PMC

(a) Survival curve of DOβCTLA-4^+/+ (n>30; ○), DOβCTLA-4^+/− (n=30; ○), DOβCTLA-4^−/− (n=27; ●), and CTLA-4^−/− (n=9; ■) mice. (b) Expression of CD62L and CD44 on splenic CD4⁺ T cells from 6-wk-old DOβCTLA-4^+/+, DOβCTLA-4^+/− and DOβCTLA-4^−/− mice. Data shown are representative of 5 mice in each group. (c) H&E-stained sections of heart, lung, and pancreas from 12-wk-old DOβCTLA-4^+/+, DOβCTLA-4^+/− and DOβCTLA-4^−/− mice (Original magnification, x200). Data shown are representative of 3 mice in each group.

(a) CD4⁺ T cells (1×10⁵ cells) purified from pancreatic lymph node of 20-day-old CTLA-4^+/+ or CTLA-4^−/− mice were cultured with (+) or without (−) Pdia2 (10 μM) in the presence of irradiated splenocytes (5×10⁵ cells). The supernatants were harvested 24 h later and IL-2 concentration was determined by ELISA. Results represent three independent experiments. (b) Serum was collected from 20-day-old CTLA-4^+/+ (n=4) or CTLA-4^−/− (n=4) mice and anti-Pdia2 antibody titers were determined by ELISA.

CD4⁺ T cells from DO11.10 CTLA-4^+/+ or DO11.10 CTLA-4^−/− mice were infected with 29TCRα Thy1.1-RV. 1×10⁶ infected cells (29TCR⁺CTLA-4^+/+ or 29TCR⁺CTLA-4^−/−) were transferred to Rag-2^−/− mice. 3 weeks after transfer, ILN, PLN, pancreas, and lung were harvested from recipient Rag-2^−/− mice. (a) The frequency of CD4⁺Thy1.1⁺ cells in ILN, PLN, pancreas, and lung was determined. (b) The number of CD4⁺Thy1.1⁺ cells in ILN, PLN, and pancreas was shown. Each bar shows mean value for three mice with SD. (c) H&E-stained sections of heart, lung, and pancreas from the recipient Rag-2^−/− mice was shown. Results are representative of two independent experiments performed with three mice in each group.

CD4⁺ T cells from DO11.10 CTLA-4^−/− mice were infected with 29TCRα Thy1.1-RV. The infected cells (0.15×10⁶) were transferred to Rag2^−/− mice with or without 0.15×10⁶ CD4⁺CD62L^hiCD25⁺ cells from CTLA-4^+/+ or CTLA-4^−/− mice. 3 weeks after transfer, pancreatic LN and pancreas were harvested from the recipient Rag-2^−/− mice. (a) The number of CD4⁺Thy1.1⁺ cells in pancreatic LN and pancreas was determined. Data show mean value for three mice with SD. (b) H&E-stained section of pancreas from the recipient Rag-2^−/− mice was shown. Results represent two independent experiments including three mice in each group.

CD4⁺ T cells (1×10⁵ cells) purified from spleen of DOβCTLA-4^+/+ or DOβCTLA-4^+/− mice, or spleen, lung, or pancreas of DOβCTLA-4^−/− mice were transferred into RAG-2^−/− mice. Pancreas, lung, and heart from individual recipients were removed 3 weeks after transfer. (a) The number of CD4⁺ T cell recovered from pancreas, lung, and heart of each RAG-2^−/− recipient mice are shown. Each bar shows the mean value for three individual mice with SD. (b) H&E-stained sections are shown of the pancreas, lung and heart from RAG-2^−/− recipients that had received CD4⁺ T cells isolated from either spleen of DOβCTLA-4^+/+ or DOβCTLA-4^+/− mice, or from spleen, lung, or pancreas of DOβCTLA-4^−/− mice as indicated in the figure. The results shown are representative of two independent adoptive transfer experiments.

