Plasmablast-like Phenotype Among Antigen-Experienced CXCR5-CD19low B Cells in Systemic Lupus Erythematosus.

OBJECTIVE: Altered composition of the B cell compartment in the pathogenesis of systemic lupus erythematosus (SLE) is characterized by expanded plasmablast and IgD-CD27- double-negative B cell populations. Previous studies showed that double-negative B cells represent a heterogeneous subset, and further characterization is needed. METHODS: We analyzed 2 independent cohorts of healthy donors and SLE patients, using a combined approach of flow cytometry (for 16 healthy donors and 28 SLE patients) and mass cytometry (for 18 healthy donors and 24 SLE patients) and targeted RNA-Seq analysis. To compare B cell subset formation during the acute immune response versus that during autoimmune disease, we investigated healthy donors at various time points after receipt of the BNT162b2 messenger RNA COVID-19 vaccine and patients with acute SARS-CoV-2 infection, using flow cytometry. RESULTS: We found that IgD-CD27+ switched and atypical IgD-CD27- memory B cells, the levels of which were increased in SLE patients, represented heterogeneous populations composed of 3 different subsets each. CXCR5+CD19intermediate , CXCR5-CD19high , and CXCR5-CD19low populations were found in the switched memory and double-negative compartments, suggesting the relatedness of IgD-CD27+ and IgD-CD27- B cells. We characterized a hitherto unknown and antigen-experienced CXCR5-CD19low subset that was enhanced in SLE patients, had a plasmablast phenotype with diminished B cell receptor responsiveness, and expressed CD38, CD95, CD71, PRDM1, XBP1, and IRF4. Levels of CXCR5-CD19low subsets were increased and correlated with plasmablast frequencies in SLE patients and in healthy donors who received BNT162b2, suggesting their interrelationship and contribution to plasmacytosis. The detection of CXCR5-CD19low B cells among both CD27+ and CD27- populations calls into question the role of CD27 as a reliable marker of B cell differentiation. CONCLUSION: Our data suggest that CXCR5-CD19low B cells are precursors of plasmablasts. Thus, cotargeting this subset may have therapeutic value in SLE.

BTLA Expression and Function Are Impaired on SLE B Cells.

B- and T-lymphocyte attenuator (BTLA/CD272) is an inhibitory checkpoint molecule expressed on T and B cells. Prior studies reported defective function of BTLA by T cells in patients with systemic lupus erythematosus (SLE), whereas nothing is known about its role on B cells in SLE, a disease with various B cell abnormalities. Peripheral blood mononuclear cells (PBMCs) from 23 healthy donors (HD) and 34 SLE patients were stained for BTLA and its expression on B cells was assessed. PBMCs or CD27-IgD+ naive B cells were stimulated together with an activating anti-BTLA antibody or an inhibitor of spleen tyrosine kinase (SYK) and differentiation as well as the expression of activation markers CD71, PD-1 and CD86 were analyzed. Our phenotypic and functional studies revealed reduced BTLA expression on CD27-IgD+ naïve B cells from SLE patients (p=0.0017) related to anti-dsDNA antibody titers (p=0.0394) and SIGLEC-1/CD169 expression on monocytes (p=0.0196), a type I interferon marker related to disease activity. BTLA engagement was found to control CpG/TLR9 activation limiting plasmablast (p=0.0156) and B cell memory induction (p=0.0078) in normal B cells in contrast to other B cell activation pathways (CD40, BCR). These BTLA functions were impaired in SLE B cells. Inhibition of SYK was found to mimic the effects of BTLA activity in vitro. Thus, is it possible that reduced BTLA expression and function of CD27-IgD+ antigen- and T cell-inexperienced SLE B cells could be overcome by SYK inhibition which should be tested in future studies as potential therapeutic principle.

Enhanced Programmed Death 1 and Diminished Programmed Death Ligand 1 Up-Regulation Capacity of Post-Activated Lupus B Cells.

