Figure 3
Figure 3. Enhanced effector functions of OVA-specific CD8+ T cells in SRA/CD204 KO mice. (A) Increased IFN-γ production by OVA-specific CTLs from SRA−/− mice. Animals (n = 5) were immunized with OVA alone, OVA-MPL, or left untreated. One week after booster immunization, splenocytes were restimulated with OVA257-264 and subjected to an ELISPOT assay (*P < .01, SRA−/− vs WT mice; error bars indicate SE). (B) Increased cytolytic activity of effector T cells from SRA−/− mice. Splenocytes were restimulated with OVA257-264 in the presence of IL-2 for 5 days, and chromium release assays were carried out using OVA-expressing EG7 cells as targets. OVA-negative EL4 cells were used as controls. Data shown as mean plus or minus SD are representative of 2 experiments. (C) Increased OVA-specific cytolytic activity in immunized SRA−/− mice. Immunized WT and SRA−/− mice (n = 3) were injected intravenously with 5 × 106 OVA257-264-pulsed, CFSEhigh splenocytes mixed with nonpulsed, CFSElow splenocytes at 1:1 ratio. Splenocytes and lymph node (LN) cells were analyzed 12 hours later by FACS. Representative histogram profiles from 2 experiments are shown. (D) Antibody response to OVA antigen not affected by SRA/CD204 deficiency. WT and SRA−/− mice (n = 5) were immunized with OVA-MPL or left untreated. Sera were collected 7 days after booster immunization and analyzed for IgG levels against OVA by ELISAs (*P > .5, WT vs SRA−/−). (E) Intrinsic T-cell function not altered by SRA/CD204 deficiency. Naive T cells (> 98% purity) were stimulated with 10 μg/mL anti-CD3, anti-CD28 mAb in precoated wells, or PHA (1 μg/mL) at 37°C for 72 hours. Cell proliferation was determined by measuring thymidine incorporation (counts per minute) using a scintillation counter (*P > .5, WT vs SRA−/−). (F) Similar in vivo proliferation of T cells from WT and SRA−/− mice. CFSE-labeled CD3+ T cells from WT or SRA−/− mice were adoptively transferred to RAG-1 mice (n = 3). The recipient mice were then immunized with OVA-MPL. CFSE intensity was measured 5 days later using FACS by gating on CD3- and CFSE-positive cells (P > .5, WT vs SRA−/−).

Enhanced effector functions of OVA-specific CD8+ T cells in SRA/CD204 KO mice. (A) Increased IFN-γ production by OVA-specific CTLs from SRA−/− mice. Animals (n = 5) were immunized with OVA alone, OVA-MPL, or left untreated. One week after booster immunization, splenocytes were restimulated with OVA257-264 and subjected to an ELISPOT assay (*P < .01, SRA−/− vs WT mice; error bars indicate SE). (B) Increased cytolytic activity of effector T cells from SRA−/− mice. Splenocytes were restimulated with OVA257-264 in the presence of IL-2 for 5 days, and chromium release assays were carried out using OVA-expressing EG7 cells as targets. OVA-negative EL4 cells were used as controls. Data shown as mean plus or minus SD are representative of 2 experiments. (C) Increased OVA-specific cytolytic activity in immunized SRA−/− mice. Immunized WT and SRA−/− mice (n = 3) were injected intravenously with 5 × 106 OVA257-264-pulsed, CFSEhigh splenocytes mixed with nonpulsed, CFSElow splenocytes at 1:1 ratio. Splenocytes and lymph node (LN) cells were analyzed 12 hours later by FACS. Representative histogram profiles from 2 experiments are shown. (D) Antibody response to OVA antigen not affected by SRA/CD204 deficiency. WT and SRA−/− mice (n = 5) were immunized with OVA-MPL or left untreated. Sera were collected 7 days after booster immunization and analyzed for IgG levels against OVA by ELISAs (*P > .5, WT vs SRA−/−). (E) Intrinsic T-cell function not altered by SRA/CD204 deficiency. Naive T cells (> 98% purity) were stimulated with 10 μg/mL anti-CD3, anti-CD28 mAb in precoated wells, or PHA (1 μg/mL) at 37°C for 72 hours. Cell proliferation was determined by measuring thymidine incorporation (counts per minute) using a scintillation counter (*P > .5, WT vs SRA−/−). (F) Similar in vivo proliferation of T cells from WT and SRA−/− mice. CFSE-labeled CD3+ T cells from WT or SRA−/− mice were adoptively transferred to RAG-1 mice (n = 3). The recipient mice were then immunized with OVA-MPL. CFSE intensity was measured 5 days later using FACS by gating on CD3- and CFSE-positive cells (P > .5, WT vs SRA−/−).

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