Phosphatase control of cytokine-mediated overproduction of galactose-deficient IgA1, the main autoantigen in IgA nephropathy

IgA nephropathy (IgAN) is an autoimmune disease characterized by the deposition of galactose-deficient IgA1 (Gd-IgA1)-containing immune complexes in the kidneys. Elevated serum levels of Gd-IgA1, the main autoantigen in IgAN, are associated with mucosal infections and poor renal outcome in IgAN patients, but little is known about the activation of IgA1-secreting cells overproducing this autoantigen. We found that in peripheral blood mononuclear cells (PBMCs), cytokine stimulation elevated Gd-IgA1 production in B cells from IgAN patients but not in those from healthy controls (p < 0.01). These results were replicated in immortalized B cells derived from PBMCs of IgAN patients and healthy controls. Using single-cell transcriptomics, we identified subsets of IgA1-secreting cells from IgAN patients, but not from healthy controls, with decreased expression of C1GALT1 in response to cytokine stimulation. The C1GALT1-encoded glycosyltransferase is responsible for addition of galactose to IgA1 O-glycans, and its reduced activity is associated with elevated serum levels of Gd-IgA1. These newly identified subsets of IgA1-secreting cells with reduced C1GALT1 expression exhibited reduced expression of several genes related to cytokine-mediated signaling, including those encoding phosphatases, such as SOCS1. siRNA knock-down of SOCS1, and the related SOCS3, increased Gd-IgA1 production in cells derived from PBMCs of healthy controls, indicating a role of these regulators in abnormal cytokine signaling and Gd-IgA1 overproduction. These results revealed that specific subsets of IgA1-secreting cells may be responsible for autoantigen production in IgAN due to abnormal regulation of cytokine-mediated signaling, a process that may occur in inflammatory responses in IgAN patients.

