Superantigen-induced CD4 memory T cell anergy. I. Staphylococcal enterotoxin B induces Fyn-mediated negative signaling

Abstract

Memory CD4 T cells must provide robust protection for an organism while still maintaining self-tolerance. Superantigens reveal a memory cell-specific regulatory pathway, by which signaling through the TCR can lead to clonal tolerance (anergy). Here we show that the src kinase Fyn is a critical regulator of anergy in murine memory CD4 T cells induced by the bacterial superantigen staphylococcal enterotoxin B (SEB). Exposure to SEB results in impaired TCR signaling due to failed CD3/ZAP-70 complex formation. Further, signal transduction through the TCR remains similarly blocked when anergic memory cells are subsequently exposed to agonist peptide antigen. Pharmacological inhibition or genetic elimination of Fyn kinase reverses memory cell anergy, resulting in SEB-induced cell proliferation. The mechanism underlying impaired TCR signaling and subsequent memory cell anergy must involve a Fyn signaling pathway given that the suppression of Fyn activity restores CD3/ZAP-70 complex formation and TCR proximal signaling.

Figure 1. ZAP-70 is not activated, but fyn is hyper-activated, in anergic memory CD4 cells

a. Memory (M) and naive (N) DO11.10 T cells were stimulated for 2 min with SEB or with OVA, or they were left unstimulated (Unstim.). Some cells, initially stimulated with SEB for 14 hrs, were then re-stimulated with OVA (SEB + OVA). ZAP-70 was immunoprecipitated (ip). Immunoblotting (blot) was performed, as indicated, with anti-phosphotyrosine and ZAP-70 Abs. b. Memory and naive DO11.10 T cells were stimulated with SEB for 14 hr or left unstimulated before cell lysis and analysis of pfyn, or c. alternatively, the SEB-stimulated cells were re-stimulated for the indicated times by OVA. Unstimulated cells are not shown in Figure 1c, but they are the same as in naive cells (Figure 1b). Fyn protein was immunoprecipitated, and immunoblotting was performed, as indicated, with anti-phosphotyrosine and anti-fyn Abs. Densitometry was performed on the immunoblots and the relative levels of phosphoprotein are shown (b) or expressed as a ratio relative to the total amount of precipitated protein (a,c). Data are representative of two independent experiments.

Figure 2. Inhibition of Fyn permits memory cells to proliferate in response to SEB

Naive and memory DO11.10 CD4 T cells were labeled with CFSE and then stimulated in vitro with OVA or SEB in the presence or absence of SU6656. After 3.5 days, proliferation was assessed by flow cytometry, measuring CFSE fluorescence on KJ1-26⁺ cells. In the histograms the number in each panel represents the percentage of cells with decreased fluorescence as compared to the undivided peak. Data are representative of three independent experiments.

Figure 3. Fyn-deficient memory cells proliferate in response to SEB

a. Memory CD4 T cells from DO11.10 and DO11.10 x Fyn^−/− mice were labeled with CFSE and then stimulated in vitro with soluble anti-CD3, SEB, or OVA. After 3.5 days, proliferation was assessed by flow cytometry. In the histograms The number in each panel represents the percentage of cells with decreased fluorescence as compared to the undivided peak. Data are representative of three independent experiments. b. Memory (gray bars) or Naive (white bars) cells from the original Fyn^−/− strain (hatched) or their wild-type litter-mates (open) were cultured with soluble anti-CD3, SEB, or ConA. After 3 days proliferation was determined by [3H]-thymidine incorporation.

Figure 4. Suppression of Fyn prevents SEB-induced memory T cell anergy and enables cell proliferation in response to agonist peptide

DO11.10 naive and memory CD4 T cells were labeled with CFSE and then stimulated for 14 hr with SEB, washed, and re-stimulated with OVA. After an additional 3.5 days, proliferation was assessed by flow cytometry. a. Cells were cultured with SEB and in the presence or absence of SU6656. b. Cultures contained cells from either wild-type (WT) or Fyn-deficient DO11.10 mice. In the histograms, the number in each panel represents the percentage of cells with decreased fluorescence as compared to the undivided peak. Data are representative of two independent experiments.

Figure 5. Suppression of Fyn restores TCR proximal signaling in response to SEB

Naive and memory T cells from (a) DO11.10, or, (b). DO11.10 x Fyn^−/− mice were stimulated for 2 min with OVA, SEB, or anti-CD3, or were left unstimulated (Unstim), and CD3ζ-chain was then immunoprecipitated. Immunoblotting was performed, as indicated, using anti-phosphotyrosine, anti-CD3ζ, anti-ZAP-70, or anti-pY319 ZAP-70 (activated ZAP-70). Data are representative of three independent experiments.

Figure 6. C-Cbl association with Fyn and activation is increased in memory cells exposed to SEB

a.,b. As in Figure 1c, memory and naive DO11.10 T cells were stimulated with SEB for 14 hr, washed, and then re-stimulated for the indicated times by OVA. Fyn protein was immunoprecipitated, and immunoblotting was performed, as indicated, with a. anti-phosphotyrosine and b. anti-c-Cbl Abs. c. Freshly isolated memory and naive DO11.10 T cells were stimulated by SEB for the indicated times and c-Cbl was then immunoprecipitated. Immunoblotting was performed, as indicated, using anti-phosphotyrosine, anti-c-Cbl, and anti-Fyn. Data are representative of two independent experiments.

Figure 7. The increase in C-Cbl activation in SEB-treated memory cells is dependent on Fyn

Naive and memory T cells from DO11.10 or DO11.10 x Fyn^−/− mice were stimulated for 10 min with OVA or SEB, or were left unstimulated (Unstim), and c-Cbl was then immunoprecipitated. Immunoblotting was performed, as indicated, using anti-phosphotyrosine or anti-c-Cbl. Data are representative of two independent experiments.