<i>Rbpj</i> deficiency in CD8⁺ T cells does not alter EAU susceptibility.

<p>Rbpj^f/f-E8I and Rbpj^+/+-E8I mice were immunized with IRBP to induce EAU and EAU clinical scores were evaluated until day 39. The data are the mean ± SD with five mice per group. The score for each mouse was the average for both eyes. The data in this Figure are representative of three independent experiments.</p

IL-22 is involved in the progression of late-phase EAU.

<p>Rbpj^f/f-CD4 and Rbpj^+/+-CD4 mice were immunized with IRBP to induce EAU and EAU clinical scores were evaluated until day 39. rIL-22 was administered to the mice according to the following schedules; the day of IRBP immunization is day 0. (A) 3-day intervals (from day 1 to day 13), (B) 3-day intervals (from day 14 to day 39), (C) 3-day intervals (from day 1 to day 39). The data are the mean ± SD with five mice per group. **<i>p</i><0.01. The data in this Figure are representative of three independent experiments.</p

<i>Rbpj</i> deficiency in T cells ameliorates the induction of EAU.

<p>(A) Rbpj^f/f-CD4 and Rbpj^+/+-CD4 mice were immunized with IRBP to induce EAU and EAU clinical scores were evaluated until day 39. The data are the mean ± SD with five mice per group. **<i>p</i><0.01. The score for each mouse was the average for both eyes. (B) Purified CD4⁺ T cells from Rbpj^f/f-CD4 and Rbpj^+/+-CD4 mice immunized with IRBP were stimulated with increasing doses of IRBP protein and their proliferative responses to antigen were measured by [³H] thymidine uptake. The results shown are the mean ± SD for triplicate cultures. **<i>p</i><0.01. The data in this Figure are representative of three independent experiments.</p

<i>Rbpj</i> deficiency in T cells reduces of IL-22 production.

<p>Rbpj^f/f-CD4 and Rbpj^+/+-CD4 mice were immunized with IRBP and CD4⁺ T cells were purified from draining lymph nodes 7 days after immunization. Purified CD4⁺ T cells were cultured with irradiated spleen cells from B6 mice in the presence of IRBP protein (50 µg/ml) for 3 days. (A) IL-17 in culture supernatants was evaluated by ELISA. (B) mRNA was purified from CD4⁺ T cells 7 days after immunization and the relative levels of <i>Rorc</i> against <i>Actin</i> were evaluated by real-time PCR. (C) IL-22 and IFN-γ in culture supernatants 72 hours after stimulation of purified CD4⁺ T cells from Rbpj^f/f-CD4 and Rbpj^+/+-CD4 mice immunized with IRBP (7 days previously) was evaluated by ELISA. The data are the mean ± SD from triplicate cultures. **<i>p</i><0.01. The data in this Figure are representative of three independent experiments.</p

GSI treatment is useful to treat EAU.

<p>B6 mice were immunized with IRBP to induce EAU and EAU clinical scores were evaluated until day 39. GSI was administered to the mice according to the following schedules; the day of IRBP immunization is day 0. 2-day intervals (from day 13 to day 39). (A) CD4 and CD8 expression in the draining lymph nodes at day 14 were evaluated by flow cytometry. (B) CD4⁺ T cells were purified from draining lymph nodes of mice that received GSI or DMSO 1 day before and expression of <i>Hes1</i> was evaluated by PCR (from left to right; 100 ng, 10 ng, 1 ng of mRNA). (C) EAU clinical scores, (D) CD4⁺ T cells were purified from draining lymph nodes on day 12. Purified CD4⁺ T cells were cultured with irradiated spleen cells from B6 mice in the presence of IRBP protein (50 µg/ml) for 3 days. IL-22 in culture supernatants was evaluated by ELISA. The data are the mean ± SD of triplicate cultures. **<i>p</i><0.01. The data in this Figure are representative of three independent experiments.</p

T cell development is normal in CD98hc^f/f-CD4 mice.

<p>(A) CD4⁺TCRβ⁺, CD8⁺TCRβ⁺, CD19⁺, and CD11c⁺ cells in the spleens and CD4⁺TCRβ⁺, CD8⁺TCRβ⁺, CD4^-CD8^- (DN) and CD4⁺CD8⁺ (DP) cells in the thymus of CD98hc^f/f-CD4 mice (solid line) and CD98hc^+/+-CD4 mice (black shadow) were stained with an anti-CD98hc mAb. CD98hc expression was evaluated by flow cytometry. Unstained cells were used as a negative control (gray shadow). (B) Thymocytes from CD98hc^f/f-CD4 (closed) and CD98hc^+/+-CD4 (open) mice were stained with anti-CD4 and anti-CD8 mAbs. Total cell numbers for CD4^-CD8^- (DN; double negative), CD4⁺CD8⁺ (DP; double positive), CD4⁺CD8^- (CD4⁺), or CD4^-CD8⁺ (CD8⁺) cells were evaluated (left). CD69 expression on CD4⁺CD8⁺, CD4⁺CD8^-, or CD4^-CD8⁺ cells was evaluated by flow cytometry (right). (C) Spleen cells (left) and lymph node cells (right) from CD98hc^f/f-CD4 (closed) and CD98hc^+/+-CD4 (open) mice were stained with anti-CD4, anti-CD8, anti-CD44, or anti-CD62L mAbs; total cells from 6 mice were counted. Results are means ± S.D. Data shown in this Figure are representative of three independent experiments.</p

T cells from CD98hc^f/f-CD4 mice cannot mount immune responses against-<i>Leishmania major</i>.