cells derived from PBMCs of healthy controls, indicating a role of these regulators in abnormal cytokine signaling and Gd-IgA1 overproduction. These results revealed that specific subsets of IgA1-secreting cells may be responsible for autoantigen production in IgAN due to abnormal regulation of cytokine-mediated signaling, a process that may occur in inflammatory responses in IgAN patients.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Introduction
IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide. It is characterized by mesangial deposition of IgA-containing immune complexes with co-deposition of IgG [1][2][3]. Complement C3 is usually also present [4]. Glomerular IgA immunodeposits in IgAN patients are of the IgA1 subclass and enriched for galactosedeficient O-glycoforms (Gd-IgA1) [5,6]. Serum levels of Gd-IgA1 are elevated in IgAN patients [7]. The current understanding of IgAN pathogenesis suggests that circulatory Gd-IgA1 is bound by IgG autoantibodies specific for Gd-IgA1, forming pathogenic immune complexes, some of which deposit in the kidneys triggering a cascade of events leading to renal injury and, in many patients, to kidney failure [8,9]. This hypothesis is supported by evidence that IgG isolated from the kidney immunodeposits is specific for Gd-IgA1 and that Gd-IgA1-IgG immune complexes are found in the circulation of IgAN patients [1,10,11]. However, despite the critical role of Gd-IgA1 in IgAN, the origin of this autoantigen and the characteristics of specific cell population(s) producing Gd-IgA1 remain unknown.
Human IgA is comprised of two subclasses, IgA1 and IgA2; IgA1 contains an extended 18-amino-acid hinge region. Circulatory IgA1 usually contains 3 to 6 core 1 O-glycans per hinge region [12]. The O-glycan synthesis is initiated by N-acetylgalactosamine (GalNAc) transferases (GalNAcTs) that attach GalNAc to some Serine (Ser)/-Threonine (Thr) residues in the IgA1 hinge region [13,14]. This is normally followed by the addition of galactose (Gal), α1,3-linked to GalNAc, by a Core 1 galactosyltransferase (C1GalT1) [14][15][16]. IgA1 with some glycans lacking Gal is termed Gd-IgA1. Genome wide association studies (GWAS) identified C1GALT1 SNPs that are associated with elevated Gd-IgA1 serum levels in IgAN patients, highlighting the role of this gene and the encoded enzyme in the autoantigen production and disease pathobiology [17,18]. In addition to genetic components, Gd-IgA1 production is impacted by some cytokines. Prior studies using IgA1-producing cell lines showed that interleukin 4 (IL-4) and IL-6 decrease C1GALT1 expression and increase Gd-IgA1 production, but only in cells derived from IgAN patients [19]. Stimulation of the IgA1-producing cell lines with single cytokines, IL-6 and leukemia inhibitor factor (LIF) resulted in enhanced and prolonged activation of STAT3 and STAT1 (signal transducer and activator of transcription 1/3), respectively, the likely mechanisms associated with Gd-IgA1 overproduction [20,21]. These studies thus showed that some cytokines can modulate expression of glycosylation enzymes and increase Gd-IgA1 production in IgAN, suggesting possible abnormalities of control mechanisms related to pro-inflammatory cytokine signaling.
It is well accepted that IgA1-secreting cells in vivo are exposed to cytokine mixtures and, usually, not to a single cytokine. The in vivo conditions that lead to elevated serum levels of Gd-IgA1 are unknown, but can be associated with mucosal infections, as IgAN patients often present with synpharyngitic hematuria [11,22]. This suggests that a cytokine milieu may promote Gd-IgA1 production, leading to formation of new immune complexes that may deposit in the glomeruli and further enhance kidney injury. We have therefore tested a mixture of cytokines (IL-4, IL-6, IL-21, CD40L) and found that it can increase Gd-IgA1 production in peripheral blood mononuclear cells (PBMCs) as well as in immortalized B cells derived from PBMCs of IgAN patients but not in those of healthy controls. In immortalized B cells, our preliminary data showed no significant difference in Gd-IgA1 or IgA1 production between single-cytokine vs. cytokine-mixture stimulation. However, based on our flow cytometry data, there appears to be substantial differences in the activation of some signaling targets, such as pSTAT1/3, between the single vs. multi-cytokine stimulation. This suggests that delineating potential in vivo targets relevant to Gd-IgA1 production should include testing with a cytokine mixture methodology.
As Gd-IgA1 constitutes a minority of total circulatory IgA1, we hypothesized that only a subpopulation(s) of IgA1-secreting cells may be responsible for Gd-IgA1 production, both at baseline and during episodes of inflammatory stimuli. To test this hypothesis, we performed single-cell transcriptomic experiments using immortalized IgA1-secreting cells derived from PBMCs of IgAN patients and healthy controls. The data were then analyzed by unsupervised UMAP (Uniform Manifold Approximation and Projection). This analysis revealed unique subpopulations of IgA1-secreting cells with decreased C1GALT1expression in the cells from IgAN donors but not healthy controls after cytokine stimulation. This reduced C1GALT1 expression thus constitutes a molecular signature of subpopulations producing Gd-IgA1 in response to cytokines. Additionally, pathway analysis of the IgAN-derived cell subpopulations with repressed C1GALT1 expression showed substantial differences in cytokine-mediated signaling pathways, as well as related phosphatase genes. Gene-specific siRNA knock-down of two phosphatase regulatory proteins identified in Gd-IgA1-secreting subpopulations, SOCS1 and SOCS3 (suppressor of cytokine stimulation 1 and 3), led to Gd-IgA1 overproduction in cells derived from PBMCs of healthy-control due to cytokine stimulation. These results support the hypothesis that subpopulations of IgA1-secreting cells may differentially respond to pro-inflammatory cytokines due to abnormal phosphatase regulation, subsequently leading to overproduction of Gd-IgA1, the main autoantigen in IgAN.

