hCPC or IFNγ-hCPC were cultured with declining concentrations of (a) anti-HLA-I W6/32 or (b) anti-HLA-II L243 mAb. The reactivity, as mean florescence intensity (MFI), for each antibody concentration was determined by flow cytometry. Results are mean MFI values ± SD obtained with hCPC (n = 6) expressing different HLA haplotypes and each tested in three different experiments. Correlation curves between MFIs and antibody concentrations for hCPC or IFNγ-hCPC along with respective R² values are indicated.

hCPC or IFNγ-hCPC were cultured with declining concentrations of anti-HLA-I W6/32 or 10 μg/ml of W6/32-F(ab’)₂ with or without complement (C) then, (a) the capacity of anti-HLA I to induce CDC was evaluated by flow cytometry as % 7AAD-positive hCPC. (b) % CDC induced by each antibody concentration in hCPC (left panel) or IFNγ-hCPC (right panel) plotted as function of respective MFIs. Results are presented as mean values ± SD from three different experiments done with each hCPC. Statistical analyses were performed using One-Way Analysis of Variance (ANOVA)-Kruskal–Wallis test-dunn’s multiple comparison (GraphPadPrism Software). P < 0.001 and P < 0.01 compared to complement.

IL-15-activated NK cells were cultured alone (medium) or with hCPC or IFNγ-hCPC (n = 6) in the presence of declining concentrations of anti-HLA-I W6/32 or 10 μg/ml of W6/32-F(ab’)₂. (a) % CD137-positive NK cells determined by flow cytometry (upper panel). % CD137-positive NK cells observed for each antibody concentration in hCPC or IFNγ-hCPC plotted as function of respective MFIs (lower panel). (b) % NK cell-mediated lysis evaluated as percentage of 7AAD-positive hCPC or IFNγ-hCPC (upper panel). % CD107a-positive NK cells plotted as function of % NK-mediated lysis (lower panel). Results are presentenced as mean values ± SD from three different experiments done with each hCPC. Statistical analyses were performed using One-Way Analysis of Variance (ANOVA)-Kruskal–Wallis test-dunn’s multiple comparison (GraphPadPrism Software). P < 0.01 and P < 0.001 compared to NK + hCPC alone.

DSA-HLA-I-A2 sera (n = 6) were incubated with HLA-A2-positive hCPC or IFNγ-hCPC (n = 4), and their reactivity was determined as MFI by flow cytometry. (a) Upper panel showing representative histograms of DSA001 (blue), DSA002 (purple), DSA003 (light green), DSA004 (dark green), DSA005 (red), and DSA006 (orange) interactions against control serum AB (black) and medium alone (gray filled). Mean MFI (geomean) values ± SD from four different experiments of each hCPC compared to serum AB and medium controls are shown in the lower panel. (b) HLA-I-A2-positive hCPC or IFNγ-hCPC (n = 4) were cultured alone, or in the presence of complement with control serum AB or with DSA-HLA-I-A2 sera and their capacity to induce CDC was evaluated by flow cytometry as % 7AAD-positive hCPC (upper panel). Results are presented as mean values ± SD from four different experiments of each hCPC. The percentages of CDC induced by DSA-HLA I-A2 sera were plotted as function of respective MFIs (lower panel). Statistical analyses were performed using Mann–Whitney test for non-paired groups. *P < 0.05, **P < 0.01, ***P < 0.001 compared to hCPC in the presence of complement alone.

IL-15-activated NK cells were cultured alone or with HLA-A2 positive hCPC or IFNγ-hCPC (n = 4) in the presence of control serum AB or DSA-HLA-I-A2 sera (DSA001-006). (a) % CD137-positive NK cells, (b) % CD107-positive NK cells, and (C) % NK cell-mediated lysis evaluated as % 7AAD-positive hCPC. Results represent mean values ± SD from four different experiments of each hCPC. The percentages of CD137-positive NK cells with hCPC or IFNγ-hCPC were plotted as function of respective MFIs of DSA-HLA-I-A2 sera (upper right panel) or as function of % CD107-positive NK cells (middle right panel), the % CD107-positive NK cells were plotted as function of % NK-mediated lysis (low right panel) for both hCPC and IFNγ-hCPC. Statistical analyses were performed using Mann–Whitney test for non-paired groups. **P < 0.01 and ***P < 0.001 compared to NK + hCPC.

