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

Experimental design

A total of 36 individuals from the 100 plus study cohort, as previously described by Holstege et al [22], were selected for this study (METC number: 2016.440, approved by the Medical Ethics Committee of the VU University Medical Center, Amsterdam, the Netherlands). In short, volunteers either had to be ≥100 years, and self-reported as cognitively healthy, which was confirmed by a family member of close relation [22], or to be the offspring or a sibling of such individuals. When including offspring, the parent had to be a confirmed p.P522R-carrier. After inclusion, peripheral blood (PB) was collected at one or multiple occasions in K3EDTA collection tubes (PB-EDTA), sodium heparin collection tubes (PB-HEP), and serum collection tubes (BD Vacutainer, BD Biosciences, San Jose, Ca, USA). To avoid influence of circadian rhythm on cell counts, samples were always collected in the morning. p.P522R-carriership was determined by Sanger sequencing or Illumina Genome Screening Array (GSA, GSAsharedCUSTOM_20018389_A2, v1, human genome build 37) as described elsewhere [23]. In the current study, we investigated quantities, immunophenotypes, and functions of circulating immune cells in three overlapping cohorts (Cohort I, II and III), described in Table 1. Immunophenotyping was performed on all individuals included in Cohort I (n = 33). Calcium flux measurements were performed on cells from donors included in Cohort I and II, with exception of two donors that showed B-cell aberrancies (n = 33). Functional evaluation (phosphorylation of PLCγ2, KREC analysis, phagocytosis assay and ROS production) was performed on Cohort II (n = 14). Lastly, vaccination responses were evaluated in Cohort III (n = 22). Family composition can be found in Table S1. In order to keep the impact of genetic diversity limited, we compared p.P552R effects between siblings whenever possible. Due to more stringent inclusion criteria (based on current health status, known co-morbidities, and medication use) for Cohort II, the effect of p.P522R could only be assessed in one family. The remainder of cohort II was age- and sex-matched.

Table 1 Description of the cohorts used in this study; Cohort I, II, IIISettings of flow cytometers

For all flow cytometers (available at the Flow cytometry Core Facility at LUMC) there was a daily quality control (QC). QC for Cytek Aurora flow cytometers (Cytek Biosciences, Fremont, Ca, USA) was performed using SpectroFlo QC Beads (Cytek Biosciences) as recommended by the manufacturer. BD FACS LSR Fortessa 4 L and BD FACS Canto II 3 L (BD Biosciences, San Jose, CA, USA) were calibrated and compensated according to EuroFlow guidelines and daily QC was performed using BD™ Cytometer Setup and Tracking (CS&T) beads (BD Biosciences) and Perfect-Count Microspheres™ (Cytognos, Spain), as described before [24, 25].

Immunophenotyping of circulating immune cells

PB-EDTA from Cohort I was used < 12 h after collection for immunophenotyping. In-depth analysis of circulating innate and adaptive immune cells was performed using previously published flow cytometry panels, or their direct prototypes, and gating strategies (Table S2). Phenotypic descriptions of each population are presented in Tables S3, S4, S5 and S6.

The dendritic cell-monocyte (DC-monocyte) panel allows identification of up to 19 different (sub) populations in the myeloid compartment [26, 27]. The CD4-T cell panel (CD4T) allows identification of at least 89 (sub) populations within the CD4 T-cell compartment, which comprise of different functionalities and maturation stages [27, 28]. The CD8 cytotoxic T-cell (CYTOX) panel allows identification of up to 50 (sub) populations within the CD8 T cells, TCRγδ T-cells and the natural killer (NK) cell compartments [27]. Lastly, the B-cell and plasma cell (BIGH) panel allows identification of up to 115 populations of B and plasma cells, distinguished based on their maturation stage-associated phenotype and the expressed Ig subclasses [27, 29, 30].

Depending on the antibody combination, samples were either processed according to the bulk lysis protocol for staining of 10 × 106 cells (DC-monocyte and BIGH) or prepared using the EuroFlow stain-lyse-wash protocol (CD4T, CYTOX); both protocols available on www.EuroFlow.org. For BIGH and CYTOX tubes, surface staining was followed by intracellular staining with the Fix & Perm reagent kit (Nordic MUbio, Susteren, The Netherlands) according to manufacturer’s protocol. In brief, 100 μL of washed sample was fixed with 100 μL of Solution A (15 min in the dark at RT), washed, and permeabilized by adding 100 μL of Solution B (15 min in the dark at RT) and antibodies against intracellular markers.

After washing, cells were re-suspended in PBS for immediate acquisition (or stored for max ~ 3 h at 4 °C).