Patients
Eligible adult patients (≥18 years of age) with biopsy-proven primary IgAN receiving care at the University of Alabama at Birmingham Nephrology clinic were approached for study participation. Healthy volunteers were recruited from the Birmingham community. Some responded to our study advertisement, others (who had previously participated in our studies) were contacted by our research team if they had indicated their interest in being included in future research opportunities.
All recruited individuals signed an informed consent. The study protocol was reviewed and approved by our institutional review board. The study was conducted in accordance with the principles of the declaration of Helsinki. For the purpose of the data generated in the current manuscript, samples from 5 healthy controls (HC) and 3 IgAN patients were included. In addition, we have over 500 IgAN patient and healthy control donor immortalized B cell lines stored in our biobank which were used for some of the experiments.

Single-cell transcriptomics
To assess transcriptional responses to cytokine mixture in IgA1-producing cells, 10X Genomics single-cell transcriptome 3′ kits were used. The data were compiled using the 10X Genomics Ranger tool. From immortalized B cells derived from PBMCs from 4 healthy controls and 4 patients with IgAN, over 52,000 cells were analyzed, with an average mean read per cell of 44,281 and a median of 2781 genes per cell. Further processing used an open-source R software tool from Dr. Satija's lab, Seurat V4.01 (https://satijalab.org/seurat/), and a software package from Alteryx for the flexible design of large-scale database analysis (www.alteryx.com). In addition, scaffold gene data (noncontiguous gene sequences not in hg38) from published sources on splice variants of IGHA1 (immunoglobulin heavy chain A1) that determine secreted (IGHA1s) vs. membranebound (IGHA1m) IgA1 isoforms were inserted into the hg38 gene reference database [25]. Based on this approach, we identified 1164 and 1400 IgA1-secreting cells (IGHA1s) from healthy-control-and IgAN-derived cells, respectively. These IGHA1s-subpopulations were then grouped based on gene co-expression using the non-linear dimension reduction algorithm UMAP. Broad Institute GO biological GSEA pathway analysis was performed on top genes from unique groupings in healthy control (HC) and IgAN UMAP. Differential gene markers for UMAP identified subgroups were identified using Seurat function calls. Analysis of UMAP subgroup gene log2 fold changes within HC and IgAN were performed by comparing a specific subgroup gene expression within either HC or IgAN to all cells in the opposing group (e.g. IgAN group #6 vs all HC cells). Analysis of specific low and high gene expressing subpopulations within the IGHA1s-subpopulations were performed with Alteryx by segregating the top 5% and bottom 5% of expressing cells.

siRNA knock-down
OnTargetPlus SmartPool siRNA sequences (Dharmacon) of SOCS1 and SOCS3 were used for knock-down. Electroporation was performed using the Amaxa Nucleofector II (Lonza) at setting X-001 with the Amaxa Nucelofector Kit C (Lonza). Immortalized B cells were suspended in electroporation buffer (2 × 106 cells in 100 μL), and siRNA added to a final concentration of 5 nM, followed by electroporation, and incubated in 2 mL of RPMI 1640 for 4 h at 37 °C. Cells were then collected by centrifugation and resuspended in 2 mL of fresh RPMI 1640 media and incubated for 48 h. Medium was replaced, and cell stimulated with cytokine mixture for 72 h, followed by IgA and Gd-IgA1 ELISA analysis, as described above.

Statistical analysis
Statistical analysis of changes in Gd-IgA1 and gene-specific subpopulations in Alteryx were performed using Data Analysis package in Excel and Alteryx, specifically the students t-test (two-tailed paired sample for means). Differential marker analysis performed on single-cell subpopulations employs non-parametric Wilcoxon rank sum test. The Kolmogorov-Smirnov test was used to determine significance for pathway analysis. Differential markers from Seurat v4.0 uses a Bonferroni adjustment for all p-values to yield a p-adj value for individual genes.