OBJECTIVE: To assess the expression of programmed death 1 (PD-1), PD ligand 1 (PD-L1), and PD-L2 by B cells from patients with systemic lupus erythematosus (SLE) at baseline and after in vitro stimulation and to analyze their functional relationship to B cell proliferation. METHODS: Peripheral blood mononuclear cells obtained from 29 SLE patients and 27 healthy donors were stimulated with interleukin-2 (IL-2)/IL-10, anti-B cell receptor (anti-BCR), CpG, and CD40L alone or in combination. Expression of PD-1, PD-L1, and PD-L2 on defined B cell subsets as well as on CD3+ T cells was analyzed by flow cytometry at baseline and after 48 hours of stimulation. Additionally, after 48 hours of stimulation, CD71 was evaluated as a proliferation marker on CD19+CD20+ B cells. RESULTS: Increased PD-1 expression was characteristic of unstimulated lupus B cells and T cells. Upon stimulation of B cells with IL-2/IL-10, anti-BCR, CpG, and CD40L for 48 hours, the capacity of SLE B cells to up-regulate PD-L1 expression was substantially diminished (P = 0.0006) along with reduced B cell proliferation (P = 0.0039). Reduced PD-L1 expression was inversely correlated with the presence of the interferon signature (r = -0.8571, P < 0.0001) and the clinical SLE Disease Activity Index score (r = -0.5696, P = 0.0087). CONCLUSION: Post-activated, hyporesponsive lupus B cells are characterized by a phenotype of increased PD-1, functionally diminished PD-L1 up-regulation capacity, and reduced proliferation upon stimulation.

Circulating Pentraxin3-Specific B Cells Are Decreased in Lupus Nephritis.

Background: Pentraxin3 (PTX3) is overexpressed in kidneys of patients developing lupus nephritis (LN). Active LN is associated with reduced anti-PTX3 antibodies. However, abnormalities of B cell differentiation against PTX3 have not been characterized in systemic lupus erythematosus (SLE). Objective: Characterization of PTX3-specific (PTX3+) B cells in peripheral blood of SLE patients with or without LN and healthy donors (HD). Patients and Methods: SLE patients without LN, biopsy-proven LN and matched HD were analyzed. Active LN was defined as proteinuria>0.5 g/day or CrCl<60 ml/min/1.73 m2 with active urinary sediment. Peripheral B cells were analyzed for direct PTX3 binding by flow cytometry using PTX3 labeled with cyanine 5 (Cy5) and phycoerythrin (PE). Results: Initially, a flow cytometry based assay to identify PTX3+ B cells was developed by demonstrating simultaneous binding of PTX3-Cy5 and PTX3-PE. Specificity of B cells was validated by blocking experiments using unlabeled PTX3. We could identify circulating PTX3+ B-cells in HD and patients. Notably, LN patients showed a significantly diminished number of PTX3+ B cells (SLE vs. LN p = 0.033; HD vs. LN p = 0.008). This decrease was identified in naïve and memory B cell compartments (naïve: SLE vs. LN p = 0.028; HD vs. LN p = 0.0001; memory: SLE vs. LN p = 0.038, HD vs. LN p = 0.011). Conclusions: Decreased PTX3+ B cells in LN within the naïve and memory compartment suggest their negative selection at early stages of B cell development potentially related to a decreased regulatory function. PTX3+ B cells could candidate for autoantigen-defined regulatory B cells as a striking abnormality of LN patients.

High maternal expression of SIGLEC1 on monocytes as a surrogate marker of a type I interferon signature is a risk factor for the development of autoimmune congenital heart block.

