Background
Autoantibodies play an important role in the pathogenesis of many autoimmune diseases, including rheumatoid arthritis (RA) [
1] and systemic lupus erythematosus (SLE) [
2]. Classical autoantibodies target the tissue directly or via formation of immune complexes [
3]. Short-lived plasmablasts and plasma cells and long-lived plasma cells can generate high titers of autoantibodies upon activation. Long-lived plasma cells reside in bone marrow and inflammatory tissue niches and produce copious amounts of autoantibodies independent of B cell activation. Studies have shown an increased number of plasmablasts or ratio of plasmablast/B cells in the blood of patients with active SLE [
4]. Although the bone marrow provides the survival niche for long-lived plasma cells [
5,
6], inflammatory tissues bear high B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL), maintain long-lived plasma cell survival, and thus contribute to the autoantibody secretion in inflammatory joints in patients with RA [
7] and nephritic kidneys in NZB/W mice [
8]. Different therapeutic agents have been developed to target antibody production in autoimmune diseases. Rituximab (anti-CD20) and belimumab (anti-BLyS) prevent short-lived plasmablasts from proliferating and reduce autoantibody production to some degree. However, non-proliferative long-lived plasma cells are not susceptible to this effect. Short-term treatment with the proteasome inhibitor bortezomib followed by B-cell target therapy (anti-CD20) decreases anti-dsDNA-secreting plasma cells and delays the development of nephritis in NZB/W mice [
9]. Clinical studies show that bortezomib depletes plasma cells and ameliorates disease in patients with refractory SLE [
10,
11]. Inhibition of plasma cell survival via blockade of BAFF and APRIL has also been tested. Atacicept (soluble TACI-Ig) has shown no clinical response in patients with RA [
12]. Though atacicept at a lower dose (75 mg) was proved non-effective in prevention of flares in SLE in patients with moderate to severe disease, a higher dose (150 mg) proved beneficial on flare rate and time to first flare. Unfortunately, the trial was terminated due to two deaths caused by pneumonia in that study group [
12]. Aside from the strategies mentioned above, it is worthwhile to determine if plasma-cell-specific targets may prove more efficacious in the treatment of autoimmune diseases, such as RA and SLE.
CD38 is a type II glycoprotein highly expressed on plasma cells, memory B cells and multiple myeloma (MM) cells [
13]. Daratumumab, a depleting monoclonal anti-CD38 antibody, has been approved for the treatment of patients with MM [
14,
15]. Plasma cell depletion in the bone marrow has been observed in patients with MM treated with daratumumab [
16]. It is therefore reasonable to hypothesize that CD38 can be a target for the treatment of autoimmune diseases by depleting plasma cells specifically. Previous studies have shown CD38 expression in the synovial biopsies of patients with established RA [
17]. A comprehensive analysis of CD38 expression on various immune cells in patients with autoimmune diseases could facilitate the dosing possibility of depleting only plasmablasts and plasma cells without affecting other potentially beneficial cells. Furthermore, despite the fact that anti-citrullinated protein antibodies (ACPA) may already exist in pre-RA stages, no studies have analyzed the dynamic role that plasma cells and plasmablasts may play during RA disease progression, namely from arthralgia to undifferentiated arthritis (UA), early RA and established RA. It is possible that targeting CD38 at the pre-disease stage could achieve greater disease suppression in patients with RA and SLE. Therefore, in this study, we investigated the potential of targeting CD38 at various stages of RA progression. Most importantly, for the first time, we evaluated the efficacy of a U.S. Food and Drug Administration-approved cancer drug targeting CD38, daratumumab, in depleting plasma cells/plasmablasts ex vivo in PBMC from patients with RA and SLE, to provide a rationale for clinical trials of daratumumab in patients with RA and SLE.
Herein, RNA sequencing (RNA-Seq) showed that various plasma cell/plasmablast-related genes are significantly up-regulated in pre-disease (arthralgia and UA) and RA (early and established) synovial tissue biopsies compared to counterpart healthy donors or patients with osteoarthritis (OA). Furthermore, the highest CD38 expression in peripheral blood was observed on plasma cells and plasmablasts, followed by NK cells, pDCs, a regulatory T cell (Treg) subpopulation and naïve T cells in healthy donors and patients with SLE and with RA. Immunohistochemistry assessment showed the presence of plasma cells and T cells in the synovial biopsies from patients with early RA. Finally, we demonstrated that daratumumab depletes plasma cells/plasmablasts in a dose-dependent manner ex vivo.