Cytokine stimulation increased Gd-IgA1 production in PBMCs from IgAN patients
Cultured PBMCs of IgAN patients produced more IgA1 than those of HC, when normalized to cell number, both at baseline (p = 0.03) and after cytokine stimulation (p = 0.02) (Fig.  1A). No change in IgA1 production was noted after cytokine stimulation within the groups. This difference between IgAN and HC PBMCs could be due to increased number of IgA1-secreting cells in circulation or enhanced production of IgA1. IgA1 production, as a percent change from control, was increased in healthy controls, but not IgAN patients, after cytokine stimulation ( Fig. 1B; p = 0.05 and p = 0.27 respectively). Baseline and post-cytokine treatmemt Gd-IgA1 production was not different between the cells from IgAN patients vs. healthy controls, when normalized to cell number. There was significant decrease in Gd-IgA1 production within the HC group after cytokine stimulation (p = 0.04), but no change within the IgAN PBMC group (p = 0.90) (Fig. 1C). However, cytokines led to a relative increase in Gd-IgA1 production by PBMCs but only within the IgAN patient group ( Fig. 1D; p = 0.03), with a relative decrease in the HC derived cell group ( Fig. 1D; p < 0.01).

Cytokine stimulation increased Gd-IgA1 production by immortalized B cells from IgAN patients
Cytokine stimulation increased IgA1 production by immortalized B cells derived from PBMCs of HC compared to those of IgAN patients ( Fig. 2A; p = 0.02), with no change within groups ( Fig. 2A). IgA1-production, as a percent change to control, found no differences between the two groups ( Fig. 2B), but did show a significant increase within the HC group, and no change within the IgAN group. Baseline Gd-IgA1 production was not different between IgAN-vs HC-derived cells, and no difference within groups (Fig.  2C). Gd-IgA1 production, as a percent change from control, showed increased Gd-IgA1 production by cells within the IgAN patient group (Fig. 2D; p = 0.03), but not those within the HC group ( Fig. 2D; p = 0.10). We did not need to normalize for cell number since all immortalized B cell experiments were plated at the same cell concentration, however, this does not account for potential differences between groups in the percent of IgA1 + cells since we are using mixtures of B cells that contain all possible classes. We did not see a decrease in Gd-IgA1 production within the immortalized HC B cell group like we did in the PBMC data, which could be due to needing a higher number of replicates since immortalization also just captures a small percentage of all the B cells found in a PBMC sample.

UMAP of single-cell transcriptomics of immortalized IGHA1sB cells after cytokine stimulation
Immortalized B cells from IgAN patients and healthy control donors were stimulated with cytokines and differential gene expression was analyzed by single-cell transcriptomics. Unsupervised UMAP analysis of immortalized IGHA1s + cells after cytokine stimulation revealed several different cell populations, some common and others unique to either HC or IgAN patients (Fig. 3). This analysis revealed that there were substantial differences within IgA1-secreting cells, and that IgAN-derived cells had unique populations (#2, #5, #6) compared to those from healthy controls (#0).

Pathway analysis of unique subgroups in IGHA1s cells from IgAN and healthy control donors
Pathway analysis using statistically significant genes of the subpopulations from the UMAP separation after cytokine stimulation found common and unique pathways within the IgANpatient and healthy-control groups. Table 1 lists for each group the unique pathways that were statistically significantly different. While we suspected that intracellular signaling related to cytokine stimulation would be important, we did not anticipate the prevalence of MAPK signaling within the IgAN groups. The Immune System Development pathway had the highest gene ratio number (k/K) of all the pathways, suggesting significant immunityspecific processes may be altered (Table 1). These results together illustrate substantial differences in cytokine-mediated signaling in cells derived from PBMCs of IgAN patients vs. healthy controls.