OBJECTIVES: Autoimmune congenital heart block (CHB) is associated with placental transcytosis of maternal autoantibodies directed against Ro/SS-A and La/SS-B. However, only about 2% of children born to mothers with the respective antibodies are affected, indicating that further risk factors exist, which are not yet fully understood. In this study, we investigated whether a maternal type I interferon (IFN) signature represents a risk factor for the development of CHB. METHODS: Blood samples, clinical data and serological parameters from 9 women with CHB pregnancies, 14 pregnant women with antibodies against Ro/SS-A but without a CHB complication and another 30 healthy pregnant women as controls were studied. SIGLEC1 expression was measured by flow cytometry and was correlated to plasma IFN-α levels measured by ELISA, and IFN-γ-induced protein 10 (IP-10) levels measured by Bio-Plex technique. RESULTS: Mothers of affected children had a significantly higher expression of SIGLEC1 (p=0.0034) and IFN-α (p=0.014), but not of IP-10 (p=0.14, all MWU) compared to mothers of unaffected children. SIGLEC1 and IFN-α expression were reduced by hydroxychloroquine and oral glucocorticoids. CONCLUSIONS: High expression of SIGLEC1 in pregnant women with autoantibodies against Ro/SS-A indicates an enhanced risk for CHB development, and these women may benefit especially from IFN-α directed therapy, for example with hydroxychloroquine.

SIGLEC1 is a biomarker of disease activity and indicates extraglandular manifestation in primary Sjögren's syndrome.

OBJECTIVES: To evaluate the interferon (IFN) biomarkers sialic acid binding Ig like lectin 1 (SIGLEC1, CD169) and IFN-γ-inducible protein-10 (IP-10) in patients with primary Sjögren's syndrome (pSS). METHODS: 31 patients fulfilling the American-European criteria for pSS were included. Disease activity was obtained by EULAR Sjögren's syndrome disease activity index (ESSDAI). SIGLEC1 expression on monocytes was analysed using flow cytometry. IP-10 concentrations were determined using Bioplex human Cytokine 27-plex kit. Spearman rank test (SRT) was used for correlation analysis and Mann-Whitney U (MWU) to test for differences between glandular and extraglandular manifestations. RESULTS: An activated IFN system was detected by an upregulation of SIGLEC1 expression in 64.5% and by elevated serum level of IP-10 in 78.9% of our patients with pSS. In a subsequent analysis SIGLEC1 expression was found to be upregulated more frequently in patients with extraglandular manifestations (16/16, 100%) compared to patients with exclusively glandular involvement (4/15, 27%). SIGLEC1 expression could significantly discriminate between these two disease subgroups (p=0.0001, MWU) with a positive predictive value (PPV) of 80% for extraglandular disease. Moreover, the expression correlated with disease activity (p=0.005, r=0.54, SRT). Serum IP-10 levels neither differed significantly between glandular and extraglandular disease nor correlated with ESSDAI. CONCLUSIONS: Our results indicate that increased SIGLEC1 expression characterises patients with systemic involvement and high disease activity. Therefore, SIGLEC1 determination might be of value for subset definition, risk stratification and differential therapeutic considerations in pSS.

Tissue distribution and dependence of responsiveness of human antigen-specific memory B cells.

Memory B cells (mBCs) are a key to immunologic memory, yet their distribution within lymphoid organs and the individual role of these for mBC functionality remain largely unknown. This study characterized the distribution and phenotype of human (Ag-specific) mBCs in peripheral blood (PB), spleen, tonsil, and bone marrow. We found that the spleen harbors most mBCs, followed by tonsils, BM, and PB, and we detected no major differences in expression of markers associated with higher maturity. Testing the distribution of tetanus toxoid-specific (TT(+)) mBCs revealed their presence in PB during steady state, yet absolute numbers suggested their largest reservoir in the spleen, followed by tonsils. To explore the role of both tissues in the maintenance of reactive B cell memory, we revaccinated controls and splenectomized and tonsillectomized individuals with TT. All donor groups exhibited comparable emergence of anti-TT IgG, TT(+) plasma cells, and TT(+) mBCs in the PB, together with similar molecular characteristics of TT(+) plasma cells. In summary, human mBCs recirculate through PB and reside in different lymphoid organs that do not reflect different mBC maturity stages. The spleen and tonsil, although harboring the largest number of overall and TT(+) mBCs, appear to be dispensable to preserve adequate responsiveness to secondary antigenic challenge.

