revealed a high production of IFN-γ and TNF-α by CD8+ T cells co-cultured with TSA-1-C4 plus Tc24-C4 stimulated cells compared to individuals TSA-1-C4 or Tc24-C4 stimulated cells for all concentration evaluated. We no observed statistical differences comparing IL-4 or IL-10 production by CD8+ T cells co-cultured with TSA-1-C4 plus Tc24-C4 stimulated cells compared to individuals TSA-1-C4 or Tc24-C4 stimulated cells for all concentration evaluated (Fig. 3,C 3,D ). All these findings indicate that macrophages stimulated from 12.5 µg/mL onwards of the bivalent recombinant protein strategy are most effective to activate CD8+ T cells compared with macrophages stimulated with individual antigens, demonstrating once again the synergistic effect of TSA-1-C4 plus Tc24-C4 recombinant protein combination; furthermore, CD8+ T cells activated were characterized by a strong production of IFN-γ and TNF-α on in-vitro assays.