Specific glycosyltransferases and phosphatases differentially expressed in IGHA1s subpopulations from IgAN patients vs. healthy controls
The gene critical for Gd-IgA1 production, C1GALT1, was significantly downregulated in two of the three IgAN-unique subpopulations (5, 6), but not in any of the HC groups ( Table 2, Fig. 4A and B). This suggests that these IgAN-unique cell populations may be producing substantial amounts of Gd-IgA1. Additionally, decreased expression in a sialyltransferase (ST6GALNAC3) was observed in the IgAN-unique subpopulations (2, 5, 6), but increased in the HC-unique group (0) ( Table 2). Sialyltransferases have been implicated in Gd-IgA1 production previously, such as ST6GALNAC2; however, this is the first time ST6GALNAC3 has been reported to be associated with Gd-IgA1 production [19]. Expression of GALNT12, a GalNAc transferase recently reported to be associated with Gd-IgA1 production [26], was also decreased in the HC-unique group (0), and increased in two of the three IgAN-unique groups (5,6). SOCS1, encoding a phosphatase and one of the primary regulators of STAT1 activation, was downregulated in one IgAN-unique group (6). SOCS3, encoding a significant regulator of STAT3, was increased in the HC-unique group (0), but no significant changes were found in any of the IgAN-unique groups ( Table 2).

B cells from IgAN donors with lowest C1GALT1 expression had highest expression of IGHA1s after cytokine stimulation
Immortalized B cells stimulated for 20 min with cytokines, were analyzed by singlecell transcriptomics, and separated into high and low C1GALT1 subpopulations. IGHA1sexpression levels were then determined for these groups. In IgAN-derived cells, IGHA1sexpression was higher in the low-expressing C1GALT1 group compared to the highexpressing C1GALT1 group (p < 0.01) and compared to the low-expressing healthy control group (p < 0.01) (Fig. 5). This observation indicates that cytokine-induced elevated secretion of IgA1 in IgAN patients may occur predominantly in the subpopulations with the lowest expression of C1GALT1.

Low expression of C1GALT1 in low-expressing SOCS1 subpopulations in IgAN IGHA1s B cells
Immortalized IGHA1s B cells after cytokine stimulation were grouped into low-and highexpressing SOCS1 and SOCS3 groups. We found significantly lower C1GALT1 expression in the low SOCS1 group compared to high SOCS1-expressing group, but only in the IgAN-derived cells (p = 0.04) (Fig. 6A). This difference in C1GALT1 expression was not seen in the SOCS3 low-and high-expressing subpopulations (Fig. 6B).

Increased expression of IGHA1s in low SOCS1 and low SOCS3 groups after cytokine stimulation
To assess the potential connection of phosphatases in IgA1-secretion, we grouped IGHA1spositive cells into low and high SOCS1 and SOCS3-expressing cell subpopulations. Expression levels of IGHA1s were higher in low-expressing SOCS1 group in IgAN-derived cells compared to the high SOCS1 group (p < 0.01) and the low SOCS1 healthy-controlderived cells (p = 0.01) (Fig. 7A). Additionally, expression of IGHA1s was higher in the low-expressing SOCS3 group from IgAN-derived cells compared to the high SOCS3 group (p < 0.01) and to the low-expressing SOCS3 group from healthy-control-derived cells (p < 0.01) (Fig. 7B).

Increased Gd-IgA1 production in healthy-control cells after siRNA-mediated genespecific knock-down of SOCS1 and SOCS3
Expression of SOCS1 and SOCS3 was altered using siRNA knockdown in immortalized B cells from HC and IgAN donors, stimulated with cytokines and Gd-IgA1 production was measured. We found increased Gd-IgA1 production after SOCS1 knock-down in healthycontrol cells without stimulation (p = 0.03), and a trend to increased Gd-IgA1 production in the SOCS3 knockdown. For IgAN-derived cells, we saw a significant increase in Gd-IgA1 without stimulation in the SOCS3 knock-down (p = 0.04), with a trend to higher Gd-IgA1 production after SOCS1 siRNA knock-down. After stimulation with cytokines, both the SOCS1 (p = 0.01) and SOCS3 (p = 0.04) knock-down increased Gd-IgA1 production in