(a) Scheme of generation of DO11.10 T cells expressing TCRα cDNA RV library. RNA was generated from pancreas-infiltrating CD4⁺ T cells from DOβCTLA-4^−/− mice. TCRα cDNA was synthesized with TCRα-specific primer and ligated with ires-Thy1.1 RV vector to generate TCRα cDNA Thy1.1 RV library. CD4⁺ T cells from DO11.10 CTLA-4^+/+ or DO11.10 CTLA-4^−/− mice were infected with TCRα cDNA Thy1.1 RV library. The infected cells (1×10⁶ cells) were transferred to Rag-2^−/− mice. (b–d) 3 weeks after transfer, lymphoid and non-lymphoid tissues were harvested from Rag-2^−/− mice. (b) The frequency of CD4⁺Thy1.1⁺ cells is shown of inguinal LN, pancreatic LN, spleen, pancreas and lung from Rag-2^−/− mice transferred with DO11.10 CTLA-4^−/− T cells that were infected with empty- or TCRα-library Thy1.1 RV. (c) The frequency or the number of CD4⁺Thy1.1⁺ cells is shown of inguinal LN (ILN), axillary LN (ALN), pancreatic LN (PLN), spleen (spleen), pancreas or lung from Rag-2^−/− mice transferred with DO11.10 CTLA-4^+/+ or DO11.10 CTLA-4^−/− T cells that were infected with TCRα Thy1.1 RV library. Data show mean value for three mice with SD. (d) H&E-stained sections of heart, lung, and pancreas from Rag-2^−/− mice transferred with DO11.10 CTLA-4^+/+ or DO11.10 CTLA-4^−/− T cells that were infected with TCRα Thy1.1 RV library. Results represent two independent experiments performed with three mice in each group.

(a) Generation of T cell hybridomas expressing TCRα library. TCRα cDNA generated from pancreas-infiltrating CD4⁺ T cells in was ligated with retrovirus vector lacking ires-Thy1.1 to generate the TCRα cDNA RV library. 58α⁻β⁻ hybridomas were infected with NFAT-GFP-hCD4 RV reporter and DOβ-mCD4 RV. Sorted hCD4⁺mCD4⁺ cells were infected with the TCRα cDNA library RV. CD3⁺ cells were sorted to obtain TCRαβ⁺ cells. (b) TCR-stimulation dependent GFP expression in TCR-reconstituted hybridomas. T cell hybridomas expressing DOβ/DOα or DOβ/TCRα cDNA library were cultured with anti-CD3 (1 μg/ml) or OVA323-339 (0.3 μM) in the presence of irradiated splenocytes. GFP expression was examined after 20 h. The data are representative of three experiments. (c) Identification of Pdia2-reactive population in the TCRα library. T cell hybridomas expressing DOβ/TCRα library were cultured with islet β-cells (1×10⁵ cells), bovine insulin (10 μM), insulin B9-23 peptide (10 μM), porcine glucagon (10 μM), or recombinant Pdia2 (10 μM) in the presence of irradiated splenocytes. GFP expression was examined after 20 h. The data are representative of three experiments. (d) Enrichment of Pdia2-reactive clones. T cell hybridomas expressing DOβ/TCRα library were cultured with Pdia2 or Insulin (10 μM) in the presence of irradiated splenocytes. GFP⁺ cells were sorted after 20 h. 6 days later, the sorted cells were re-stimulated with Pdia2 or Insulin and GFP expression was examined after 20 h. (e) Generation of Pdia2-specific hybridoma clones. Limiting dilution was performed to generate Pdia2-specific clones. Pdia2-specific GFP expression of 4 representative clones is shown. (f) Pdia2-specific response of DOβ⁺29TCRα⁺ hybridomas. TCRα cDNA was isolated from clone #29 (29TCRα). DOβ⁺ cells expressing NFAT-GFP reporter were infected with retroviral 29TCRα and CD3⁺ cells (DOβ⁺29TCRα⁺) were sorted. DOβ⁺ cells or DOβ⁺29TCRα⁺ cells were cultured with medium alone or 5 μM Pdia2 and GFP expression was examined after 20 h.