The anti-CD74 humanized monoclonal antibody, milatuzumab, which targets the invariant chain of MHC II complexes, alters B-cell proliferation, migration, and adhesion molecule expression.

INTRODUCTION: Targeting CD74 as the invariant chain of major histocompatibility complexes (MHC) became possible by the availability of a specific humanized monoclonal antibody, milatuzumab, which is under investigation in patients with hematological neoplasms. CD74 has been reported to regulate chemo-attractant migration of macrophages and dendritic cells, while the role of CD74 on peripheral naïve and memory B cells also expressing CD74 remains unknown. Therefore, the current study addressed the influence of milatuzumab on B-cell proliferation, chemo-attractant migration, and adhesion molecule expression. METHODS: Surface expression of CD74 on CD27- naïve and CD27+ memory B cells as well as other peripheral blood mononuclear cells (PBMCs) obtained from normals, including the co-expression of CD44, CXCR4, and the adhesion molecules CD62L, ß7-integrin, ß1-integrin and CD9 were studied after binding of milatuzumab using multicolor flow cytometry. The influence of the antibody on B-cell proliferation and migration was analyzed in vitro in detail. RESULTS: In addition to monocytes, milatuzumab also specifically bound to human peripheral B cells, with a higher intensity on CD27+ memory versus CD27- naïve B cells. The antibody reduced B-cell proliferation significantly but moderately, induced enhanced spontaneous and CXCL12-dependent migration together with changes in the expression of adhesion molecules, CD44, ß7-integrin and CD62L, mainly of CD27- naïve B cells. This was independent of macrophage migration-inhibitory factor as a ligand of CD74/CD44 complexes. CONCLUSIONS: Milatuzumab leads to modestly reduced proliferation, alterations in migration, and adhesion molecule expression preferentially of CD27- naïve B cells. It thus may be a candidate antibody for the autoimmune disease therapy by modifying B cell functions.

Epratuzumab targeting of CD22 affects adhesion molecule expression and migration of B-cells in systemic lupus erythematosus.

INTRODUCTION: Epratuzumab, a humanized anti-CD22 monoclonal antibody, is under investigation as a therapeutic antibody in non-Hodgkin's lymphoma and systemic lupus erythematosus (SLE), but its mechanism of action on B-cells remains elusive. Treatment of SLE patients with epratuzumab leads to a reduction of circulating CD27(negative) B-cells, although epratuzumab is weakly cytotoxic to B-cells in vitro. Therefore, potential effects of epratuzumab on adhesion molecule expression and the migration of B-cells have been evaluated. METHODS: Epratuzumab binding specificity and the surface expression of adhesion molecules (CD62L, ß7 integrin and ß1 integrin) after culture with epratuzumab was studied on B-cell subsets of SLE patients by flow cytometry. In addition, in vitro transwell migration assays were performed to analyze the effects of epratuzumab on migration towards different chemokines such as CXCL12, CXCL13 or to CXCR3 ligands, and to assess the functional consequences of altered adhesion molecule expression. RESULTS: Epratuzumab binding was considerably higher on B-cells relative to other cell types assessed. No binding of epratuzumab was observed on T-cells, while weak non-specific binding of epratuzumab on monocytes was noted. On B-cells, binding of epratuzumab was particularly enhanced on CD27(negative) B-cells compared to CD27(positive) B-cells, primarily related to a higher expression of CD22 on CD27(negative) B-cells. Moreover, epratuzumab binding led to a decrease in the cell surface expression of CD62L and ß7 integrin, while the expression of ß1 integrin was enhanced. The effects on the pattern of adhesion molecule expression observed with epratuzumab were principally confined to a fraction of the CD27(negative) B-cell subpopulation and were associated with enhanced spontaneous migration of B-cells. Furthermore, epratuzumab also enhanced the migration of CD27(negative) B-cells towards the chemokine CXCL12. CONCLUSIONS: The current data suggest that epratuzumab has effects on the expression of the adhesion molecules CD62L, ß7 integrin and ß1 integrin as well as on migration towards CXCL12, primarily of CD27(negative) B-cells. Therefore, induced changes in migration appear to be part of the mechanism of action of epratuzumab and are consistent with the observation that CD27(negative) B-cells were found to be preferentially reduced in the peripheral blood under treatment.

