Figure 3. Characterization of WT and Klrc1^−/− CD8⁺ T cell and NK cell responses to ECTV.

Mice were infected with ECTV and spleens analyzed for CD8⁺ T cell responses 7 d.p.i. (A) CD3⁺CD8⁺B8R-H2-K^b+ T cell numbers were quantified from spleens. (B) WT or Klrc1^−/− splenocytes were pulsed ex vivo with irrelevant SIINFEKL peptide or poxvirus-specific B8R_20-27, then assessed for CD107a and IFN-γ expression by intracellular staining of CD3⁺CD8⁺ lymphocytes. The frequency of CD107a⁺IFN-γ⁺ CD8⁺ T cells and MFI of IFN-γ⁺ cells were quantified from WT (black bars) and Klrc1^−/− splenocytes (white bars). (C) Splenocytes were incubated overnight with irrelevant or B8R_20-27 peptides to restimulate CD8⁺ T cells. IFN-γ accumulation in the supernatant was quantified by bead array. (D) IFN-γ concentration was measured from the sera of ECTV-infected mice by bead array. (E) Splenocytes were treated as in panel A, then assessed for GM-CSF intracellular content. GM-CSF⁺ cell frequency and MFI of GM-CSF⁺ cells are shown. (F) Splenocytes were treated as in panel C. GM-CSF accumulation in the supernatant was quantified by ELISA. (G) Splenocytes were treated as in panel A, and TNF or IL-2 intracellular content was determined. (H) CD3⁺CD8⁺ lymphocyte blasting was assessed by FSC and SSC. (I) Total CD3⁺CD8⁺ lymphocytes were analyzed for KLRG1 and CD127 expression. Frequency of SLEC (KLRG1⁺CD127⁻) and MPEC (KLRG1⁻CD127⁺) populations are shown. (J) Expression of IFN-γ and CD107a in splenic NK cells were measured 5.5 d.p.i. Splenocytes were incubated with or without 1×10⁵ YAC-1 targets cells, then stained for NK cell markers and intracellular IFN-γ. Dot plots show CD107a and IFN-γ expression on CD3⁻NK1.1⁺ NK cells. (K) Viral load in popliteal lymph nodes was assessed by plaque assay 3 days p.i. All data are pooled from at least 2 independent experiments and were analyzed by multiple t tests. Further supporting evidence can be found in Supplemental Figure 3.