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).

附加文件 1：文本 S1. 包含 Cohort II 受捐者内容的翻译问卷。图 S1. 遗传背景对循环免疫细胞数量的影响。每种颜色和符号代表一个家族的成员。全开放符号表示百岁老人。半开放符号（橙色）表示百岁老人的兄弟姐妹。虚线表示使用与本研究中使用的面板高度相似的流式细胞术面板生成的可用年龄匹配参考值（这些面板的早期或后期原型）。对于 B 细胞和 T 细胞亚群，参考线表示 60-79 岁的队列。对于先天髓系细胞群，参考线表示 55 岁以上的队列。在没有虚线的图表中，没有可用的与高度相似的流式细胞术面板的已发表参考值。图 S2. 评估 B 细胞受体（BCR）在 IgG 和 IgM Fab 片段刺激下的钙流。图 S2A 和 S2B 分别测量了 IgM Fab 在预生发中心 B 细胞（CD27-IgA-IgG-）或未切换记忆 B 细胞（CD27+IgA-IgG-）中的钙释放（‘流’）以及在 CD27-IgG+ 记忆 B 细胞（CD27-IgD-IgA-）或 CD27+IgG+ 记忆 B 细胞（CD27+IgD-IgA-）中的钙释放。加入离子霉素以计算最大钙释放。N=14。Pre-GC：生发中心之前，MBC：记忆 B 细胞。图 S3. 评估所有 p.P522R 带因者和非带因者在 BCR 刺激下的 B 细胞活化。在队列 I 中，通过 IgM Fab 刺激预生发中心 B 细胞（CD27-IgG-IgA-）（图 S3A）或未切换记忆 B 细胞（CD27+IgG-IgA-）（图 S3B）测量钙释放（‘流’）。加入离子霉素以计算最大钙释放。通过比较总 Fab 刺激下的曲线下面积（AUC）（从刺激开始直到加入离子霉素，约 10 分钟，流强度和持续时间）、Fab 刺激后反应的峰值（加入 Fab 到细胞后 5 个最高点；流强度）以及加入离子霉素后的峰值（以确定最大流强度）来评估带因者和非带因者之间的差异。仅计算高于基线值（未刺激样本）的点。N=31（由于技术故障丢失了两个样本）。未观察到显著差异。Pre-GC：生发中心之前。图 S4. 在 pHRodo™ Green 大肠杆菌生物颗粒（FcR/PLCγ2 依赖性刺激）刺激下，先天免疫细胞亚群的内吞作用和活性氧（ROS）的产生。为了评估内吞作用实验的结果，每个种群使用了三种不同的读数：进行内吞作用的细胞百分比、每个细胞内吞的颗粒的平均数量以及内吞作用后的 ROS 产生。这三个读数进一步合并为一个值：标准化 ROS。这些值分别表示 CD62L+ 经典单核细胞（cMo）亚群（图 A）、中间单核细胞（iMo）（图 B）、中性粒细胞（图 C）、CD14- 和 CD14dim 髓系树突状细胞（mDCs）（图 D,E）以及非内吞作用的浆细胞样树突状细胞（pDCs）（图 F）。最后，还展示了 T 细胞（阴性对照）的结果（图 G）。使用 Mann-Whitney U 检验评估了带因者和非带因者之间的差异，但未发现具有统计学意义的差异。N=14。在两位捐献者中，由于制备的抗体制剂中缺乏区分抗体，无法将单核细胞分为亚群，因此，在图 A 和 B 中，只显示了 5 位非带因者和 7 位带因者。虚线表示 ROS 的背景水平（阴性对照组的 ROS 产生；T 细胞）。所有结果都通过从激活样本（37°C 孵育）中减去对照样本（冰上孵育）的值来校正背景或基线激活。由于对照样本在未进行内吞作用的细胞群中比激活样本的背景更高，因此设置了负值。图 S5. 在 p.P522R 带因者和非带因者刺激下，先天免疫细胞亚群中 ROS 的产生（FcR/PLCγ2 非依赖性刺激）。N=14。在两位捐献者中，由于制备的抗体制剂中缺乏抗体，无法将单核细胞分为亚群，因此，在图 A-D 中，只显示了 5 位非带因者和 7 位带因者。虚线表示 ROS 的背景水平（阴性对照组的 ROS 产生；T 细胞）。所有结果都通过从激活样本（37°C 孵育）中减去对照样本（冰上孵育）的值来校正背景或基线激活。表 S1. 所有纳入家族和额外捐献者的描述。表 S2. 本研究中使用的流式细胞术面板概述。表 S3. 使用 EuroFlow PERISCOPE B 细胞和浆细胞面板（BIGH）通过人工分析定义的 B 细胞亚群的表型描述。表 S4. 使用 EuroFlow PERISCOPE CD4 T 细胞（TCD4）面板通过人工分析定义的 T 细胞亚群的表型描述。表 S5. 使用 EuroFlow PERISCOPE CD8 细胞毒性 T 细胞（CYTOX）面板通过人工分析定义的 T 细胞和 NK 细胞亚群的表型描述。表 S6. 使用 EuroFlow PERISCOPE DC-Monocyte 面板通过人工分析定义的先天免疫细胞（亚）群的表型描述。表 S7. 免疫相关单核苷酸多态性（SNP）的 polygenic 风险评分和 SNP 注释。表 S8. 吞噬作用实验中的对照和实验管。表 S9. 检测反应性氧物种（ROS）产生时的对照和实验管。