GITR co-stimulation activates classical NF-κB and the Akt-mTORC1 signaling axis to regulate CD4 T cell accumulation post-priming.

<p>(A, B) C57BL/6 mice received a 1:1 mixture of GITR^+/+ and GITR^-/- SMARTA, and were infected the following day with LCMV cl 13. At day eight p.i., proportions of GITR⁺ and GITR- cells were evaluated, with gating strategy shown in B. (C) 10⁶ or (D) 10⁴ GITR^+/+ and GITR^-/- CFSE-labeled CD45.1⁺ SMARTA from F2 littermates were co-transferred into naïve CD45.2 C57BL/6 mice one day prior to LCMV cl 13 infection. The total numbers of GITR⁺ and GITR- SMARTA cells in the spleen at different time points following LCMV cl 13 infection are shown. Each symbol in C shows mean ± SEM of at least two to three mice per group, representative of at least two experiments per time point. (E) Proportions of GITR⁺ and GITR- of: total, CD44^hi, T-bet⁺ Th1, and Tfh SMARTA were evaluated in the spleen at day eight p.i. (F) The proportions of IFNγ⁺ or IFNγ⁺IL-2⁺, and the quantity of IFNγ produced per cell were evaluated in GITR⁺ and GITR^- SMARTA CD4 T cells following five hours of GP_61–80 peptide restimulation, with representative staining shown in G. (H) 10⁶ GITR^+/+ and GITR^-/- CFSE-labeled CD45.1⁺ SMARTA from F2 littermates were co-transferred into naïve CD45.2 C57BL/6 mice. At days three and five p.i., CFSE dilution was evaluated. (I) 10⁶ GITR^+/+ and GITR^-/- CD45.1⁺ SMARTA from F2 littermates were co-transferred into naïve CD45.2 C57BL/6 mice as in A. At day three post-infection, cells were stained directly <i>ex vivo</i> for (p) p65 NF-κB pSer529 and (p)S6 ribosomal protein pSer235/236. (J) Activated GITR^+/+ SMARTA were expanded for two days and then serum starved for 12 hours prior to engaging with 10μg/mL anti-GITR (cone DTA-1) or Rat IgG, followed by evaluation of phosphorylation at Thr308 of Akt. MFI of pThr308 following stimulation is shown, representative of three independent experiments. Numbers adjacent to the representative histograms of I and J are median MFI values. Each symbol in D–G represents an individual mouse, with bars indicating mean ± SEM. Data are pooled from two experiments with a total of eight mice. Data in H show a single representative experiment with two-three mice per group, with at least two independent repeats. Data in I are pooled from two independent experiments with a total of seven mice.