Figure 1
Figure 1. C1q is required for optimal IFN-γ production by HY-specific CD4+ T cells upon activation by DCs pulsed with male apoptotic cells. (A-C) WT and C1qa−/− mice immunized with intraperitoneal male splenocytes were killed and splenic cells restimulated in vitro for 7 days with irradiated male splenocytes. IFN-γ and IL-2 were detected by intracellular staining after another round of restimulation with peptide-pulsed (APC + pep) or unpulsed (APC) female APCs. (D) Immature DCs alone (DC) or pulsed with female/male apoptotic cells (DC + F apo and DC + M apo, respectively) were cultured with HY-specific CD4+ T-cell clone (B9 cells) for 48 hours and supernatants tested for IFN-γ. Male apoptotic cells (M apo) cultured with B9 cells in the absence of DCs were used as control. (E) B9 cells were cultured with WT or C1qa−/− DCs pulsed with male or female apoptotic cells and production of IFN-γ was evaluated by ELISA. (F) B9 cells were cultured with WT or C1qa−/− DCs pulsed with male or female apoptotic cells as described for panel E. C1q (25 μg/mL) was added to the coculture as indicated. (G) Marilyn T cells were stained with CFSE and cocultured with DCs (WT and C1q−/−) pulsed with male apoptotic cells for 72 hours. T-cell proliferation was assessed by CFSE dilution using flow cytometry. (H,I) WT and C1qa−/− DCs pulsed with male or female apoptotic thymocytes (DC + M apo and DC + F apo, respectively) were cultured with Marilyn cells for 48 hours and supernatants tested for IFN-γ (H) and IL-2 (I). (J) Marilyn T cells were cultured with WT or C1qa−/− DCs pulsed with male or female apoptotic cells. C1q (25 μg/mL) was added to the coculture when indicated. (K) WT and C1qa−/− DCs generated using GM-CSF and IL-4 were pulsed with male or female apoptotic thymocytes (DC + M apo and DC + F apo, respectively) and were cultured with Marilyn T cells for 48 hours. IFN-γ was measured in the supernatants. One representative experiment (mean ± SD of duplicate samples) of at least 3 independent experiments performed with cells from different mice is shown. (L,M) WT and C1qa−/− splenic DCs pulsed with male or female apoptotic thymocytes (DC + M apo and DC + F apo, respectively) were cultured with Marilyn cells for 48 hours and supernatants tested for IFN-γ and IL-2. *P < .05, statistically different from control.

C1q is required for optimal IFN-γ production by HY-specific CD4+ T cells upon activation by DCs pulsed with male apoptotic cells. (A-C) WT and C1qa−/− mice immunized with intraperitoneal male splenocytes were killed and splenic cells restimulated in vitro for 7 days with irradiated male splenocytes. IFN-γ and IL-2 were detected by intracellular staining after another round of restimulation with peptide-pulsed (APC + pep) or unpulsed (APC) female APCs. (D) Immature DCs alone (DC) or pulsed with female/male apoptotic cells (DC + F apo and DC + M apo, respectively) were cultured with HY-specific CD4+ T-cell clone (B9 cells) for 48 hours and supernatants tested for IFN-γ. Male apoptotic cells (M apo) cultured with B9 cells in the absence of DCs were used as control. (E) B9 cells were cultured with WT or C1qa−/− DCs pulsed with male or female apoptotic cells and production of IFN-γ was evaluated by ELISA. (F) B9 cells were cultured with WT or C1qa−/− DCs pulsed with male or female apoptotic cells as described for panel E. C1q (25 μg/mL) was added to the coculture as indicated. (G) Marilyn T cells were stained with CFSE and cocultured with DCs (WT and C1q−/−) pulsed with male apoptotic cells for 72 hours. T-cell proliferation was assessed by CFSE dilution using flow cytometry. (H,I) WT and C1qa−/− DCs pulsed with male or female apoptotic thymocytes (DC + M apo and DC + F apo, respectively) were cultured with Marilyn cells for 48 hours and supernatants tested for IFN-γ (H) and IL-2 (I). (J) Marilyn T cells were cultured with WT or C1qa−/− DCs pulsed with male or female apoptotic cells. C1q (25 μg/mL) was added to the coculture when indicated. (K) WT and C1qa−/− DCs generated using GM-CSF and IL-4 were pulsed with male or female apoptotic thymocytes (DC + M apo and DC + F apo, respectively) and were cultured with Marilyn T cells for 48 hours. IFN-γ was measured in the supernatants. One representative experiment (mean ± SD of duplicate samples) of at least 3 independent experiments performed with cells from different mice is shown. (L,M) WT and C1qa−/− splenic DCs pulsed with male or female apoptotic thymocytes (DC + M apo and DC + F apo, respectively) were cultured with Marilyn cells for 48 hours and supernatants tested for IFN-γ and IL-2. *P < .05, statistically different from control.

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