Figure 3
Therapeutic MR-DCs carrying donor-Ag fail to directly modulate antidonor T cells in vivo. (A) On the left, FACS analysis of CFSE-labeled 2C CD8+ T-cell proliferation when transferred into host MHC class I−/− B6 mice injected (or not, control) the next day with 5 × 106 or 15 × 106 BALB/c MR-DCs, or 5 × 106 BALB/c LPS-matured DCs. Analysis was performed 3 days after DC administration. Numbers in histograms are percentages of dividing 2C CD8+ T cells. On the right, bar diagram shows absolute numbers of 2C CD8+ T cells in the spleen. (B) On the left, FACS analysis of CFSE-labeled 1H3.1 CD4+ T-cell proliferation when transferred into host MHC class II−/− B6 mice injected (or not, control) the next day with 5 × 106 or 15 × 106 B6 MR-DCs pulsed with the BALB/c IEα52-68 allopeptide, or 5 × 106 B6 LPS-matured DCs pulsed with IEα52-68. On the right, bar diagram shows absolute numbers of splenic 1H3.1 CD4+ T cells. (C) In vivo analysis of donor-reactive CD4+ T-cell anergy. MHC II−/− B6 mice reconstituted with CFSE-labeled 1H3.1 CD4+ T cells were injected (or not, control) with B6 MR-DCs pulsed with BALB/c IEα52-68. After 7 days, mice were challenged in vivo, or not, with B6 LPS-matured DCs pulsed with IEα52-68. Three days later, proliferation of splenic 1H3.1 CD4+ T cells (in parentheses) and FoxP3 expression (in gated regions) were analyzed by FACS. (D) In vivo analysis of antidonor CD8+ T-cell anergy. MHC I−/− B6 mice reconstituted with CFSE-labeled 2C CD8+ T cells were injected intravenously (or not, control) with BALB/c MR-DCs. After 7 days, mice were challenged in vivo, or not, with B6 LPS-matured DCs pulsed with the 2C TCR agonistic peptide SIYRYYGL (SIYRL). Three days later, proliferation and CD28 expression of splenic 2C CD8+ T cells were analyzed by FACS, and IFN-γ and IL-2 secretion by splenic 2C T cells was assessed by ELISA after ex vivo restimulation (24 hours) with B6 LPS-matured DCs pulsed with SIYRL. Numbers in dot plots indicate percentages of cells. (A-D) Results represent 2 or more independent experiments with at least 3 animals per group. NS indicates not significant.

Therapeutic MR-DCs carrying donor-Ag fail to directly modulate antidonor T cells in vivo. (A) On the left, FACS analysis of CFSE-labeled 2C CD8+ T-cell proliferation when transferred into host MHC class I−/− B6 mice injected (or not, control) the next day with 5 × 106 or 15 × 106 BALB/c MR-DCs, or 5 × 106 BALB/c LPS-matured DCs. Analysis was performed 3 days after DC administration. Numbers in histograms are percentages of dividing 2C CD8+ T cells. On the right, bar diagram shows absolute numbers of 2C CD8+ T cells in the spleen. (B) On the left, FACS analysis of CFSE-labeled 1H3.1 CD4+ T-cell proliferation when transferred into host MHC class II−/− B6 mice injected (or not, control) the next day with 5 × 106 or 15 × 106 B6 MR-DCs pulsed with the BALB/c IEα52-68 allopeptide, or 5 × 106 B6 LPS-matured DCs pulsed with IEα52-68. On the right, bar diagram shows absolute numbers of splenic 1H3.1 CD4+ T cells. (C) In vivo analysis of donor-reactive CD4+ T-cell anergy. MHC II−/− B6 mice reconstituted with CFSE-labeled 1H3.1 CD4+ T cells were injected (or not, control) with B6 MR-DCs pulsed with BALB/c IEα52-68. After 7 days, mice were challenged in vivo, or not, with B6 LPS-matured DCs pulsed with IEα52-68. Three days later, proliferation of splenic 1H3.1 CD4+ T cells (in parentheses) and FoxP3 expression (in gated regions) were analyzed by FACS. (D) In vivo analysis of antidonor CD8+ T-cell anergy. MHC I−/− B6 mice reconstituted with CFSE-labeled 2C CD8+ T cells were injected intravenously (or not, control) with BALB/c MR-DCs. After 7 days, mice were challenged in vivo, or not, with B6 LPS-matured DCs pulsed with the 2C TCR agonistic peptide SIYRYYGL (SIYRL). Three days later, proliferation and CD28 expression of splenic 2C CD8+ T cells were analyzed by FACS, and IFN-γ and IL-2 secretion by splenic 2C T cells was assessed by ELISA after ex vivo restimulation (24 hours) with B6 LPS-matured DCs pulsed with SIYRL. Numbers in dot plots indicate percentages of cells. (A-D) Results represent 2 or more independent experiments with at least 3 animals per group. NS indicates not significant.

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