(A) Expression profile of Drak2 mRNA in mouse tissue. Each bar represents the normalized intensity of the amount of Drak2 transcript in each tissue.
(B) The various subsets of cells were purified from thymus, spleen, and lymph nodes and analyzed by Western blot for DRAK2 expression. All subsets were greater than 94% pure. For a loading control, the blots were stripped and then blotted with an anti-actin antibody. These data are representative of two separate sorts.

(A) A sample of the culture supernatant was removed from each well of the experiment described in Figure 2C after 24 hr of stimulation. The amount of IL-2 in each sample was measured by ELISA. This experiment is representative of at least three experiments.
(B) Purified CD4⁺ T cells were stimulated with anti-CD3 and anti-CD28 in the presence of Th1, Th2, or nonskewing conditions for 3 days. The cells were then rested in the presence of IL-2 for 3 days, then restimulated with anti-CD3 in the presence of Brefeldin A for 4 hr and stained for intracellular cytokine and analyzed by flow cytometry. The data are representative of two separate experiments.
(C) Purified T cells were stimulated with 20 ng of plate bound anti-CD3 or 100 ng of plate bound anti-CD3 with soluble anti-CD28 for either 24 or 72 hr, harvested, and stained with antibodies to the various costimulatory molecules. The filled histograms represent electronically gated CD4⁺, Drak2^+/+ T cells and the open represent CD4⁺, Drak2^−/− T cells. Data are representative of three separate experiments.

(A) T cells were purified from the lymph nodes of wt mice and stimulated with anti-CD3 and anti-CD28 for either 2 min, 1 hr, or 24 hr. The cells were then lysed and separated into cytoplasmic and nuclear fractions and analyzed by Western for DRAK2 expression. To confirm that each fraction was pure, the blot was stripped and reprobed with anti-AKT (cytoplasmic) and anti-PARP (nuclear). These data are representative of two separate experiments.
(B) T cells were purified from the lymph nodes of wt mice and stimulated with anti-CD3 or anti-CD3 and anti-CD28 for 24 hr. RNA was isolated and hybridized to Affymetrix GeneChip arrays. Each sample was done in duplicate, and the average of each normalized intensity is shown.
(C) T cells from Drak2^+/+ (solid lines) and Drak2^−/− (dashed lines) mice were purified and labeled with Fluo-4 and Fura red, stained with antibodies against CD4 and CD8, then stimulated with biotin anti-CD3 and streptavidin. Calcium flux was measured by taking a ratio of the change in fluorescence of the two dyes as measured by flow cytometry. Data represent the calcium flux from CD4⁺ cells.
(D) Purified OT-I;Drak2^+/+ and OT-I;Drak2^−/− CD8⁺ T cells were stimulated with peptide-pulsed, magnetically labeled APC for 5 min at 37°C. Each sample was then run over a magnet to separate the APC and T cells. The T cell fraction was lysed and analyzed by Western blot analysis. The amount of ZAP70 is displayed to show that lysates from an equal number of T cells were analyzed. The data represent three separate experiments.

(A) OT-I;Drak2^+/+, Drak2^−/−, and Drak2^−/− splenocytes were labeled with CFSE and incubated with various amounts of the strong agonist peptide, OVAp, or the weak agonist, G4. After 2 days, the number of CFSE^lo, CD8⁺ T cells was determined by flow cytometry. These data are representative of three separate experiments.
(B) T cells were purified by negative depletion from the spleen of Drak2^+/+ (solid lines) and Drak2^−/− (dashed lines) littermates (N3 to C57BL/6), labeled with CFSE, and stimulated with plate bound anti-CD3. The numbers represent the percent of cells that have divided at least once. The percentage for Drak2^+/+ is shown above the line and the percentage for Drak2^−/− is below the line.
(C) Lymph node T cells from wt, Drak2^−/−, and Cbl-b^−/− mice (≥N6 to C57BL/6) were purified by negative depletion and subsequently separated with CD62L magnetic beads. The CD62L⁺ fraction was CFSE labeled and stimulated with plate bound anti-CD3. The cells were greater than 95% Thy1⁺, CD62L⁺. The data are representative of three experiments.

(A) Purified T cells were labeled with CFSE and stimulated with plate-bound anti-CD3 in the absence or presence of varying amounts of anti-CD28. After 4 days, cells were harvested and labeled with antibodies against CD4 and CD8, and stained with Annexin V. Data for CD4⁺ cells from Drak2^+/+ and Drak2^−/− littermates (N3 to C57BL/6J) are shown. The numbers represent the percentage of cells in each quadrant.
(B) The number of live cells recovered from these cultures is shown in the graphs. The solid lines represent Drak2^−/− T cells, and the dashed lines represent Drak2^+/+ T cells. The CD4⁺ cells are denoted by the open square (□), and the CD8⁺ by the closed circle (●).
(C) Drak2^+/+ and Drak2^−/− littermates (>N6 to C57BL/6J) were infected with 2 × 10⁵ PFU of LCMV. T cells were isolated from the spleen and stimulated in vitro for 5 hr in the presence of gp33 or gp61 and Brefeldin A, and stained for intracellular IFN-γ to detect LCMV-specific T cells. The number of CD8⁺, CD44⁺, IFN-γ⁺ (top) and CD4⁺, CD44⁺, IFN-γ⁺ (bottom) cells is plotted. Each point is the average of three mice. The data are representative of four separate infections.
(D) Serum was isolated from the blood of LCMV-infected mice at each time point, and the amount of IFN-γ present was measured by ELISA. These data are representative of three experiments.

(A) EAE was induced with immunization in the hind flanks with a MOG peptide emulsified in complete Fruend’s adjuvant. The mice were monitored daily for signs of disease and given a score according to the severity of disease: 0, no signs of disease; 0.5, altered gait and/or hunched appearance; 1, limp tail; 2, partial hind limb paralysis; 3, complete hind limb paralysis; 4, complete hind limb paralysis and partial forelimb paralysis, and/or moribund. The experiment was performed two times, and the data were pooled together. Each line represents the average score for ten Cbl-b^−/−, ten Drak2^−/−, eight wt mice. The Cbl-b^−/− mice were N12 to C57BL/6J and the Drak2^−/− mice were at least N6 to C57BL/6, and littermates of the Drak2^−/− mice were the wt mice.
(B) Spinal cords were removed from mice 21 days after EAE immunization, fixed, decalcified, and stained with Hematoxylin and Eosin. The images are 40× magnification, and the arrows indicate an example of mononuclear infiltrate.