Circulating plasmablasts/plasma cells: a potential biomarker for IgG4-related disease

Background Immunoglobulin G4 (IgG4)-related disease (IgG4-RD) is a multisystem fibroinflammatory disease. We previously reported that a circulating cell population expressing CD19+CD24−CD38hi was increased in patients with IgG4-RD. In this study, we aimed to document that this cell population represented circulating plasmablasts/plasma cells, to identify the detailed phenotype and gene expression profile of these IgG4-secreting plasmablasts/plasma cells, and to determine whether this B-cell lineage subset could be a biomarker in IgG4-related disease (IgG4-RD). Methods A total of 42 untreated patients with IgG4-RD were evaluated. Peripheral B-cell subsets, including CD19+CD24−CD38hi plasmablasts/plasma cells, CD19+CD24+CD38− memory B cells, CD19+CD24intCD38int naïve B cells, and CD19+CD24hiCD38hi regulatory B cells, were assessed and sorted by flow cytometry. Microarray analysis was used to measure gene expression of circulating B-cell lineage subsets. Further characterization of CD19+CD24−CD38hi plasmablasts/plasma cells was carried out by evaluating additional surface markers, including CD27, CD95, and human leukocyte antigen (HLA)-DR, by flow cytometric assay. In addition, various B-cell lineage subsets were cultured in vitro and IgG4 concentrations were measured by cytometric bead array. Results In untreated patients with IgG4-RD, the peripheral CD19+CD24−CD38hi plasmablast/plasma cell subset was increased and positively correlated with serum IgG4 levels, the number of involved organs, and the IgG4-related Disease Responder Index. It decreased after treatment with glucocorticoids. Characterization of the plasmablast/plasma cell population by gene expression profiling documented a typical plasmablast/plasma cell signature with higher expression of X-box binding protein 1 and IFN regulatory factor 4, but lower expression of paired box gene 5 and B-cell lymphoma 6 protein. In addition, CD27, CD95, and HLA-DR were highly expressed on CD19+CD24−CD38hi plasmablasts/plasma cells from patients with IgG4-RD. Furthermore, CD19+CD24−CD38hi plasmablasts/plasma cells secreted more IgG4 than other B-cell populations. Conclusions Circulating CD19+CD24−CD38hi plasmablasts/plasma cells are elevated in active IgG4-RD and decreased after glucocorticoid treatment. This IgG4-secreting plasmablast/plasma cell population might be a potentially useful biomarker for diagnosis and assessing response to treatment.

IgG4-RD is characterized by a number of abnormalities in the differentiation of cells of the B-cell lineage, including increased serum levels of IgG, IgG4, and often IgE; infiltration of affected tissues by IgG4-secreting plasma; and the presence of increased frequencies of circulating plasma cells/plasmablasts [13][14][15]. B cells and plasma cells may play important roles in the development of this disease [16]. Our previous studies revealed that, compared with healthy control subjects and patients with primary Sjögren's syndrome, patients with IgG4-RD expressed an increased circulating population of CD19 + CD24 − CD38 hi cells that appeared to be circulating plasmablasts/plasma cells and correlated positively with serum IgG4 levels [17]. The full characterization of this circulating population was not carried out, but its appearance and correlation with serum IgG4 suggested that it might be an important biomarker of IgG4-RD. To begin to address these questions, we initially characterized the circulating plasmablasts/plasma cells in IgG4-RD in greater detail.

Patients
Forty-two untreated patients with IgG4-RD fulfilling the 2011 comprehensive IgG4-RD diagnostic criteria were enrolled in this study. The diagnosis of IgG4-RD was based on the following three manifestations: (1) clinical examination showing characteristic diffuse/localized swelling or masses in single or multiple organs; (2) hematological examination showing elevated serum IgG4 concentration (>135 mg/dl); and (3) histopathologic examination showing (a) marked lymphocyte and plasma cell infiltration and fibrosis or (b) infiltration of IgG4 + plasma cells (ratio of IgG4 + /IgG + cells >40% and >10% IgG4 + plasma cells per highpower field). Patients with cancer or lymphoma and other autoimmune diseases were excluded.

