Figure 4.
C4d and C3c deposition and inhibition in HC and CM-TMA samples. (A-L) All experiments were done in the presence of C5 inhibitor, eculizumab (100 μg/mL). WT, PIGAKO, or CD46KO cells were treated with 20% serum in GVB++ for 30 minutes and then stained singly for either C4d or C3c and deposition quantified by flow cytometry. The same healthy control (n = 4) and CM-TMA (n = 4 for WT; n = 5 for PIGAKO and CD46KO) were used across all cell types. Serum was treated, as indicated, with the sut (30 μg/mL) or ACH-5548 (1 μM; FDi). (A) Comparison of C4d deposition across cell types by percent positive cells determined by gating on heat-inactivated HC sample. (B) Relative median fluorescence intensity (MFI) of C3c deposition induced by CM-TMA serum vs healthy control serum. (C) Comparison of C3c deposition across cell types by percent positive cells determined by gating on heat-inactivated HC sample. (A-C) Intra–cell line differences between HC and CM-TMA P values were calculated with unpaired Mann-Whitney test. (D,G,J) C4d deposition on WT cells (D), PIGAKO (G), and PIGAKO (J) in HC or CM-TMA with or without addition of sut or FDi. (E,H,K) C3c MFI on WT cells (D), PIGAKO (G), PIGAKO (J) in HC or CM-TMA with or without addition of sutimlimab or FDi. (D-E,G-H,J-K) P values calculated using Friedman test for Dunn multiple comparisons. (F) Example histograms of CM07 on WT cells with or without HI, sut, or FDi. (I) Example histograms of CM12 on PIGAKO cells with or without HI, sut, or FDi. (L) Example histograms of CM06 on CD46KO cells with or without HI, sut, or FDi. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, not significant.

C4d and C3c deposition and inhibition in HC and CM-TMA samples. (A-L) All experiments were done in the presence of C5 inhibitor, eculizumab (100 μg/mL). WT, PIGAKO, or CD46KO cells were treated with 20% serum in GVB++ for 30 minutes and then stained singly for either C4d or C3c and deposition quantified by flow cytometry. The same healthy control (n = 4) and CM-TMA (n = 4 for WT; n = 5 for PIGAKO and CD46KO) were used across all cell types. Serum was treated, as indicated, with the sut (30 μg/mL) or ACH-5548 (1 μM; FDi). (A) Comparison of C4d deposition across cell types by percent positive cells determined by gating on heat-inactivated HC sample. (B) Relative median fluorescence intensity (MFI) of C3c deposition induced by CM-TMA serum vs healthy control serum. (C) Comparison of C3c deposition across cell types by percent positive cells determined by gating on heat-inactivated HC sample. (A-C) Intra–cell line differences between HC and CM-TMA P values were calculated with unpaired Mann-Whitney test. (D,G,J) C4d deposition on WT cells (D), PIGAKO (G), and PIGAKO (J) in HC or CM-TMA with or without addition of sut or FDi. (E,H,K) C3c MFI on WT cells (D), PIGAKO (G), PIGAKO (J) in HC or CM-TMA with or without addition of sutimlimab or FDi. (D-E,G-H,J-K) P values calculated using Friedman test for Dunn multiple comparisons. (F) Example histograms of CM07 on WT cells with or without HI, sut, or FDi. (I) Example histograms of CM12 on PIGAKO cells with or without HI, sut, or FDi. (L) Example histograms of CM06 on CD46KO cells with or without HI, sut, or FDi. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, not significant.

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