An additional flowcytometry panel was used to evaluate expression of non-phosphorylated PLCγ2 in various cell populations (Table S2). Here, samples were processed according to the bulk lysis protocol for staining of 2.5 × 106 cells (www.EuroFlow.org).

For precise enumeration of cells, we used Perfect-Count Microspheres™ (Cytognos) according to the EuroFlow SOP (protocol available on www.EuroFlow.org). In short, 50 μL of well-mixed Perfect Count Microspheres were added to 50 μL of peripheral blood. This mixture was incubated for 30 min with antibodies directed against CD19, CD3 and CD45. Next, 500 μL of NH4Cl was added to lyse the red blood cells. After a 10 min incubation with NH4Cl, samples were immediately acquired on a BD FACS LSR Fortessa 4 L, or a BD FACS LSR Fortessa X-20 4 L (both BD Biosciences, San Jose, CA, USA).

Comparison of immune-related SNPs and calculation of polygenic risk score of immune-related SNPs between carriers and non-carriers

To investigate whether other immune-related genetic differences may explain the observed differences between the carriers and non-carriers, we calculated an immune-AD polygenic risk score (PRS). We included 59 SNPs (including the CD33 SNP) that were identified by a recent GWAS [31] and were predicted to have an effect on immune-function by snpXplorer (http://snpxplorer.net/, Table S7 [32]). We excluded the rs72824905 SNP in PLCG2. PRSs were compared between p.P522R PLCG2 carriers and non-carriers. We used logistics regression models to compare AD-PRS and immune-PRS between cases (p.P522R PLCG2-carriers) and controls (PLCG2 wild-type). For the single-SNP association, we used logistic regression models using SNP dosages, and associations were corrected with a False Discovery Rate (FDR < 0.1).

Measurement of phosphorylated PLCγ2

For in vitro stimulation assays, PB-HEP from Cohort II was used < 12 h after collection. Intracellular expression of phosphorylated PLCγ2 (pPLCγ2) was measured to assess B-cell activation upon stimulation with IgM or IgG Fabs (being F (ab)2 fragment Goat anti-human IgM Heavy Chain secondary antibody (Southern Biotech), and F (ab)2 fragment Goat anti-human IgG (Jackson ImmunoResearch), respectively). First, PB-HEP was subjected to a bulk lysis to remove red blood cells (protocol at www.EuroFlow.org). Then, cells were incubated with an antibody cocktail for surface staining (45 min, RT in the dark) (Table S2), washed with PBS, incubated with 1 μL 1:10 Zombie NIR™ Fixable Viability Dye (BioLegend) for 30 min at RT and washed again. Subsequently, cells were resuspended in 230 μL PBS + 0.5% BSA and incubated with 10 μg anti-IgM F (ab)2 or anti-IgG F(ab)2 fragments (10 min at 37 °C in a shaking water bath). The reaction was stopped by adding 62.5 μL Inside fix (Cell signaling buffer set A; Miltenyi) and incubating for 10 min in the dark. Afterwards, cells were washed and permeabilized (Cell signaling buffer set A, Miltenyi), washed again and resuspended in PBS + 0.5% BSA. Next, antibodies to detect pPLCγ2 and immunoglobulins were added (30 min at RT in the dark). After a final washing step, cells were resuspended in PBS and acquired on a BD FACS Canto II 3 L. Integrated MFI (iMFI) was calculated according to the following Eq. (E1) [33]:

$$integrated\ MFI=\% pPLC\gamma 2\ positive\ cells\times MFI\ pPLC\gamma 2\ positive\ cells$$

(1)

As the stimulated B-cell receptor could not be targeted for antibody stain to identify cells, the following marker combinations were used to identify B-cell subsets: For IgM stimulation: pre-GC B cells, CD20+CD27-IgG-IgA-; unswitched MBCs, CD20+CD27+IgG-IgA-; class-switched MBCs, CD20+CD27+IgG+ or CD20+CD27+ IgA+. For IgG stimulation: pre-GC B cells, CD20+CD27-IgD+IgA-; unswitched MBCs, CD20+CD27+IgD+IgA-; class-switched IgG MBCs, CD20+CD27+IgD-IgA-.