Clinical data and inflammatory parameters
Clinical data, including age, gender, disease duration, and manifestations, were obtained for all patients. Laboratory findings were recorded, including erythrocyte sedimentation rate (ESR); C-reactive protein (CRP); and serum immunoglobulin IgG, IgA, IgM, and IgG subsets. IgG4-related Disease Responder Index (IgG4-RD RI) was calculated for each patient [18].

In vitro cell culture
Purified B-cell populations from patients with IgG4-RD were resuspended in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics (penicillin 100 IU/ml, streptomycin 100 μg/ml; Life Technologies, Carlsbad, CA, USA) in 96-well U-bottomed plates (Nunc, Langenselbold, Germany) in a humidified atmosphere of 5% CO 2 at 37°C with 1 × 10 5 cells in each well. For each group, 100 ng/ml recombinant human CD40L (Abcam, Cambridge, MA, USA) and 0.1 μg/ml cytosine phosphate guanosine oligodeoxynucleotide 2006 (CpG ODN 2006; InvivoGen, San Diego, CA, USA) was added at the beginning. After 7 days of culture, the levels of Ig secretion in collected supernatants were tested.

Cytometric bead array analysis
Culture supernatant samples from different B-cell subsets were collected and stored at −80°C until used. Cytometric bead array (CBA) analysis for IgG, IgA, IgM, IgG4, and IgE in supernatants was performed according to the manufacturer's instructions (BD Biosciences). Data were analyzed using CBA analysis software obtained from BD Biosciences. The concentrations of IgG, IgA, IgM, IgG4, and IgE in supernatants were determined by reference to a standard curve.

Microarray analysis of gene expression
Sorted B-cell subpopulations were placed in TRIzol reagent (Life Technologies) for RNA extraction following the manufacturer's instructions. Isolated RNA was further purified with the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and processed for microarray analysis using the standard Affymetrix protocols (www.affymetrix.com; Affymetrix, Santa Clara, CA, USA). Briefly, 1-10 μg of RNA was reversetranscribed into complementary DNA (cDNA) (Life Technologies). The template cDNA was purified for amplification and in vitro transcription to cRNA using the BioArray™ HighYield™ RNA Transcript Labeling Kit (T7) (Enzo Life Sciences, Inc., Farmingdale, NY, USA). cRNA was biotin-labeled, purified, and hybridized to Human Genome U133A GeneChips® (Affymetrix). GeneChips® were scanned on a high-resolution scanner using GCOS version 1.2 software (Affymetrix). Data analysis was conducted after standard Affymetrix algorithm analysis (MAS5).

Statistical methods
Statistical analyses were performed using IBM SPSS Statistics version 19.0 software (IBM, Armonk, NY, USA) and Prism software version 5.0 (GraphPad Software, La Jolla, CA, USA). A P value <0.05 was considered significantly different. Data are reported as mean ± SD. Normal distribution data between two groups were analyzed using independent-samples t tests or pairedsamples t tests, and one-way analysis of variance (ANOVA) was used to compare groups. The relationships between CD19 + CD24 − CD38 hi plasmablasts/plasma cells and clinical features were analyzed by Pearson's rank correlation test, and a P value <0.05 was considered significant. The level of gene expression was standardized by robust multiarray average and detection above background. One-way ANOVA was used to test different levels of gene expression. The Benjamini-Hochberg method was employed to determine the false discovery rate after multiple hypothesis testing. A false discovery rate <0.3 was used.