Steady-state generation of mucosal IgA+ plasmablasts is not abrogated by B-cell depletion therapy with rituximab.

The anti-CD20 antibody rituximab depletes human B cells from peripheral blood, but it remains controversial to what extent tissue-resident B cells are affected. In representative patients with rheumatoid arthritis, we here demonstrate that recently activated presumably short-lived plasmablasts expressing HLA-DR(high) and Ki-67 continuously circulate in peripheral blood after B-cell depletion by rituximab at 26%-119% of their initial numbers. They circulate independent of splenectomy, express immunoglobulin A (IgA), ß7 integrin, and C-C motif receptor 10 (CCR10) and migrate along CCL28 gradients in vitro, suggesting their mucosal origin. These plasmablasts express somatically hypermutated V(H) gene rearrangements and spontaneously secrete IgA, exhibiting binding to microbial antigens. Notably, IgA(+) plasmablasts and plasma cells were identified in the lamina propria of patients treated with rituximab during peripheral B-cell depletion. Although a relation of these "steady state"-like plasmablasts with rheumatoid arthritis activity could not be found, their persistence during B-cell depletion indicates that their precursors, that is, B cells resident in the mucosa are not deleted by this treatment. These data suggest that a population of mucosal B cells is self-sufficient in adult humans and not replenished by CD20(+) B cells immigrating from blood, lymphoid tissue, or bone marrow, that is, B cells depleted by rituximab.

Secondary immunization generates clonally related antigen-specific plasma cells and memory B cells.

Rechallenge with T cell-dependent Ags induces memory B cells to re-enter germinal centers (GCs) and undergo further expansion and differentiation into plasma cells (PCs) and secondary memory B cells. It is currently not known whether the expanded population of memory B cells and PCs generated in secondary GCs are clonally related, nor has the extent of proliferation and somatic hypermutation of their precursors been delineated. In this study, after secondary tetanus toxoid (TT) immunization, TT-specific PCs increased 17- to 80-fold on days 6-7, whereas TT-specific memory B cells peaked (delayed) on day 14 with a 2- to 22-fold increase. Molecular analyses of V(H)DJ(H) rearrangements of individual cells revealed no major differences of gene usage and CDR3 length between TT-specific PCs and memory B cells, and both contained extensive evidence of somatic hypermutation with a pattern consistent with GC reactions. This analysis identified clonally related TT-specific memory B cells and PCs. Within clusters of clonally related cells, sequences shared a number of mutations but also could contain additional base pair changes. The data indicate that although following secondary immunization PCs can derive from memory B cells without further somatic hypermutation, in some circumstances, likely within GC reactions, asymmetric mutation can occur. These results suggest that after the fate decision to differentiate into secondary memory B cells or PCs, some committed precursors continue to proliferate and mutate their V(H) genes.

Activated memory B cell subsets correlate with disease activity in systemic lupus erythematosus: delineation by expression of CD27, IgD, and CD95.