Methods
Human subjects
The human donors (healthy controls and people with OA, arthralgia, UA, early RA and established RA) in this study have been described previously [
18]. Briefly, synovial biopsy specimens from healthy and donors with OA were obtained from a group of patients attending a sports medicine facility with knee pain. Healthy subjects were defined as those who had no arthritis, cartilage damage or synovitis on knee arthroscopy. Donors with OA had clinical history and/or examination findings suggestive of OA in addition to supportive arthroscopic findings. The average age of healthy donors was 35.2 years. The average age of donors with OA was 49.0 years. There were 13 women (59.1%) and 9 men (40.9%). Patients with arthralgia (
n = 10) were subjects with symptoms of aches and pains, without clinical signs of synovitis or significantly raised C-reactive protein (CRP) (mean = 4.49 mg/l) at first assessment, but with positive circulating rheumatoid factor (RF
+) and ACPA. There were eight women and two men, and the mean age was 51.6 years (range 34–66 years). Patients with undifferentiated arthritis/inflammatory arthritis (UA/IA) (
n = 6) were defined as subjects presenting with clinical signs of synovitis, but who did not meet the 2010 American College of Rheumatology (ACR) criteria for RA. All six patients were female, the mean age was 46 years, and they had significantly raised CRP (mea
n = 17.66 mg/l). Early RA (
n = 57) was defined as within 12 months of disease diagnosis without prior small or large molecule disease modifying anti-rheumatic drugs (DMARDs) usage (mean disease duratio
n = 5 months). Early RA synovial tissue biopsies were collected from two different cohorts of patients identified at Flinders University and Queen Mary University of London. There were 33 women and 22 men, and the mean age was 55.9 years. Established RA (
n = 95; disease duration >1 year) synovial tissue biopsies were collected from two different cohorts of patients identified at Queen Mary University of London and St. Vincent’s Hospital. The average disease duration in this patient population was 68 months. The average age in the group was 54.0 years. All patients with established RA had received small molecule DMARDs or anti-TNFα treatments. More details about the clinical characteristics of these subjects included in this analysis can be found in a report of a previous study [
18].
PBMC samples for CD38 expression analysis from patients with SLE and RA and from healthy donors were acquired from Bioreclamation (Westbury, NY, USA) and Precision for Medicine (Frederick, MD, USA). All donors with SLE were clinically active at the most recent visit into the clinic before the blood draw and under standard of care treatments, including prednisone, benlysta (n = 1), rituximab (n = 1) and other small molecule therapeutics. One patient was treatment-naïve at the time of the blood draw. The mean SLE disease activity index (SLEDAI) scores in these subjects was 18.7 (range 6–32), which indicates active disease. All donors with RA were clinically active at the time of blood draw and had received various DMARD treatments. The biologic treatments included Orencia (n = 1), Simponi (n = 1), Simponi Aria (n = 1), Humira (n = 1), and Enbrel (n = 3). All subjects had received small molecule DMARD treatments before the blood draw.
All protocols for collecting synovial biopsies and blood/serum were approved by the Institutional Review Board. All patients signed the consent form for participating in the study.
RNA-Seq gene expression analysis
RNA-Seq analysis was performed on the same synovial biopsies using the same algorithm as reported previously and the RNA-Seq data were deposited in the Gene Expression Omnibus (GEO) database as part of a previous study [GEO:GSE89408] [
18].
Total RNA was extracted from frozen synovial biopsies from patients with RA and only RNA samples that passed quality control by an Agilent Bioanalyzer were further analyzed. RNA-Seq was performed by Q2 Solutions (Morrisville, NC, USA). Sequencing libraries were prepared using TruSeq Stranded Total RNA RiboZero protocol from Illumina. Libraries were pooled and sequenced with an Illumina HiSeq 2000 with paired-end 100-bp flow cells. FastQC was used to evaluate raw read quality.
Reads were trimmed for adaptors and sequence quality. The average number of clusters (post-trimming) per sample was 8.9 × 10
7. Trimmed reads were aligned to the human b37.3 reference genome using the STAR v2.4 aligner [
19]. Aligned reads were quantified using RSEM v1.2.14 algorithm [
20] with UCSC transcriptome model (accessed on 17 March 2014) that included long intergenic non-coding RNAs (lincRNAs) from Ensembl v75. This transcriptome model has a total of 34,495 genes and 88,933 isoforms. Aligned data were evaluated for quality using several metrics (e.g., mapping rate, coverage) and visually inspected for deviation from the population across multiple metrics and principal components analysis. Statistical testing of RNA-Seq data was performed in R with the “limma” package [
21]. Counts were converted to log2 counts per million, quantile normalized and precision weighted. A linear model was fitted to each gene, and empirical Bayes moderated
t statistics were used to assess differences in expression.
