Responses of C57BL/6 WT and Daf1^−/− mouse lymphocytes to antigen restimulation. (A–D) LN cells from four mice in each group were pooled and restimulated with antigen in vitro (in triplicate assays) 12 d after immunization with OVA (A and B) or MOG 38–50 (C and D). Cell proliferation (A and C) and IFN-γ production (B and D) were determined. Similar results were obtained with splenocytes (not depicted). Results are representative of three independent experiments. (E–H) Spleen and LN cells from each mouse were combined and restimulated with antigen in vitro (in triplicate assays) 60 d after immunization with OVA, and the production levels of four cytokines were determined (each bar represents a single mouse, and four mice were used in each group). The x axis represents antigen concentration during restimulation assays. Asterisks designate levels that were below the detection limits. Results are representative of three independent experiments.

Enhanced restimulation response of Daf1^−/− spleen and LN cell tracks with T cells. (A) CD4⁺ T cells and APCs were purified from spleen and LN cells of immunized C57BL/6 WT and Daf1^−/− mice (pooled from four animals in each group and immunized with OVA for 12 d) and cross-matched in antigen-restimulation assays in vitro (2 × 10⁵ CD4⁺ cells/well or 10⁶ APCs/well alone or in combination with or without 25 μg/ml OVA, triplicate assays for each treatment). IFN-γ levels in the cell culture medium were assayed after 48 h of culture. B shows ELISPOT assays of IFN-γ–secreting T cells in lymphocyte cultures of C57BL/6 WT and Daf1^−/− mice. Spleen and LN cells from immunized C57BL/6 WT and Daf1^−/− mice (pooled from four animals in each group and immunized with OVA for 12 d) were restimulated with 25 μg/ml OVA for 40 h (six replicate assays, 10⁵cells/well). Results are representative of two independent experiments.

Daf1^−/− but not Daf1^−/−-C3^−/− mice developed exacerbated EAE. MOG 38–50 was used to induce EAE in groups of C57BL/6 WT, C3^−/−, Daf1^−/−, and Daf1^−/−-C3^−/− mice. Compared with WT mice, Daf1^−/− but not Daf1^−/−-C3^−/− mice developed markedly exacerbated EAE disease (A). There was no significant difference between WT and C3^−/− mice in daily clinical scores. One animal each in the WT and C3^−/− group died, at days 36 and 14, respectively. 8 of the 10 animals in the Daf1^−/− group died, 1 each at days 16, 25, 31, 36, 37, and 39, and 2 at day 35. B–E show representative histological pictures of spinal cords of WT (B), Daf1^−/− (C), C3^−/− (D), and Daf1^−/−-C3^−/− (E) mice harvested from a separate experiment at 15 d after disease induction, demonstrating that there were significantly more inflammatory cell infiltrates in the Daf1^−/− mouse spinal cords (a magnification of 400). F–H show that compared with that of WT (F) and Daf1^−/−-C3^−/− (H) mice, the spinal cords of Daf1^−/− (G) mice stained less intensely with luxol fast blue, suggesting increased demyelination (a magnification of 100).

Responses of BALB/c WT and Daf1^−/− mouse lymphocytes to antigen restimulation. Spleen and LN cells from each mouse were combined and restimulated with OVA in vitro (in triplicate assays) 12 d after immunization, and cytokine production was determined (each bar represents a single mouse, and four mice were used in each group). The x axis represents OVA concentration used for restimulation. Asterisks designate levels that were below the detection limits. Results are representative of two independent experiments.

Hyper T cell response in Daf1^−/− mice is largely dependent on complement. In the experiment shown in A–D, C57BL/6 WT, C3^−/−, Daf1^−/−, and Daf1^−/−-C3^−/− mice were immunized with OVA. After 105 d, spleen and LN cells (pooled from four animals in each group) were restimulated in vitro (triplicate wells) and assayed for IFN-γ (A) and IL-2 (B) production. The frequency of IFN-γ–secreting T cells in the lymphocyte cultures was also determined by ELISPOT (C; six replicate assays, 10⁵ cells/well, stimulated with 25 μg/ml OVA for 40 h). The average number of IFN-γ–secreting T cells per well is shown in D. Results are representative of two independent experiments. Experiments using splenocytes or LN cells alone gave similar results to those shown in A and B (not depicted). In the experiments shown in E and F, C57BL/6-Df1^−/− mice were treated with an anti-C5 mAb or an isotype-matched IgG control antibody. As another control, C57BL/6 WT mice were also treated with the isotype-matched IgG. 12 d after immunization, spleen and LN cells (pooled from four mice in each group) were restimulated with 25 μg/ml OVA in culture for 40 h. IFN-γ production was assessed by ELISA (E; triplicate wells) or ELISPOT (F; six replicate wells at 10⁵ cells/well).

Daf1^−/− mouse T cells responded similarly to SEB stimulation and had no defect in T reg cells. In the experiments shown in A–D, splenocytes from C57BL/6 WT and Daf1^−/− mice (n = 4 mice in each group) were stimulated with SEB at the indicated concentrations and their proliferative response (A), CD69 expression on CD4⁺ (B) and CD8⁺ (C) T cells, and IFN-γ production (D) were compared. No significant difference was detected between the two groups in any of the measurements. Results are representative of two independent experiments. E and F show that there was no significant difference between WT and Daf1^−/− mice in the number of naturally occurring T reg cells. Percentage of CD25⁺ cells among spleen CD4⁺ T cells was determined by FACS analysis in seven WT and eight Daf1^−/− mice (E). Foxp3 expression in purified spleen CD4⁺ T cells (n = 3 samples in each group, with each sample representing splenocytes pooled from two mice before CD4⁺ cell selection) was determined by real-time RT-PCR (F). Foxp3 levels are shown as relative expression (R.E.) to the housekeeping gene L32. G shows that there was no difference between CD4⁺ CD25⁺ T cells from WT (W) or Daf1^−/− (K) mice in their ability to inhibit anti-CD3–stimulated proliferation of CD4⁺ CD25⁻ T cells. W⁻, CD4⁺ CD25⁻ T cells from WT; K⁻, CD4⁺ CD25⁻ T cells from Daf1^−/−; W⁺, CD4⁺ CD25⁺ T cells from WT; K⁺, CD4⁺ CD25⁺ T cells from Daf1^−/−; APC, irradiated WT splenocytes.