FIG. 5.
Effect of C. neoformans supernatant fractions and mannosylated inhibitors on activation of the T-cell hybridoma cell line X5A3. (A) Irradiated naive splenocytes (2 × 105/well) and X5A3 hybridoma cells (105/well) were incubated for 24 h in the presence of the indicated protein concentrations of C. neoformans mannoprotein and flowthrough culture supernatant fractions. (B) The contribution of mannose receptors to the X5A3 response to MP was assessed by using the following inhibitors: no inhibitor (control), 50 mM methyl α-d-galactopyranoside (GalP), 50 mM methyl α-d-mannopyranoside (ManP), or 1 mg of S. cerevisiae mannans/ml (mannan). For both panels, supernatants were collected, and the activation of X5A3 was measured by assaying for IL-2 production by using the CTLL-2 bioassay, as described in Materials and Methods. Data are the means ± the standard error of the means of three representative independent experiments, each performed in duplicate or triplicate. (A: *, P < 0.03, by Student t test, comparing MP versus FT; B: *, P < 0.03 comparing control or GalP versus ManP or mannan).