Calcium flux assay

Peripheral blood mononuclear cells (PBMCs) from Cohort I and II were isolated from fresh PB-HEP by means of a density gradient (in-house; Ficoll-amidotrizoate, density 1.077 g/mL) and stored in liquid nitrogen (freeze medium; RPMI + 40% FCS + 10% DMSO). At the day of analysis, PBMCs were thawed and washed twice with loading buffer (HBSS + 10 mM HEPES + 5%  FCS). Next, cells (10 × 106 PBMCs/mL) were loaded with Indo-1 Calcium Sensor Dye (Fisher Scientific, final concentration 2 μg/mL) for 30 min at 37 °C, labeled with antibodies directed against cell surface markers and incubated for additional 15 min at 37 °C (Table S2). Subsequently, cells were washed with 10-20x labeling volume in Flux buffer (HBSS + 10 mM HEPES + 5% FCS + 1 mM CaCl2) and resuspended in 500 μL Flux buffer. Samples were measured immediately on a Cytek Aurora 5 L flow cytometer.

After establishing a ~ 2.5 min baseline, cells were stimulated with 20 μL (10 μg) anti-IgM F(ab)2 Fragments (Southern Biotech) or 8.3 μL (10 μg) anti-IgG F(ab)2 Fragments (Jackson ImmunoResearch) and measured for ~ 10 min. Finally, ionomycin was added (5 μL, 1 mg/mL in DMSO) and samples were measured for ~ 2.5 minutes. Acquisition was performed at medium speed (~ 30 μL/min). During stimulation and measurement, samples were kept at 37 °C in a tube heating device. Indo-Free and Indo-Bound signal was detected in the UV7 and UV1 detector, respectively. Samples were analyzed by dividing the sample into 30 time slots (of equal time, ~ 30 sec) with the Infinicyt Software (Cytognos) and determining the UV1/UV7 ratios in each time slot. Time slots 1–5 represent baseline signal, time slots 6–25 represent Fab-stimulated signal, and time slots 26–30 represent ionomycin-stimulated signal. MFIs of all time slots were used to plot a curve per B-cell population, from which the area under the curve (AUC) was determined for each individual sample using the GraphPad PRISM Software (v8.1.1). To calculate AUC for the ‘total Fab stimulation’, time slots 6–25 were used. When calculating the AUC for the ‘peak of Fab stimulation’, time slots 7–12 were used, as these were the highest values in all donors and presented the peak of calcium release. ‘Ionomycin stim’ AUC was calculated using time slots 26–30. In all cases, AUC was only calculated for points higher than baseline signal (unstimulated sample). As the stimulated B-cell receptor could not be targeted for antibody stain to identify cells, the following marker combinations were used to identify B-cell subsets: For IgM stimulation: pre-GC B cells, CD20+CD27-IgG-IgA-; unswitched MBCs, CD20+CD27+IgG-IgA-; class-switched MBCs, CD20+CD27+IgG+ or IgA+; and for IgG stimulation: pre-GC B cells, CD20+CD27-IgD+IgA-; unswitched MBCS, CD20+CD27+IgD+IgA-; class-switched IgG MBCs, CD20+CD27+IgD-IgA-.

KREC analysis to determine cell proliferation history

PB-HEP from Cohort II was used < 24 h after collection for high-speed cell sorting of pre-GC B cells (CD19+CD27-IgM+IgG-IgA-), unswitched MBCs (CD19+CD27+IgM+IgG-IgA-) and class-switched MBCs (CD19+CD27+IgM-IgG+ or CD19+CD27+IgM-IgA+) using a BD FACS Aria III 4 L (BD Biosciences, San Jose, CA, USA). On average, a purity of 98% was reached for pre-GC B cells, unswitched MBCs, and class-switched MBCs. KREC numbers were determined as previously described [34, 35]. In short, DNA of sorted populations and the KREC control cell line (DB01) was isolated with a QIAmp DNA Micro Kit (QIAGEN) and DNA concentrations were determined by NanoDrop 2000 (Thermo Fisher Scientific). Next, we performed qPCR (Quantstudio qPCR Machine, Thermo Fisher Scientific) to quantify the average amount of coding joints (Cj), signal joints (Sj) and Albumin (Alb) in each B-cell population and the control cell line (DB01), using the primers that were previously described by van Zelm and colleagues [34]. The number of cell divisions (ΔCt) for each B-cell population was calculated with the following Eq. (E2):

$$\varDelta Ct= Ct(Sj)- Ct(Cj)-\varDelta Ct(control)$$

(2)

Where ΔCt (control) is a standard correction for primer efficacy based on the Ct(sj) and Ct(Cj) from DNA from the DB01 control cell line.