Fluorescence-activated cell sorting (FACS) analysis
PBMC samples were analyzed in three different staining panels for CD38 expression as follows: Panel 1: CD38-FITC, CD14-PE, HLA-DR-PerCPCy5.5, CD11b-PECy7, CD33-APC, BDCA2-VioBlue, CD16-BV510, Lineage (CD3/CD8/CD4/CD19)-BV605, CD45-BV650, CD11c-BV711 and CD56-BV786. Panel 2: CD38-FITC, CD62L-PE, CCR7-PerCPCy5.5, CD27-PECy7, CD4-APC, CD127-BV421, CD8-BV510, CD3-BV605, CD25-BV650 and CD45RA-BV786. Panel 3: CD38-FITC, BCMA-PE, CD24-PerCPCy5.5, IgD-PECy7, CD20-APC, CD27-BV421, IgM-BV510, CD138-BV605, CD3-BV650, CD56-BV650 and CD19-BV711. For the ex vivo depletion assay, a different panel was used to measure NK cells and plasma cells/plasmablast in one panel as follows. Panel: CD38-FITC, CD138-PE, IgD-PECy7, CD20-APC, Live-Dead/Near-IR, CD27-Pacific Blue, CD3-BV605, CD56-BV650, and CD19-BV711. All antibodies were purchased from BD Bioscience except for the following: CD27-BV421, CD138-PE, CD56-BV650, BCMA-PE (Biolegend) and BDCA2-VioBlue (Miltenyi). For the analysis of CD38 expression on PBMC, CD38-FITC (Catalog number: CYT-38F2) was purchased from Cytognos (Salamanca, Spain). In the depletion assay, CD38 expression was analyzed using HuMax-003-FITC (Genmab/Janssen R&D), a monoclonal antibody (Ab) that binds to a different epitope than daratumumab, as described previously [
22]. Isotype controls and/or FMOs (Fluorescence Minus One) were used to determine gating boundaries. Samples were acquired using the LSRII (BD Bioscience) and analyzed using FlowJo software (Treestar Inc.).
Immunohistochemistry analysis (IHC)
Frozen sections of synovial biopsies were in optimal cutting temperature compound and taken at 4-μm thickness for evaluation of protein markers in the samples by IHC. Tissue sections were briefly fixed in methanol, air dried and incubated with either anti-CD38 (SP149) (Cell Marque, Rocklin, CA, USA), anti-CD3 (2GV6) (Ventana, Tuscon, AZ, USA) or anti-CD138 (B-A38) (Abcam, Cambridge, MA, USA). The markers were visualized with 3,3′-diaminobenzidine and the relative expression of each marker was noted by JA and MS while blinded to the specimen group information. The scoring paradigm describes the observed expression of each marker as either abundant, moderate or low as determined by the number of positive cells counted within a total magnification of × 100 across the entire biopsy section. Abundant denotes strongly positive clusters of more than 20 cells in any field, moderate presence if between 5 and 20 and low if fewer than 5 cells were observed per field. The observation in the highest density field in a sample was recorded as the score. A pathologist, JA, reviewed indicated areas of inflammation and hyperplasia.
Daratumumab depletion assay
PBMC samples from healthy controls and patients with RA or SLE were thawed in complete Roswell Park Memorial Institute medium (RPMI) and rested overnight before the assay. Cells were then cultured in 96-well U-bottom plates at 2.5 × 10
5 cells/well. Human serum (Complement Technology, TX, USA) was applied at 10% to the culture as a source of complement. Daratumumab was added to the wells at concentrations indicated in the text. Isotype control, IgG1-b12 (Genmab), a human monoclonal Ab against an innocuous antigen (HIV1-gp120) [
22], was applied at 1 μg/ml. Cells were cultured for 72 h before FACS, quantitative (q)PCR or branced DNA (bDNA) analysis. CountBright absolute counting beads (Life Technologies, CA, USA) were added to the samples before acquisition on A flow cytometer for quantification of each cell population. RNA was prepared from the same culture for qPCR analysis of immunoglobulin J (
IGJ).
RNA, qPCR and bDNA assay
RNA was prepared using Qiagen RNeasy kits (Qiagen) for qPCR analysis of IGJ. Complementary DNA (cDNA) was prepared using SuperScript IV VILO MasterMix and TaqMan qPCR assay with TaqMan Universal PCR Master Mix and samples were run on the VIIA7 instrument (Applied Biosystems). Primers include IGJ (Hs00376160_m1) and housekeeping genes IPO8 (Hs00183533_m1) and GUSB (4332655). All cDNA and TaqMan reagents were purchased from Life Technologies (CA, USA).
