FIG. 4.

Increased IL-2 production and T-cell proliferation in ALX-deficient mice. (A) Purified T cells from wild-type and ALX-deficient mice were stimulated with 3 μg/ml plate bound anti-CD3 alone, with 0, 0.1, 0.3, or 1 μg/ml anti-CD28 in solution, or with 50 ng/ml PMA and 1 μM ionomycin as indicated in the figure. Culture supernatants were collected after 48 h and examined for IL-2 production by ELISA. The results shown are averages from three wild-type and three ALX-deficient mice and are normalized to the amount of IL-2 produced by ALX-deficient T cells stimulated with plate-bound anti-CD3 and anti-CD28. Error bars show the standard deviations. (B) Purified T cells from wild-type and ALX-deficient mice either were left unstimulated (shaded area), were stimulated for 48 h with 3 μg/ml plate-bound anti-CD3 with 0.3 μg/ml anti-CD28 in solution (darker line), or were stimulated with 50 ng/ml PMA and 1 μM ionomycin (lighter line) as for panel A and examined for expression of CD25 by FACS. Shown are representative data from four separate experiments. (C) Purified T cells from wild-type (shaded gray) and ALX-deficient (darker line) mice were CFSE labeled and stimulated with plate-bound anti-CD3 with or without anti-CD28. After 3 days, the cells were harvested. TOPRO-3 (100 nM) was added to exclude dead cells from further analysis, along with CD4-PE to analyze proliferation within the T-cell population. A fixed number of 6-μm beads were added to each sample to permit calculation of the absolute number of live cells within each sample. The y axis represents absolute numbers of live T cells. The arrow indicates the undivided peak. (D) Quantification of the absolute number of live T cells recovered from four sets of CSFE experiments with wild-type and ALX-deficient purified T cells as described for panel C. Error bars reflect the standard deviations for four mice within each group. (E) Ex vivo hyperresponsiveness to antigen in ALX-deficient mice. Three wild-type and three ALX-deficient mice were immunized with 100 μg of OVA plus complete Freund's adjuvant. After 2 weeks, splenocytes of the same genotype were pooled and cultured ex vivo with 10 μg/ml OVA. Cells restimulated for 48 h were given 1μCi/well [³H]thymidine for an additional 24 h. The supernatant was assessed for IL-2 production by ELISA at 48 hours after OVA restimulation. The standard deviations reflect triplicate ELISA wells. ND, not detected. (F) Results of [³H]thymidine incorporation at 72 h. The standard deviations reflect six wells per condition. WT, wild type; ALX KO, ALX deficient.