FIG. 5.

Impaired antigen receptor-mediated activation of Sly1^d/d T cells. (a) Splenocytes from wild-type (white bars) or Sly1^d/d mice (solid bars) were stimulated with CD3 (2 μg/ml) alone or a combination of CD3 (0.4 μg/ml) and CD28 (2.5 μg/ml) antibodies and cytokine production was measured after 18 h. Additionally, IL-2 release was determined upon stimulation with the indicated concentrations of phorbol myristate acetate and ionomycin (n = 4 to 6 mice per group). (b) Splenocytes from wild-type (white bars) or Sly1^d/d mice (solid bars) were stimulated by the addition of antibodies against CD3. CD28 antibodies (2.5 μg/ml) or IL-2 (10 ng/ml) were added as indicated. After 66 h [³H]thymidine was added and incorporation was measured after additional 16 h (n = 12 mice per group). (c) Total splenocytes or purified splenic CD4 and CD8 T cells were prepared from Sly1^d/d or wild-type (WT) mice and stimulated with allogeneic BALB/c splenocytes (B/c) or autologous cells (n = 5 mice per group). (d) Mice were immunized with ovalbumin in the presence or absence of CpG DNA (CpG) as an adjuvant. Draining lymph node cells were harvested 4 days later and cultured in vitro with recombinant IL-2 for an additional 4 days. Cytotoxic T-cell activity was assayed using EL4 target cells pulsed with the SIINFEKL peptide (n = 4 independent experiments). *, P < 0.05, and **, P < 0.01 (Student's t test), for all experiments.