Ablation of SLP-76 signaling after T cell priming generates memory CD4 T cells impaired in steady-state and cytokine-driven homeostasis

Abstract

The intracellular signaling mechanisms regulating the generation and long-term persistence of memory T cells in vivo remain unclear. In this study, we used mouse models with conditional deletion of the key T cell receptor (TCR)-coupled adaptor molecule SH2-domain-containing phosphoprotein of 76 kDa (SLP-76), to analyze signaling mechanisms for memory CD4 T cell generation, maintenance, and homeostasis. We found that ablation of SLP-76 expression after T cell priming did not inhibit generation of phenotypic effector or memory CD4 T cells; however, the resultant SLP-76-deficient memory CD4 T cells could not produce recall cytokines in response to TCR-mediated stimulation and showed decreased persistence in vivo. In addition, SLP-76-deficient memory CD4 T cells exhibited reduced steady-state homeostasis and were impaired in their ability to homeostatically expand in vivo in response to the gamma(c) cytokine IL-7, despite intact proximal signaling through the IL-7R-coupled JAK3/STAT5 pathway. Direct in vivo deletion of SLP-76 in polyclonal memory CD4 T cells likewise led to impaired steady-state homeostasis as well as impaired homeostatic responses to IL-7. Our findings demonstrate a dominant role for SLP-76-dependent TCR signals in regulating turnover and perpetuation of memory CD4 T cells and their responses to homeostatic cytokines, with implications for the selective survival of memory CD4 T cells following pathogen exposure, vaccination, and aging.

Fig. 1.

Downmodulation of SLP-76 expression after T cell activation using a conditional knockout mouse model. (A) Kinetics of SLP-76 knockdown. Splenic CD4⁺ T cells from SLP-76^F/nullR26R^yfp (F/null) and SLP-76^F/+R26R^yfp (F/+) mice were activated with anti-CD3/anti-CD28 antibodies and cultured with TATCre protein, and SLP-76 expression was determined by flow cytometry 6–24 h later. Histograms show intracellular SLP-76 expression gated on total CD4⁺ T cells or YFP⁺CD4⁺ T cells (for 19 and 24 h). Shaded histograms represent isotype controls. (B) SLP-76 deletion occurs in the YFP⁺ fraction of cKO^TATCre cells. (Left) Western blot of SLP-76 and LAT expression in CD4 T cells from F/null and F/+ mice before stimulation (Prestim) and sorted, CD4⁺YFP⁺ cKO or cHET cells after TATCre. The line separates the noncontiguous lanes. (Right) Flow cytometric analysis of YFP versus SLP-76 expression in cKO^TATCre and cHET^TATCre CD4 T cells analyzed 48 h post-TATCre administration, gated on CD4⁺ T cells, representative of seven experiments. (C) Surface expression of CD44, CD25, and IL-7Rα on cKO^TATCre (Upper) and cHET^TATCre (Lower) primed cells gated on CD4⁺YFP⁺cells. Results are representative of six experiments. (D) Intracellular phospho-PLC-γ1 expression of cKO^TATCre and cHET^TATCre primed CD4 T cells gated on CD4⁺YFP⁺cells, with shaded histograms denoting control. Results are representative of three experiments.

Fig. 2.

In vivo transfer of primed SLP-76-deficient CD4 T cells. CD4 T cells from cKO and cHET mice were primed as in Fig.1, transferred into RAG2^−/− adoptive hosts, and recovered 2 weeks posttransfer. (A) SLP-76 and YFP expression of persisting cKO^TATCre and cHET^TATCre CD4 T cells, representative of three independent experiments. (B) IL-2 and IFN-γ production by persisting cKO^TATCre and cHET^TATCre CD4 T cells stimulated for 6 h with anti-CD3/anti-CD28 antibodies (Upper) or PMA/ionomycin (Lower) as determined by intracellular cytokine staining. Results are expressed as mean percentage of cytokine⁺YFP⁺ CD4 T cells (n = 6), from two experiments.