OBJECTIVE: Analysis of peripheral B cell subsets in patients with systemic lupus erythematosus (SLE) has provided evidence of specific alterations, such as an expansion of CD27++ plasma cells/blasts and transitional B cells. However, memory B cells in lupus have not been thoroughly investigated, and only recently a CD27- memory B cell subset was identified in the peripheral blood of lupus patients. Focusing on CD27- B cells, this study aimed to identify abnormalities in peripheral B cell subsets in patients with SLE. METHODS: Three independent cohorts of lupus patients were used to characterize CD27- memory B cells, using multiparameter flow cytometry and single-cell reverse transcription-polymerase chain reaction of heavy-chain transcripts. RESULTS: We identified a homogeneous subset of CD27-,IgD-,CD95+ memory B cells with an activated phenotype that was increased in patients with disease flares and that correlated with disease activity and serologic abnormalities. In contrast, the entire subset of CD27-,IgD- B cells was found to be heterogeneous, did not correlate significantly with lupus activity, and was also increased in patients with bacterial infections. CONCLUSION: We conclude that CD95 is a useful marker to identify CD27- memory B cells with an activated phenotype, which might serve as a biomarker for lupus activity and as a target of further investigations aiming to elucidate the pathogenic potential of these cells and the mechanisms involved in the generation as well as regulation of this CD27-,IgD-,CD95+ memory B cell subset.

Cryopreserved human B cells as an alternative source for single cell mRNA analysis.

Reverse transcription-polymerase chain reaction (RT-PCR) of individual B-lymphocytes has been shown to be a powerful tool for the simultaneous analysis of different mRNA specificities in both malignant and non-malignant B cell subpopulations. However, especially for longitudinal studies, this may also require analyses of cryopreserved cells. Therefore, the current study assessed whether cryopreserved (liquid nitrogen, dimethyl sulfoxide [DMSO]-stored) viable B cells are an alternative source for single cell RT-PCR analysis. Fresh (non-frozen) and post-thawed human peripheral blood B cells were analyzed by fluorescence-activated cell sorting (FACS). As a result, different B cell subpopulations could be reliably stained and separated from both fresh and post-thawed cells by four-color flow cytometry, although slightly diminished fluorescence intensities of some subpopulation markers were observed when analyzing cryopreserved cells. Subsequently, viable individual CD19+CD27+ memory B cells were sorted into single wells and analyzed for the expression of mRNA transcripts of the 'house-keeping gene' glyceraldehyde phosphate dehydrogenase (GAPD), the constitutive B cell homing receptor CXCR4, and immunoglobulin heavy chain variable region (IgVH) genes by nested RT-PCR protocols. Comparing both B cell sources, RT-PCR analysis revealed comparable yields of cells expressing transcripts for the three mRNA specificities tested (GAPD, CXCR4, IgVH) indicating the integrity of the respective mRNAs in cryopreserved B cells. In conclusion, these data indicate that optimally cryopreserved B cells may be an alternative source for single-cell RT-PCR analysis, especially in longitudinal B cell studies. However, the settings for both FACS analysis and RT-PCR should be re-evaluated for each distinct subpopulation and target mRNA of interest when analyzing post-thawed cells.

Dysregulation of chemokine receptor expression and function by B cells of patients with primary Sjögren's syndrome.

OBJECTIVE: To assess whether abnormal chemokine receptor expression and/or abnormal responsiveness to the cognate ligands might underlie some of the disturbances in B cell homeostasis characteristic of primary Sjögren's syndrome (SS). METHODS: Chemokine receptor expression by CD27- naive and CD27+ memory B cells from patients with primary SS and healthy control subjects was analyzed using flow cytometry, single-cell reverse transcriptase-polymerase chain reaction (RT-PCR), and migration assays. RESULTS: In contrast to healthy subjects, significantly higher expression of both surface CXCR4 and CXCR4 messenger RNA (mRNA) was seen in peripheral blood B cells from patients with primary SS. These differences were most prominent in CD27- naive B cells (P < or = 0.0006). In addition, significantly higher frequencies of CD27- naive B cells from patients with primary SS expressed mRNA for the inhibitory regulator of G protein signaling 13 (P = 0.001). Expression of CXCR5 by peripheral CD27+ memory B cells was moderately diminished in patients with primary SS compared with healthy controls (P = 0.038). No significant differences were noted in the expression of CXCR3, CCR6, CCR7, and CCR9 between B cells from healthy controls and those from patients with primary SS. Transmigration assays of blood B cells from patients with primary SS and healthy controls showed comparable responses of CD27- naive B cells but significantly diminished responses of activated primary SS CD27+ memory B cells to the ligands of CXCR4 and CXCR5, CXCL12 (P = 0.032), and CXCL13 (B lymphocyte chemoattractant; B cell-attracting chemokine 1; P = 0.018), respectively, when compared with those from healthy controls. Finally, compared with controls, peripheral reduction but glandular accumulation of CXCR4+,CXCR5+,CD27+ memory B cells was identified in patients with primary SS. CONCLUSION: In primary SS, overexpression of CXCR4 by circulating blood B cells does not translate into enhanced migratory response to the cognate ligand, CXCL12. This migratory response may be modulated by intracellular regulators. Retention of CXCR4+,CXCR5+, CD27+ memory B cells in the inflamed glands seems to contribute to diminished peripheral CD27+ memory B cells in primary SS.