Characteristics of patients with IgG4-RD
All 42 patients were newly diagnosed, untreated patients with IgG4-RD. Their demographic features as well as clinical and laboratory manifestations are listed in Table 1. Their average age was 55 (41.5-60) years old, and the male-to-female ratio was 2.23:1. Thirty-two (76.2%) of the patients were characterized as definite IgG4-RD, 1 (2.4%) was classified as probable IgG4-RD, and 9 (21.4%) were categorized as possible IgG4-RD. The majority of patients had multiple organ involvement. Forty-one (97.6%) patients had elevated serum IgG4.
Differences in gene expression profiling among CD19 + C24 − CD38 hi plasmablasts/plasma cells, naïve B cells, memory B cells, and regulatory B cells To further analyze the characteristics of CD19 + CD24 − CD38 hi plasmablasts/plasma cells, we collected PBMCs from 15 newly diagnosed patients with IgG4-RD, and the following B cell subsets were sorted by flow cytometry: Bregs (CD19 + CD24 hi CD38 hi ), memory B cells (CD19 + CD24 + CD38 − ), naïve B cells (CD19 + CD24 int CD38 int ), and plasmablasts/plasma cells (CD19 + CD24 − CD38 hi ). Because the numbers of each B-cell subset were small, the subsets of B cells from individual patients were pooled. Total RNA was extracted for microarray analysis. Hierarchical clustering visualized by heat mapping indicated significant differences in gene expression by the four B-cell subsets (Fig. 2). Importantly, CD19 + CD24 − CD38 hi plasmablasts/plasma Among genes that were differentially upregulated in CD19 + CD24 − CD38 hi plasmablasts/plasma cells compared with naïve B cells and memory B cells, we found those that are known to alter the B-cell program and commit a B cell to plasma cell differentiation, such as interferon regulatory factor 4 (IRF4), PR domain containing 1 (PRDM1), and Xbox binding protein 1 (XBP1). Ig heavy and light chains as well as J chain were also significantly upregulated, in addition to those previously reported to be upregulated in plasma cell genes, such as VDR, CAV1, SDC1, SSR4, ERP70, PPIB, ITGA6, CD38, and others listed in Tables 2  and 3 [19][20][21][22][23][24][25][26]. Both IGK and IGL genes were upregulated, implying the polyclonal nature of the plasma cell expansion. Genes involved in plasma cell homing to tissue niches such as SDC1 were also upregulated, although CXCR4, which is also involved in homing of plasma cells to niches, was downregulated. Finally, tumor necrosis factor receptor superfamily member 17 (TNFRSF-17), which encodes BCMA, a BAFF-R involved in plasma cell survival, was Columns from left to right represent CD19 + CD24 − CD38 hi plasmablasts/plasma cells, CD19 + CD24 int CD38 int naïve B cells, CD19 + CD24 + CD38 − memory B cells, and CD19 + CD24 hi CD38 hi Bregs, respectively. Red represents overexpressed genes, and blue represents underexpressed genes greatly upregulated in CD19 + CD24 − CD38 hi plasmablasts/ plasma cells. As expected, BCR complex, CD19, CD20, CIITA, human leukocyte antigen (HLA)-DRA, and HLA-DRB1 gene expression was downregulated in CD19 + CD24 − CD38 hi plasmablasts/plasma cells. Likewise, the genes characteristic of earlier stages of B-cell development or differentiation, such as SPIB, BCL6, BLK, FYN, BCL11A, CD37, CD1C, VAV3, CCR6, and CD22, were downregulated significantly.
B-cell differentiation-related transcription factor genes in CD19 + CD24 − CD38 hi plasmablasts/plasma cells in patients with IgG4-RD The transcription factors regulating B-cell differentiation into plasmablasts/plasma cells were analyzed in the various B-cell subsets in greater detail (Fig. 3). In patients with   CD19 were significantly downregulated, compared with memory and naïve B cells (Fig. 4, Tables 2 and 3). High expression of IGHD, IGJ, IGK, and IGHM are important indicators for B-cell differentiation into plasma cells. In CD19 + CD24 − CD38 hi plasmablasts/plasma cells from patients with IgG4-RD, IGH gene expression was also markedly increased, as was that of IGK and IGL genes. In addition, the expression of CD27 and Fas/CD95 genes was significantly higher than in the other three B-cell subsets, whereas CD40 gene expression was decreased. CD23 was also markedly decreased in CD19 + CD24 − CD38 hi plasmablasts/plasma cells of patients with IgG4-RD.