Discussion
The role of the autoantigen, Gd-IgA1, in the pathobiology of IgAN has been widely understood as necessary but not sufficient for the development of this disease [14]. However, the origins and mechanisms that mediate the overproduction of Gd-IgA1 during states of disease activity are poorly understood. The correlation between autoantigen production and inflammation can be seen in IgAN patients who present with synpharyngitic hematuria with concurrent elevated levels of Gd-IgA1 and its circulating immune complexes [22,27]. Furthermore, GWAS have supported this connection with inflammation, based on discovery of IgAN-risk-associated genetic alleles, i.e., SNPs, such as those in HORMAD2 locus with genes encoding also LIF and OSM (oncostatin M) [28]. Moreover, several studies revealed enhanced Gd-IgA1 production in response to IL-6 in IgAN-derived cells, a process involving aberrant signaling leading to reduced expression of C1GALT1 [19]. Here we show that abnormal responses to cytokines are not limited to single-cytokine exposures, and thus may not be due to only one abnormal proinflammatory pathway (Figs. 1 and 2). Our findings highlight the importance of experimenting with in vivo mimicking conditions where IgA1+ B cells are exposed to a multi-cytokine milieu.
Gd-IgA1 in the circulation, mostly bound in immune complexes, is predominantly in the polymeric form, i.e., the molecular form thought to be produced mainly by plasma cells in mucosal tissues [16]. Polymeric IgA1 constitutes only ~10% of the total circulatory IgA1 [29] and Gd-IgA1 in immune complexes represents <1% of total serum IgA1. Thus, we hypothesize that only a small fraction of IgA1-secreting cells is responsible for production of polymeric Gd-IgA1. Single-cell transcriptomic data revealed, for the first time, several unique subpopulations of IGHA1s-expressing cells derived from PBMCs of IgAN patients. These cells exhibited reduced C1GALT1 expression after cytokine stimulation ( Table 2, Fig. 4). Of note, the comparison of healthy-control-derived cells before and after cytokine stimulation showed no difference in C1GALT1 expression, whereas the same pairwise analysis of IgAN-derived samples showed changes in C1GALT1 expression after cytokine exposure. (S.Tables 1 and 2, S. Figs. 1 and 2). Another glycosyltransferase gene, ST6GALNAC2, has been reported to be overexpressed in immortalized B cells from IgAN patients after cytokine stimulation. However, we have not observed differences among cell subpopulations in our analyses [19]. Conversely, we found that ST6GALNAC3 was decreased in IgAN subpopulations, compared to a relative increase in HC supopulations ( Table 2). This may be important in the disease pathogenesis because the Gd-IgA1 in circulating Gd-IgA1-IgG immune complexes lacks a sialic acid on the terminal GalNAc, thus allowing for the binding of anti-Gd-IgA1 IgG autoantibodies [10].
The IgAN-unique subpopulations (Fig. 3) had altered cell-signaling pathways (Table  1), as well as differential phosphatase expression of SOCS1 and SOCS3 compared to healthy control cells after cytokine stimulation (Table 2). To determine if these specific phosphatases correlated with changes in C1GALT1, we separated data for cells with low vs. high C1GALT1-expression, and found that low SOCS1-expressing subpopulations in IgAN-derived IGHA1s cells had decreased expression of C1GALT1. Similar analyses were performed for low vs. high expression SOCS3 groups, but no such correlation was found (Fig. 6). These low SOCS1-expressing cells had higher expression of IGHA1s and lower expression of C1GALT1, indicating a potential for overproduction of Gd-IgA1 (Fig. 7). This is supported by the knockdown of SOCS1 and SOCS3 data showing increased Gd-IgA1-production responses to cytokine stimulation in healthy-control-derived cells (Fig. 8). Additionally, other phosphatases, such as PTPN2 and PTPN6 (S. Table 3), were differentially regulated in IgAN-unique cells compared to healthy-control-derived cells after cytokine stimulation. These changes in expression of phosphatases may be responsible for abnormal responses to pro-inflammatory processes, as indicated by decreased NFKBIA (NF-kappa-B inhibitor A) expression in IgAN-unique groups compared to healthy-control-derived groups (S. Table 3). We pose that multiple phosphatases may act in concert and mediate abnormal cytokine-induced responses that lead to reduced C1GALT1 expression and, thus, overproduction of Gd-IgA1 in IgAN.