Involvement of inducible costimulator in the exaggerated memory B cell and plasma cell generation in systemic lupus erythematosus.

OBJECTIVE: In systemic lupus erythematosus (SLE), the increased generation of memory B cells and plasma cells leads to autoimmune hypergammaglobulinemia and destructive immunoglobulin deposits in the kidneys. We undertook this study to determine the biologic mechanism driving this overactivation of the B cell compartment, which is the central issue in SLE. METHODS: We used flow cytometry to analyze expression of the T cell-specific inducible costimulator (ICOS) and its ligand (ICOS-L) on B cells obtained from the peripheral blood of SLE patients. We correlated ICOS-L expression with the differentiation status of the B cells using a large panel of surface antigens. In addition, SLE kidneys were analyzed by immunohistology. RESULTS: We found an increased expression of ICOS on CD4+ as well as CD8+ T cells in SLE. At the same time, we documented a down-regulation of ICOS-L on a high proportion of peripheral blood memory B cells. Based on in vitro experiments, we inferred that this ICOS-L down-regulation on B cells was a signature of recent interaction with ICOS+ T cells in vivo. In the kidneys of SLE patients, we found clusters of B cells and plasma cells in close contact with ICOS+ T cells. CONCLUSION: Detailed analysis of B cells with down-regulated ICOS-L suggests that ICOS is one of the forces driving the formation of memory B cells and plasma cells in SLE. Furthermore, our identification of plasma cells in areas of T cell-B cell interaction in kidneys suggests that components of a T cell-driven B cell activation process may take place in peripheral tissues in SLE.

Abnormalities in peripheral B cell memory of patients with primary Sjögren's syndrome.

OBJECTIVE: To delineate disturbances in peripheral B cell memory in primary Sjögren's syndrome (SS). METHODS: Isotype-specific immunoglobulin (Ig) heavy-chain transcripts were analyzed in single-sorted CD19+,CD27- naive and CD19+,CD27+ memory B cells from patients with primary SS and normal healthy control subjects. RESULTS: A significantly higher frequency of B cells expressing mu-, alpha-, and/or gamma-chain transcripts were found in patients with primary SS compared with controls (58.0% versus 14.3%; P < 0.0001). Notably, 30.5% of individual B cells (for primary SS, 38.7%; for controls, 12.7% [P < 0.0001]) simultaneously expressed transcripts for different Ig heavy-chain isotypes using identical V(H)-D-J(H) rearrangements. However, these cells lacked surface expression of more than one of the respective Ig heavy-chain isotypes as well as messenger RNA (mRNA) transcripts for 2 germinal center markers, activation-induced cytidine deaminase, and Bcl-6. In contrast with the findings in normal healthy controls, peripheral B cell memory in patients with primary SS was characterized by 1) circulating CD27+ B cells expressing heavily mutated Ig V(H) transcripts (mutational frequency 8.6% versus 4.3%; P < 0.0001), 2) significantly enhanced mutational frequencies of C mu transcripts (9.6% versus 2.5%; P < 0.0001), 3) a high proportion (61.2%) of CD27+ B cells expressing transcripts for multiple Ig heavy-chain isotypes, and 4) a CD27- memory-type B cell subpopulation expressing mutated C mu transcripts. CONCLUSION: Altogether, both B cell hyperactivity and striking abnormalities in peripheral B cell memory are indicated at the single-cell mRNA level in patients with primary SS. Detection of multiple Ig heavy-chain transcripts in peripheral CD19+,CD27+ memory B cells of patients with SS may represent the abnormal retention of pre-switch mRNA transcripts in circulating post-switch B cells.