Analysis of B-cell proliferation-regulating genes and homing factor genes
Marker of proliferation Ki-67 (MKI67), encoding the Ki-67 protein and denoting recent cellular proliferation, was markedly increased in CD19 + CD24 − CD38 hi plasmablasts/plasma cells compared with the other three B-cell subsets (Fig. 5). We also found that a number of genes involved in cell homing, including SELL, which encodes L-selectin (CD62L), was markedly increased, whereas expression of CCR7, CXCR5, and CXCR4 was greatly decreased, in CD19 + CD24 − CD38 hi plasmablasts/plasma cells compared with the other three B-cell subsets. CCR10 expression was also markedly increased compared with memory B cells and naïve B cells (Fig. 5).
Expression of activating factors of CD19 + CD24 − CD38 hi plasmablasts/plasma cells By flow cytometric analysis, we compared the expression of B-cell-activating factors and homing factors in different Bcell subsets in patients with IgG4-RD (Fig. 6). This analysis revealed that CD86, CD62L, HLA-DR, and CD95 were highly expressed in CD19 + C24 − CD38 hi plasmablasts/ plasma cells compared with other B-cell subsets. In particular, the majority of CD19 + CD24 − CD38 hi B cells expressed CD27 (76.46 ± 7.16%), which demonstrated that the CD19 + CD24 − CD38 hi population was a larger population that included CD19 + CD20 − CD27 + plasmablasts/plasma cells. Whereas CD138, BAFF-R, and BCMA were expressed by a subset of CD19 + CD24 − CD38 hi plasma/plasma cells, there was almost no expression of TACI, CD23, CD40, IgE, and IgD by this population.

Immunoglobulin secretion by different B-cell subsets in IgG4-RD
The CD19 + C24 − CD38 hi plasmablasts/plasma cells, Bregs, memory B cells, and naïve B cells from patients with IgG4-RD were isolated by cell sorting and cultured in vitro. At day 7, the supernatants were collected, and the secretion of immunoglobulin was tested.