Fig. 3.

Reduced persistence and homeostasis of SLP-76–deficient memory CD4 T cells. CD4 T cells from cKO and cHET mice were primed in vitro for 72 h, TATCre treated, and transferred into B6.CD45.1 hosts. Persisting cKO^TATCre and cHET^TATCre memory CD4 T cells were harvested from spleen and lymph nodes 1–5 weeks posttransfer. (A) Surface expression of CD44, CD25, IL-7Rα, and TCRβ gated on CD45.2⁺CD4⁺YFP⁺ cells of cKO^TATCre and cHET^TATCre memory CD4 T cells 5 weeks posttransfer. Shaded histograms are controls. Results are representative of three experiments (n = 5 mice per group). (B) Persistence of cKO^TATCre and cHET^TATCre CD4⁺YFP⁺ memory CD4 T cells in adoptive hosts calculated by dividing the average number of YFP⁺ memory T cells at >5 weeks posttransfer by the average number of YFP⁺ cells at day 6 posttransfer, with n = 12–14 mice for cKO^TATCre and n = 9–10 mice for cHET^TATCre. (C) Flow cytometry plots of BrdU incorporation in cKO^TATCre and cHET^TATcre memory CD4 T cells gated on YFP⁺CD45.2⁺ CD4⁺ cells.

Fig. 4.

Impaired homeostasis of SLP-76–deficient memory CD4 T cells to IL-7/anti-IL-7 complexes. (A) (Left) Absolute numbers of host splenocytes in untreated and IL-7/M25-treated hosts of cKO^TATCre and cHET^TATCre memory cells. (Right) Absolute numbers of CD45.2⁺ YFP⁺CD4⁺ cKO^TATCre and cHET^TATCre memory T cells. (B) BrdU incorporation by cKO^TATCre and cHET^TATCre memory CD4 T cells in untreated and IL-7/M25-treated hosts. (Left) BrdU incorporation of lymph node cKO^TATCre and cHET^TATCre memory CD4 T cells gated on CD45.2⁺YFP⁺SLP-76^lo or CD45.2⁺YFP⁺, respectively, with the percentage of BrdU incorporation indicated in the upper right quadrant. (Right) Average BrdU incorporation of splenic cKO^TATCre (n = 6) and cHET^TATCre (n = 5) memory CD4 T cells compiled from two experiments. (C) cKO^TATCre and cHET^TATCre memory CD4 T cells were incubated for 30 min with media alone (Ctrl), 0.1 ng/mL recombinant IL-7, or 10 ng/mL IL-15 and phosphorylation of STAT5 (pSTAT5) was analyzed by intracellular staining. Histograms show pSTAT5 expression gated on CD45.2⁺YFP⁺CD4⁺ T cells (n = 3–4).

Fig. 5.

Homeostasis and cytokine responses in SLP-76-deficient memory CD4 T cells generated by drug-induced deletion. (A) Tamoxifen-treated mice (Fig. S1) were administered IL-7/M25 complexes or left untreated and in vivo proliferation was assessed by BrdU incorporation. Representative plots show CD4 versus BrdU incorporation gated on CD44hiYFP⁺SLP-76^lo or CD44hiYFP⁺ cells, for cKO^CreT2 and cHET^CreT2 groups, respectively. (B) Compiled BrdU incorporation (mean ± SD) of cKO^CreT2 and cHET^CreT2 groups after IL-7/M25 treatment. (n = 3). (C) cKO^CreT2 and cHET^CreT2 CD4 T cells were incubated for 30 min with media alone (Ctrl), 0.1 ng/mL recombinant IL-7, or 10 ng/mL IL-15 and phosphorylation of STAT5 (pSTAT5) was analyzed by intracellular staining. Histograms show pSTAT5 expression gated on YFP⁺CD44hi CD4 T cells (n = 3–4 mice per group).