In conclusion, we discovered unique IgA1-secreting cells with differential responses to cytokine stimulation, and showed that some IgAN-unique cell types exhibited reduced expression of C1GALT1, leading to Gd-IgA1 overproduction. Our data revealed that abnormal phosphatase regulation is involved in cytokine-mediated autoantigen overproduction. Moreover, a minority of IgA1-secreting cells may be responsible for Gd-IgA1 overproduction. We do not know if the unique IgAN subpopulations (2,5,6) are downstream from a more "normal" subset that occurs due to cytokine stimulation, or if they represent a critical starting subpopulation in IgAN patients. We anticipate that assessing these subpopulations for Gd-IgA1 production will require a combination of cell-surface phenotyping and single-cell transcriptomics of PBMCs in IgAN patients. These findings suggest a possibility of a direct targeting of pro-inflammatoiy regulatory pathways in Gd-IgA1-producing cells and their subsets. The evolution of single-cell transcriptomics and direct antigen-targeting technologies will enable a more details analyses of IgA1 + cells to further elucidate inflammatory drivers of dysregulated glycosylation and autoantigen production in IgAN.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material. IgA1 and Gd-IgA1 production after cytokine stimulation differs between immortalized B cells from IgAN patients (n = 3) and healthy controls (n = 3): IgA1 production by immortalized B cells was lower in samples from IgAN patients compared to those of healthy controls (HC) (p = 0.02) (A). HC had a higher increase in IgA1 production after cytokine stimulation compared to IgAN-derived B cells (B). There was no difference in baseline Gd-IgA1 production between HC and IgAN PBMCs (C), but IgAN-derived cells had higher increase in Gd-IgA1 production in response to cytokines compared to HC (p = 0.03), with no statistically significant change for HC (p = 0.10) (D).  Differential regulation of C1GALT1 in IGHA1s subpopulations: Immortalized B cells from HC (n = 4) and IgAN (n = 4) donors were stimulated with cytokines for 20 min, and then subjected to single-cell transcriptomic analysis. IgA1-secreting cell (IGHA1s + cells) subpopulations were identified using UMAP (Fig. 3), followed by differential gene marker analysis to determine differences between groups. (A) Average C1GALT1 expression between all subgroups in HC-and IgAN-derived B cells with cytokine stimulation, and (B) a breakdown of C1GALT1 expression for each subpopulation.   siRNA gene-specific knock-down of SOCS1 and SOCS3 increased Gd-IgA1 production: siRNA knock-down of SOCS1 and SOCS3 increased Gd-IgA1 production in cytokinestimulated B cells from HC donors. Knockdown of SOCS1 in HC-derived B cells after siRNA-mediated knock-down of SOCS3 in IgAN-derived B cells also increased Gd-IgA1 production without cytokine stimulation.  Table 2 Differential regulation of glycosyltransferase and phosphatase genes in IGHA1s subpopulations: Immortalized B cells from HC (n = 4) and IgAN (n = 4) donors were stimulated with cytokines for 20 min, and then subjected to single-cell transcriptomic analysis. IgA1-secreting cell (IGHA1s + cells) subpopulations were identified using UMAP (Fig. 3), followed by differential gene marker analysis to determine differences between groups. Listed are some glycosyltransferase and phosphatase genes found to be differentially regulated between IgA1-secreting groups in HC and IgAN. ND = genes with no differential expression from other groups. LFC = log2 fold change.