Discussion
The data derived from the present study indicate that CD19 + CD24 − CD38 hi plasmablasts/plasma cells are significantly elevated in the peripheral blood of patients with IgG4-RD, and this B-cell population notably correlates with the number of organs involved, serum IgG4, IgG4-RD RI, and the ratio of IgG4/IgG. In addition, peripheral CD19 + CD24 − CD38 hi plasmablasts/ plasma cells decreased remarkably after treatment of glucocorticoids, and the clinical manifestations of patients improved accordingly. These findings are consistent with previous observations [17] and indicate that the level of CD19 + CD24 − CD38 hi plasmablasts/ plasma cells could be an important biomarker in IgG4-RD. By gene expression analysis, we found that CD19 + CD24 − CD38 hi plasmablasts/plasma cells differed greatly from the other three subsets of B cells, including naïve B cells, memory B cells, and Bregs, and expressed many genes typical of plasma cells. Considering that the gene expression of CD19 + CD24 − CD38 hi plasmablasts/plasma cells differed from that of other known B-cell subsets, we attempted to use gene expression analysis to determine their stage of plasma cell maturation. A number of typical plasma cell genes were overexpressed, including XBP1, PRDM1, IRF4, and TNFRSF17, whereas BCL6, SPIB, and PAX5 were underexpressed, which is typical of mature plasma cells. However, expression of these genes does not permit a more detailed analysis of the stage of maturation of plasma cells. It is known, however, that expression of HLA-DR can be employed to identify newly generated cells from more mature plasma cells; in this regard, CD19 + CD24 − CD38 hi plasmablasts/ plasma cells from patients with IgG4-RD exhibited bright expression of HLA-DR protein, as is typical of newly formed plasma cells [27]. Notably, however, messenger RNA (mRNA) levels of various major histocompatibility complex class II molecules were decreased, as has been noted in systemic lupus erythematosus (SLE) [22], suggesting that these plasma cells were transitioning from newly formed to more mature plasma cells that had decreased mRNA expression but retained protein expression of HLA-DR. Consistent with this, IgG4-RD plasma cells greatly overexpressed Mki-67 mRNA, which encodes the Ki-67 protein, a marker of recently divided immunoglobulin-secreting plasma cells [28] and which is increased in plasmablasts compared with memory B cells [29]. Together, the increased expression of HLA-DR protein but not mRNA and the increase in Mki-67 mRNA identify IgG4-RD plasma cells as newly generated and similar to those found in the circulation of patients with SLE [30].
A number of other mRNAs and proteins were assessed to further understand the maturation status of the IgG4-RD plasma cells. The presence of mRNA for both lambda and kappa light chains suggests that these CD19 + CD24 − CD38 hi plasmablasts/plasma cells are polyclonal, although more detailed analysis on a single-cell level would be required to confirm this. An interesting feature of CD19 + CD24 − CD38 hi plasmablasts/plasma cells was their decreased expression of CXCR4. This chemokine Fig. 8 The association between CD19 + CD24 − CD38 hi plasmablasts/plasma cells and disease activity. a and b Correlations of the ratio of CD19 + CD24 − CD38 hi cells/CD19 + B cells and the absolute number of CD19 + CD24 − CD38 hi plasmablasts/plasma cells with IgG4-related Disease Responder Index (IgG4-RD RI) before treatment. c Change of CD19 + CD24 − CD38 hi plasmablasts/plasma cells before and after treatment. d-h Ratios of CD19 + CD24 − CD38 hi cells/ CD19 + B cells, absolute number of CD19 + CD24 − CD38 hi plasmablasts/plasma cells, IgG4-RD RI, IgG, and IgG4 before and after treatment. *** P < 0.001, **** P < 0.0001 receptor uniquely recognizes CXCL12 and plays an important role in plasma cell homing to bone marrow and other niches [31,32]. The decreased expression of CXCR4 could contribute to their persistence in the circulation. A similar abnormality has been noted in SLE plasma cells [22].
To further identify the characteristics of CD19 + CD24 − CD38 hi plasmablasts/plasma cells, we analyzed the surface markers by flow cytometry, and we found that CD19 + CD24 − CD38 hi plasmablasts/plasma cells highly expressed CD86, CD62L, IL-6R, CD27, and CD95, which was consistent with those observed in gene levels. CD86 (B7-2) signaling plays a pivotal role in activating T cells [33]. Expression of CD86 on B-lineage cells has been shown to foster B-cell-T-cell collaboration and facilitate immunoglobulin production, including IgE and IgG4 [34][35][36][37][38][39]. In IgG4-RD, the CD19 + CD24 − CD38 hi plasmablasts/plasma cell subset highly expressed the CD86 molecule, suggesting the intriguing possibility that this cell subset may maintain some features of earlier B cells and, by virtue of persistent expression of CD86, chronically stimulate T-cell help and in so doing enhance the likelihood of classswitching recombination to the downstream heavy-chain isotypes IgG4 and IgE. The physiologic role of other molecules expressed by CD19 + CD24 − CD38 hi plasmablasts/ plasma cells requires delineation.
As previously reported, circulating plasmablasts identified using CD19 low CD38 + CD20 − CD27 + phenotypic markers are significantly elevated in active IgG4-RD, even in patients with normal serum IgG4 concentrations, suggesting that plasmablast counts are a potentially useful biomarker for diagnosis of IgG4-RD as well as for assessing response to treatment [13]. In our study, we found that 76.46% of CD19 + CD24 − CD38 hi cells expressed CD27, indicating that the CD19 + CD24 − CD38 hi plasmablasts/plasma cell population is a larger one that contained CD19 + CD20 − CD27 + CD38 hi cells. Similarly, the ratio of CD19 + CD24 − CD38 hi cells correlated positively with IgG4-RD RI, as did the absolute number of CD19 + CD24 − CD38 hi plasmablasts/plasma cells [17]. Although there was no statistical correlation between the magnitude of change in circulating CD19 + CD24 − CD38 hi plasmablasts/plasma cells and the change in the IgG4-RD RI, this may emerge when more patients are studied. Moreover, the significant correlation between disease activity and circulating plasmablasts/plasma cells, as well as the decrease in circulating plasma cells with therapy, suggests that this might be useful in assessing these patients and their response to treatment.

Conclusions
In this study, we found that CD19 + CD24 − CD38 hi plasmablasts/plasma cells are prominently increased in peripheral blood of untreated patients with IgG4-RD, correlating positively with serum IgG4, IgG4/IgG ratio, IgG4-RD RI, and the number of involved organs. This population decreased significantly after steroid treatment, suggesting that CD19 + CD24 − CD38 hi plasmablasts/plasma cells may be a biomarker of IgG4-RD and potentially could be useful in confirming a diagnosis, monitoring response to therapy, or assessing disease activity.