Phagocytosis of pHRodo™ Green E. coli bioparticles

Assessment of the phagocytic capacity was performed with opsonized E. coli (pHRodo™ Green E. coli Bioparticles, ThermoFisher). Polystyrene FACS tubes (4 mL) were filled with 200 μl PB-HEP (Cohort II, < 12 h after collection) and placed on ice for at least 10 minutes. Then, 40 μL of pHRodo™ Green E. coli was added, samples were mixed and incubated at 37 °C for exactly 20 min. All further steps were performed on ice or at 4 °C to stop the reaction. Cells were washed and stained (30 min on ice in the dark) with an antibody cocktail (Table S2). Next, samples were lysed with BD lyse (10 min, rolling at 4 °C). Lastly, cells were washed, resuspended in cold PBS with 0.5% BSA and acquired at a Cytek Aurora 3 L flow cytometer. To prevent shedding of CD62L, TAPI-2 (final concentration of 20 μM) was always present. To account for background activation or signal, several control tubes were measured in addition to the sample tubes (Table S8).

Production of reactive oxygen species (ROS)

Production of ROS upon stimulation was assessed with the phagoBURST kit (PHAGOBURST™ CE/IVD kit, BD Biosciences) according to manufacturer’s protocol with some modifications, as described further in this paragraph. In short, each tube was filled with 200 μL PB-HEP (Cohort II, < 12 h after collection) and placed on ice for at least 10 minutes. Then, 20 μL of wash buffer (reagent A), Phorbol 12-Myristate 13-acetate (PMA) (Sigma, P8139-1 mg, final concentration 600 ng/mL), or E. coli (reagent B, well-mixed by pipetting) was added to each tube. After proper mixing, tubes were incubated for exactly 10 min at 37 °C in a shaking water bath. Next, 20 μL of substrate (dihydrorhodamine -DHR123, reagent E) was added to each tube and mixed. Tubes were incubated for another 10 min at 37 °C. Subsequently, the assay was stopped by lysing the cells (reagent F, 15 min, RT, dark). Cells were washed twice, stained for 15 min in the dark at RT with antibody cocktail (Table S2) and PBS with 0.5% BSA, washed again, resuspended in PBS with 0.5% BSA and stored on ice until acquisition on a Cytek Aurora 3 L flow cytometer (< 1 h). To prevent shedding of CD62L, TAPI-2 (final concentration of 20 μM) was always present. The rhodamine 123 (R123) signal was used as a readout for ROS production (conversion from DHR123 to R123). To account for background activation or signal, several control tubes were measured in addition to the sample tubes (Table S9).

Data integration phagocytosis and ROS production

The phagocytosis assay and the ROS production were considered complementary assays in which we evaluated three aspects of phagocytosis. First, we evaluated the percentage of phagocytosing cells, then we combined this – together with the pHRodo™ Green signal- into the integrated MFI (iMFI) [33], which was used as a measure of how many particles each cell has phagocytosed (E3). For ease of interpretation, this value was divided by 100,000.

$$integrated\ MFI=\%E.\ coli\ positive\ cells\times MFI\ E.\ coli\ positive\ cells$$

(3)

Lastly, we measured the production of ROS upon phagocytosis and combined this with the iMFI into one output; the normalized ROS production, which was defined as the ROS generation per given number of phagocytosed particles (E4).

$$normalized\ ROS\ production=\frac$$

(4)

In vivo evaluation

We collected additional blood samples from donors (Cohort III), 7–14 weeks after their second vaccination against SARS-CoV-2. Serum antibodies directed against the Spike (S) protein, Receptor Binding Domain (RBD) and Nucleocapsid (N) protein were determined by a fluorescent-bead-based multiplex immunoassay (MIA), as previously described [36]. In short, the stabilized pre-fusion conformation of the ectodomain of the Spike protein, the Receptor Binding Domain of the S-protein (RBD) and the Nucleocapsid (N) protein were each coupled to beads or microspheres with distinct fluorescence excitation and emission spectra. Serum samples were diluted and incubated with the antigen-coupled microspheres. Following incubation, the microspheres were washed and incubated with phycoerythrin-conjugated goat anti-human, IgG, IgA, and IgM. The data were acquired on the Luminex FlexMap3D System and MFI was converted to international units per milliliter (IU/mL), using Bioplex Manager 6.2 (Bio-Rad Laboratories) software.

Data analysis and statistical analysis

Flow cytometry data was analyzed with Infinicyt software (version 2.0.3.a. and 2.0.4.b., Cytognos, Spain). Statistical analysis was performed in GraphPad Prism 8.1.1 software (GraphPad, San Diego, CA, USA). Differences between p.P522R-carriers and non-carriers, were evaluated using the Mann-Whitney U test. Impact of age was assessed using Spearman’s correlation. P < 0.05 was considered significant.

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