Additional file 1 of Carriers of the p.P522R variant in PLCγ2 have a slightly more responsive immune system

Additional file 1: Text S1. Translated questionnaire to include donor in Cohort II. Fig. S1. Impact of genetic background on numbers of circulating immune cells. Each color and symbol indicate members of one family. Fully open symbols represent a centenarian. Semi-open symbol (orange) represents a sibling of a centenarian. Dashed lines indicate the available age-matched reference values produced with flow cytometry panels highly similar to the panels used in this study (earlier or later prototypes of these panels). For B- and T-cell subsets, reference lines indicate a cohort aged 60–79 years. For innate myeloid populations, reference lines indicate a cohort > 55 years old. In plots without dashed lines, no published reference values from highly similar flow cytometry panels were available. Fig. S2. Results of the pilot study to evaluate the calcium flux upon stimulation of the B-cell receptor (BCR) with IgG and IgM Fab fragments. (A) Measurement of calcium release (‘flux’) after B-cell stimulation with IgM Fabs in pre-GC B cells (CD27-IgA-IgG-) or unswitched memory B cells (CD27+IgA-IgG-). (B) Measurement of calcium release (‘flux’) after B-cell stimulation with IgG Fabs in CD27-IgG+ memory B cells (CD27-IgD-IgA-) or CD27+IgG+ memory B cells (CD27+IgD-IgA-). Ionomycin was added to calculate maximum calcium release. N = 14. Pre-GC; pre-Germinal Center, MBC; memory B cell. Fig. S3. Assessment of B-cell activation in all p.P522R-carriers and non-carriers upon BCR stimulation. Measurement of calcium release (‘flux’) after B-cell stimulation with IgM Fabs in pre-GC B cells (CD27-IgG-IgA-) (A) or unswitched memory B cells (CD27+IgG-IgA-) (B) in cohort I. Ionomycin was added to calculate maximum calcium release. Differences between carriers and non-carriers were evaluated by comparing the area under the curve (AUC) of the total Fab stimulation (from stimulation until the moment ionomycin was added, ~ 10 min, flux intensity and duration), the peak of the response after Fab stimulation (the 5 highest points after the Fabs were added to the cells; flux intensity), and after ionomycin was added (to determine the maximum flux). AUC was calculated only for points that were higher than baseline value (unstimulated sample). N = 31 (two samples were lost due to technical failure). No significant differences were observed. Pre-GC; pre-Germinal Center. Fig. S4. Phagocytosis and ROS production in innate immune cell subsets after stimulation with pHRodo™ Green E. coli Bioparticles (FcR/PLCγ2-dependent stimulation). To evaluate the outcome of the phagocytosis assays, three different readouts were used per population: % of cells that were phagocytosing, the average amount of particles phagocytosed per cell, and the ROS production upon phagocytosis. These three readouts were further combined into one value: the normalized ROS. These values are presented for the CD62L+ classical monocyte (cMo) subset (A), intermediate monocytes (iMo) (B), neutrophils (C) CD14- and CD14dim myeloid dendritic cells (mDCs) (D,E), and non-phagocytosing plasmacytoid dendritic cells (pDCs) (F). Lastly, the outcomes for T cells (negative control) are shown (G). Mann-Whitney U test was used to evaluate differences between carriers and non-carriers, but no statistically significant differences were found. N = 14. In two donors, monocytes could not be divided into subsets due to absence of a differentiating antibody in the prepared antibody cocktail, therefore, in panel A and B, only 5 non-carriers and 7 carriers are shown. Dashed lines indicate the background level of ROS (ROS production in negative control population; T cells). All outcomes were corrected for background or baseline activation by subtracting the value of the control (incubated on ice) from the activated (incubated at 37 °C) sample. Negative values (caused by higher background in control samples than activated samples in cell populations that did not perform phagocytosis) were set to 0. Fig. S5. ROS generation in innate immune cell subsets in p.P522R-carriers and non-carriers upon stimulation with PMA (FcR/PLCγ2-independent stimulation). N = 14. In two donors, monocytes could not be divided into subsets due to absence of an antibody in the prepared antibody cocktail, therefore, in panel A-D, only 5 non-carriers and 7 carriers are shown. Dashed lines indicate the background level of ROS (ROS production in negative control population; T cells). All outcomes were corrected for background or baseline activation by subtracting the value of the control (incubated on ice) from the activated (incubated at 37 °C) sample. Table S1. Description of all included families and additional donors. Table S2. Overview of the flow cytometry panels that were used in this study. Table S3. Phenotypic descriptions used to define B-cell subsets stained with EuroFlow PERISCOPE B-cell and plasma cell panel (BIGH) panel by manual analysis. Table S4. Phenotypic descriptions used to define T-cell subsets stained with EuroFlow PERISCOPE CD4 T-cell (TCD4) panel by manual analysis. Table S5. Phenotypic descriptions used to define T-cell and NK-cell subsets stained with the EuroFlow PERISCOPE CD8 cytotoxic T-cell (CYTOX) panel by manual analysis. Table S6. Phenotypic descriptions used to define innate immune cell (sub) sets stained with the EuroFlow PERISCOPE DC-Monocyte panel by manual analysis. Table S7. Polygenic Risk Score and SNP annotation for immune-related SNPs. Table S8. Control and assay tubes measured in the phagocytosis experiment. Table S9. Control and assay tubes measured when detection production of reactive oxygen species (ROS).