IL-15-activated NK cells from healthy donors (n = 3) or from patients with MI (n = 3) were cultured alone or with HLA-A2-positive hCPC or -IFNγ-hCPC (n = 2) in the presence of control serum AB or DSA-HLA-I-A2 sera (DSA001, DSA004, DSA006). (a) Percentages of NK cells present in total PBMC (left panel) and % NK cell-mediated lysis in NK-target k562 cells evaluated as % 7AAD-positive k562 (right panel). (b) % NK cell-mediated lysis evaluated as % 7AAD-positive hCPC (right panel) or IFNγ-hCPC (left panel) in the presence of mAb and (c) in the presence of DSA-HLA I. Results represent mean values ± SD from 3 different experiments. Statistical analyses were performed using Mann–Whitney test for non-paired groups. NS: non-significant **P < 0.01compared to NK + hCPC.

DSA-HLA-II-DR4 sera (n = 2) were incubated with HLA-DR4-positive hCPC or IFNγ-hCPC (n = 2) then their reactivity was determined as MFI by flow cytometry. (a) Left panel showing representative histograms of DSA017 (blue) and DSA021 (red) interactions against control serum AB (black) and medium alone (filled gray). Mean MFI (geomean) values ± SD from three different experiments of each hCPC compared to serum AB and medium controls (right panel). (b) HLA-DR4-positive hCPC or IFNγ-hCPC (n = 2) were cultured alone, with control serum AB, or with DSA-HLA-II-DR4 sera (DSA017, 021), in the presence or absence of complement, then their capacity to induce CDC was evaluated as % 7AAD-positive hCPC. Results are presented as mean values ± SD from four different experiments of each hCPC. (c–e) IL-15-activated NK cells were cultured alone or with HLA-DR4-positive hCPC or IFNγ-hCPC (n = 2) in the presence of control serum AB or DSA-HLA-II-DR4 sera. (c) % CD137-positive NK cells, (d) % CD107-positive NK cells and (e) % NK-mediated lysis evaluated as % 7AAD-positive hCPC. Results represent mean values ± SD from three different experiments of each hCPC. (f) % Healthy- or patient-NK-mediated lysis evaluated as % 7AAD-positive hCPC. Results represent mean values ± SD from three different experiments. Statistical analyses were performed using Mann–Whitney test for non-paired groups and were non-significant.

(a) Left panel DSA-HLA-I (n = 15) and right panel DSA-HLA-II (n = 6) sera flow cytometry-detected MFIs values were plotted as function of their Luminex-detected MFIs. (b) Left panel % CDC and right panel % ADCC against hCPC or IFNγ-hCPC plotted as function of their Luminex-detected MFIs. Correlations curves along with their respective R² values are indicated. (c) Schematic representation of DSA-HLA-sensitization risk for hCPC-based therapy. DSA-HLA-I of Luminex-detected high mean florescence intensity (MFI) present an absolute risk of humoral rejection inducing both CDC and ADCC through complement activation and the formation of the membrane attack complex (MAC) or by bridging NK cells CD16 receptor with HLA-I molecules at the surface of hCPC, respectively. DSA-HLA-I with Luminex-detected MFIs ranging from 5000 to 10000 present a relative risk of humoral rejection bridging NK cells CD16 receptor with HLA-I molecules at the surface of hCPC will induce only ADCC. DSA-HLA-I having Luminex-detected MFIs < 5000 and DSA-HLA-II with MFI up to 16000 are safe.