<p>CD98hc^+/+-CD4 or CD98hc^f/f-CD4 mice under a C57BL/6 background were infected with <i>Leishmania majo</i>r in the footpad. (A) Footpad swelling in CD98hc^+/+-CD4 (closed) or CD98hc^f/f-CD4 (open) mice was measured after infection. Results are means ± S.D. of 8 mice; *or ** significant difference (<i>p</i> < 0.05 or p < 0.01, respectively). (B) Total popliteal lymph nodes from <i>Leishmania major</i>-infected CD98hc^+/+-CD4 or CD98hc^f/f-CD4 mice (day 30) were cultured for 5 days. Then, parasite numbers were counted and parasites/lymph node cells were determined. Results are means ± S.D. of 8 mice; ** significant difference (<i>p</i><0.01). (C) Purified CD4⁺ T cells (5 x 10⁵/well) from lymph node cells from CD98hc^f/f-CD4 (closed) or CD98hc^+/+-CD4 (open) mice infected with <i>Leishmania major</i> (day 10 and day 70) were stimulated with irradiated spleen cells (2 x 10⁵/well) and parasite-derived antigens for 3 days. Then, IFN-γ and IL–4 in culture supernatants were determined by ELISA. Results are means ± S.D. of 8 mice; * or ** significant difference (<i>p</i><0.05 or p < 0.01, respectively). Purified CD4⁺ T cells (5 x 10⁵/well) from lymph node cells from CD98hc^f/f-CD4 (closed circle) or CD98hc^+/+-CD4 (open circle) mice infected with <i>Leishmania major</i> (day 20) were stimulated with irradiated spleen cells (2 x 10⁵/well) and parasite-derived antigens for 3 days. Then, [³H]-thymidine incorporation was determined. Results are means ± S.D. from 8 mice; ** significant difference (<i>p</i><0.01). Data shown in this Figure are representative of three experiments.</p

Functional differentiation of CD4⁺ T cells is impaired in CD98hc^f/f-CD4 mice.

<p>CD98hc^f/f-CD4 or CD98hc^+/+-CD4 mice were immunized with OVA protein emulsified in CFA. (A) Serum anti-OVA specific IgG, IgG1, IgG2a, IgG2c, and IgM titers at eight days after OVA immunization were determined by ELISA. As negative controls, sera from unimmunized CD98hc^f/f-CD4 and CD98hc^+/+-CD4 mice were used. Results are means ± S.D. of 7 mice; * significant difference (<i>p</i><0.05). (B) Total lymph node cells from OVA-immunized CD98hc^f/f-CD4 or CD98hc^+/+-CD4 mice were stimulated with OVA protein for 3 days. [³H]-thymidine incorporation during the final 6 hours was determined. Results are means ± S.D. of 5 mice; * significant difference (<i>p</i><0.05). (C) After stimulating T cells from OVA immunized CD98hc^f/f-CD4 and CD98hc^+/+-CD4 mice with OVA protein (50 μg/ml) for 3 days, culture supernatant concentrations of IL–4, IL–17, and IFN-γ were determined by ELISA. Results are means ± S.D. of 3 mice; * significant difference (<i>p</i><0.05). Data shown in this Figure are representative of three experiments.</p

IFN-γ secretion is disturbed in CD98hc^f/f-CD4 mice.

<p>(A) CFSE labeled spleen cells from CD98hc^f/f-CD4-OT11 or CD98hc^+/+-CD4-OT11 mice were stimulated with OVA peptides under Th1 culture conditions for 3 days. Cells were stained with anti-CD4, anti-CD98hc and anti-IFN-γ mAbs and evaluated by flow cytometry. Data shown are gated on CD98hc^- cells. (B) Spleen cells from CD98hc^f/f-CD4-OT11 or CD98hc^+/+-CD4-OT11 mice were stimulated with OVA peptides under Th1 culture conditions for 3 days. Cells were stained with anti-CD4 and AnnexinV, and evaluated by flow cytometry. (C) Cells from CD98hc^f/f-CD4-OT11 (Thy1.2) or CD98hc^+/+-CD4-OT11 (Thy1.1⁺/Thy1.2⁺) mice were labeled with CFSE and transferred into CD45.1 C57BL/6 mice. Mice were then immunized with OVA protein. CFSE dilution and intracellular INF-γ expression were evaluated by flow cytometry gated on CD98hc⁺ (CD98hc^+/+-CD4-OT11) or CD98^- cells (CD98hc^f/f-CD4-OT11) cells four days after immunization (left). The percentages of INF-γ⁺ cells among total CD4⁺ T cells are shown (middle). The percentages of INF-γ⁺ cells among total CD4⁺ cells that underwent cell division at the indicated times were counted (right). Results are means ± S.D. of 4 mice; * significant difference (<i>p</i><0.05). Data shown in this Figure are representative of three experiments.</p