Alternatively, a bDNA assay was used to measure IGJ messenger RNA (mRNA). After incubation, cells were pelletted and triplicate wells were pooled in 100 μl PBS and then pelleted again. Following the QuantiGene sample processing protocol for PBMC, each well was resuspended in 200 μl working lysis buffer and incubated for 30 min at 50–55 °C. Cell lysates were then stored at − 80 °C until needed for assay. A custom Quantigene Plex assay containing endogenous controls TBP and HPRT1 and the target gene IGJ was run according to the manufacture’s manual and samples were analyzed on a Luminex 200 instrument (BioRad). For the fold-change (FC) of IGJ mRNA in the presence of daratumumab, the relative expression level of IGJ was normalized to IGJ mRNA in the isotype control group in some analyses.
Statistics
For high-dimensional RNA-Seq, features were considered differentially expressed if they satisfied a 1.5-fold change and 0.05 adjusted p value cutoff unless otherwise specified. The Benjamini-Hochberg method was used to calculate p values adjusted for multiple hypotheses. Fold changes were calculated from log2(FC) estimates and reported with a positive sign for ratios greater than 1 (log2(FC) >0) and with a negative sign for ratios less than 1 (log2(FC) <0). Data from flow cytometry phenotyping and depletion experiments were analyzed using GraphPad™ Prism software (v7). One way analysis of variance (ANOVA) with Tukey’s test for multiple comparisons was performed to compare cohorts and cell types.
Discussion
CD38 has been extensively studied as a target for the treatment of patients with MM and daratumumab has shown outstanding efficacy in this patient population [
14‐
16]. However, the application of this potential plasma cell/plasmablast-depleting agent such as daratumumab in the treatment of autoimmune disease has not been studied, especially in early stage disease. In this study, using integrative analysis of synovial tissue biopsies obtained from different stages of RA disease and PBMC, we have shown that 1)
CD38 and plasma cell/plasmablast-related genes are up-regulated in ACPA
+RF
+ arthralgia and UA disease stages before the onset of RA; 2) CD38 is expressed at the highest level on plasma cells compared to other immune cell populations in RA, SLE and healthy donors in the peripheral blood; and most importantly 3) daratumumab effectively depletes plasma cells/plasmablast in SLE and RA PBMC ex vivo.
Plasma cells and autoantibodies are important in RA pathogenesis as indicated by ACPA production in patients with RA and the correlation between ACPA titer and disease activity score [
30]. Staining for CD38 has also been observed in synovial biopsies from patients with established RA [
31]. However, there has been no focus on subjects with pre-RA disease (arthralgia and UA) and treatment-naïve patients with early RA. Herein, we showed that the expression of major genes related to plasma cells and plasmablast development and survival is significantly heightened in synovial biopsies from donors with arthralgia and UA compared to healthy donors and those with OA. These include
CD38,
XBP1,
IGJ and
TNFSF13B,
IRF4 and
PRDM1, and their up-regulation is observed in synovial biopsies from donors with UA, early RA and established RA. These data indicate that even before the onset of clinically classifiable RA disease, there may be autoantibody-producing plasma cells in the joints, which may drive the progression of the disease. This is in keeping with previous data showing the presence of autoreactive plasma cells in synovial ectopic lymphoid structures (ELS) driving local production of ACPA antibodies [
32]. In addition, in the same paper it was shown that ELS act as self-sustained survival niches for plasma cells, as ELS-positive but not ELS-negative synovial tissue displayed a sustained capacity to produce class-switched ACPA when transplanted into SCID mice in the absence of new cells infiltrating the grafts [
32]. Thus, the functional presence of plasmablasts/cells in synovial niches together with the high expression of genes involved in their development and survival indicates that therapeutic targeting of antibody-producing plasma cells/plasmablasts with these molecules, in particular CD38, could be a viable strategy for disease intervention in patients with arthralgia to prevent progression to established RA. This notion is further supported by recent observations demonstrating increased levels of circulating IgA plasmablasts in seropositive (ACPA
+) subjects without any clinical signs of disease [
33]. The absence of
IRF4 and
PRDM1 up-regulation in the joints of patients with arthralgia may indicate three possibilities. First, there are not yet enough plasma cells in the joints of these patients to allow for the detection of these changes in transcription factor expression compared to healthy controls. Second, modest changes in these master transcription factors may indeed reflect a decent number of plasma cells/plasmablasts to initiate the disease progression. Third, at this early stage of the disease progression, plasma cells/plasmablasts may migrate into the joints from the periphery and de novo differentiation/development has not yet occurred. Further studies are warranted to determine if plasma cells/plasmablasts are the earliest detectable immune cells in the joints of arthralgia patients and justify the clinical testing of CD38-depleting agents in this patient population.
