Fig 6.

Spleen cells from 5-w.p.i. LP-BM5 B6 mice inhibit CD4⁺ and CD8⁺ T-cell proliferation as alternatively assessed by flow cytometry. Responder CD8⁺ T-cell IFN-γ production is inhibited in vitro by Ly6G⁺-depleted CD11b⁺-enriched MDSCs. (A) CFSE-labeled B6 responder cells were cultured for 72 h with ConA in the presence of either nonfractionated splenic suppressor cells from uninfected mice or nonfractionated or Ly6G⁺-depleted CD11b⁺-enriched cells from 5-w.p.i. LP-BM5-infected mice. At the termination of incubation, the cells were stained with α-CD4 and α-CD8 fluorochrome-conjugated MAbs, and CFSE dilution was assessed by FACS. The percentages of CD4⁺ and CD8⁺ cells divided and proliferation indices were obtained by the FlowJo proliferation platform (see Materials and Methods), with the Δ values obtained by subtracting the medium-stimulated responder values for the percentages of cells divided or the division index values. The presented pattern of results is representative of two additional experiments. (B) Naïve unfractionated B6 responder cells were cultured with plate-bound anti-CD3 and soluble anti-CD28 in the presence of splenic control cells from uninfected mice or spleen cells from 5-w.p.i. LP-BM5 mice which were Ly6G⁺ depleted and CD11b⁺ enriched. After 72 h, ICCS was performed (see Materials and Methods) to assess responder cell IFN-γ production (% denotes the percentage of total CD8⁺ T cells that are IFN-γ⁺).