Correlation between circulating CD27high plasma cells and disease activity in patients with systemic lupus erythematosus.

OBJECTIVE: Disease activity in systemic lupus erythematosus (SLE) is usually assessed with complex disease activity scores comprising a variety of different parameters. In order to determine whether SLE disease activity correlates with abnormal B lymphocyte activity, B cell subsets were analyzed, and their relationship to clinical and humoral measures of disease activity was assessed. METHODS: The distribution of B cell subsets was determined by fluorescence-activated cell sorting analysis and assessed in relation to the autoantibody profile, disease activity measured by the SLE Disease Activity Index (SLEDAI) and the European Consensus Lupus Activity Measure scores, disease duration, and therapy. RESULTS: The number and frequency of CD27(high) plasma cells were significantly correlated with the SLE disease activity indices and with the titer of anti-double-stranded DNA (anti-dsDNA) autoantibodies. Circulating B cell subsets were not influenced by age or sex, but appeared to relate to the duration of disease and the therapeutic regimen, with the number and frequency of CD27(high) plasma cells increasing and those of CD27- naive B cells decreasing over time. Patients were divided into those with a SLEDAI score of 0-8 (low disease activity) and those with SLEDAI score >8 (high disease activity). Patients with high disease activity had an increased frequency of both CD19+ B cells and CD27(high) plasma cells. By using a nonparametric data sieving algorithm, we observed that these B cell abnormalities provided predictive values for nonactive and active disease of 78.0% and 78.9%, respectively. The predictive value of the B cell abnormalities (78.9%) was greater than that of the humoral/clinical data pattern (71.4%), including anti-dsDNA antibody levels, circulating immune complexes, increased erythrocyte sedimentation rate, mucocutaneous involvement, and acute renal involvement. CONCLUSION: Flow cytometric monitoring of B cell subsets in the peripheral blood provides new insights into abnormalities of B cell function in SLE and may also be a diagnostically valuable option for monitoring the activity of this autoimmune disease.

Diminished peripheral blood memory B cells and accumulation of memory B cells in the salivary glands of patients with Sjögren's syndrome.

OBJECTIVE: To delineate the mechanism of the abnormalities in B cell biology found in patients with primary Sjögren's syndrome (SS). METHODS: The distribution of peripheral B cell subpopulations in 21 patients with primary SS was analyzed by immunofluorescence labeling and flow cytometry. Immunoglobulin rearrangements were analyzed in single B cells isolated from the peripheral blood and parotid glands by fluorescence-activated cell sorting. RESULTS: A significant reduction in the number of peripheral CD27+ memory B cells was found in SS patients, including a significantly reduced number of CD27+/IgD+/IgM+/CD5+ memory B cells. Remarkably, SS patients with secondary lymphoma uniquely exhibited an increase in CD27-expressing peripheral B cells, including CD27(high) plasmablasts. Molecular analysis for mutated Ig gene rearrangements confirmed that CD27 expression distinguished naive and memory cells in SS. In contrast to the peripheral blood, the majority of parotid B cells from 1 patient examined exhibited both the mutational status and phenotype of memory B cells. Accordingly, the mutational frequencies of V(H) rearrangements were significantly greater in parotid B cells than in peripheral blood B cells, whereas the V(H) gene repertoire appeared to be very similar between the compartments. CONCLUSION: These data indicate that there is an accumulation/retention of memory B cells in the inflamed salivary glands of SS patients. It is possible that preferential accumulation of CD27+ memory B cells in the inflamed parotid gland explains their reduction in the peripheral blood.