The above reported IHC results indicate that plasma cells and T cells are dominant in the synovial tissues of treatment-naïve patients with early RA. The data also show that CD38 staining is in the same region as cells that are neither T cells nor plasma cells, which could be macrophages. Therefore, CD38-targeted therapeutics may decrease other pathogenic cells in the synovial tissue while preferentially depleting plasma cells. More careful monitoring of clinical data is warranted to determine the target immune populations in patients with RA or SLE.
We have also shown that plasma cells and plasmablasts can be effectively depleted ex vivo in PBMC samples from donors with SLE or RA and healthy donors. This provides the rationale for depleting the plasma cells/plasmablasts specifically in patients with RA and SLE. NK cells have been reported to decrease significantly in patients with cancer treated with daratumumab [
34]. Although it is possible that NK cells become depleted in patients with RA, we saw more robust plasma cell/plasmablast depletion ex vivo in donors with SLE or RA and healthy donors. Therefore, we may be able to find a dose of daratumumab, potentially much lower than the clinical dose in MM, which selectively depletes plasma cells/plasmablasts without significantly affecting NK cells in the same patient. The remarkable decrease in plasma cells and plasmablasts in peripheral blood may serve as a surrogate biomarker for the pharmacodynamic efficacy measurement. Furthermore, the down-regulation of
IGJ mRNA confirms the depletion of plasma cells upon daratumumab treatment. The dose-response with daratumumab shown in donor PBMCs suggests
IGJ as a potential surrogate biomarker for plasma cell depletion. Consistent with previous studies showing
IGJ as a hallmark gene for plasma cells [
23], we observed positive correlation between the number of plasma cells and
IGJ mRNA in all conditions ex vivo (Fig.
6a and
b). The positive correlation between the changes in plasma cell number and change in
IGJ mRNA reinforces the notion of using
IGJ mRNA as a pharmacodynamic biomarker for daratumumab (Fig.
6f). The donors with more plasma cells at baseline and thus higher
IGJ mRNA had more readily measurable dose response to daratumumab-mediated depletion ex vivo than the donors with fewer plasma cells and lower
IGJ mRNA (Figs.
5 and
6, and data not shown). However, the lack of correlation between the number of plasma cells and maximal
IGJ mRNA down-regulation level may be due to the high antibody-dependent cellular cytotoxicity (ADCC) activity of daratumumab and the small number of cells
, and therefore very efficient depletion in all donors ex vivo. Nevertheless, the dose response suggests that
IGJ mRNA level may also serve as a patient stratification marker as we enter the era of personalized medicine, i.e. only patients with higher
IGJ mRNA and thus a more plasma cells may be treated with daratumumab or other anti-CD38 therapeutic antibodies. The application of
IGJ as biomarker can potentially facilitate clinical development of anti-CD38 in autoimmune diseases. This is consistent with the finding that identified
IGJhi as a biomarker in patients with RA who may not benefit from B cell depletion therapy with rituximab [
29]. Plasma cell depleting agents such as daratumumab may prove efficacious in this patient population.
Given the high unmet need for deeper and long-lasting responses in RA and SLE, it is important to develop therapies with more specificity for patient subpopulations based upon selective dysregulated pathways. Patient stratification based on plasma cell and plasmablast analysis may prove more efficacious in clinical testing of anti-CD38 therapeutic agents in RA and SLE. Studies are warranted to determine if daratumumab or other anti-CD38 antibody-based therapies have efficacy in pre-disease states, such as arthralgia and UA, by delaying or preventing the clinical course of disease progression. In summary, our results indicate that plasma cell/plasmablast depleting mechanisms can be used not only for the treatment of established RA but also for the prevention of the disease progression to RA. Furthermore, a plasma cell/plasmablast depleting agent, such as daratumumab, may also show efficacy in other autoantibody-dependent indications, like SLE, IgG4-related disease, multiple sclerosis, myasthenia gravis, primary Sjogren’s syndrome and so on. Finally, in a manner analogous to pre-RA disease, SLE is also considered to have a natural course of evolution with pre-disease state(s) exhibiting asymptomatic autoimmunity [
35]. An anti-CD38 or other plasma cell/plasmablast targeting agent may also prevent/delay the progression of asymptomatic autoimmunity to a clinically classifiable lupus stage.