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.