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Systemic lupus erythematosus is a complex autoimmune disease with significant morbidity that demands further examination of tolerance-inducing treatments. Short-term treatment of lupus-prone NZB/WF1 mice with combination CTLA4Ig and anti–CD40 ligand, but not single treatment alone, suppresses disease for >6 mo via modulation of B and T cell function while maintaining immune responses to exogenous Ags. Three months after a 2-wk course of combination costimulatory blockade, we found a modest decrease in the number of activated T and B cells in both combination and single-treatment cohorts compared with untreated controls. However, only combination treatment mice showed a 50% decrease in spare respiratory capacity of splenic B and T cells. RNA sequencing and gene set enrichment analysis of germinal center (GC) B cells confirmed a reduction in the oxidative phosphorylation signature in the combination treatment cohort. This cohort also manifested increased expression of BCR-associated signaling molecules and increased phosphorylation of PLCγ in GC B cells after stimulation with anti-IgG and anti-CD40. GC B cells from combination treatment mice also displayed a signature involving remodeling of GPI-linked surface proteins. Accordingly, we found a decrease in cell surface expression of the inhibitory molecule CD24 on class-switched memory B cells from aged NZB/W mice that corrected in the combination treatment cohort. Because both a profound decrease in BCR signaling and remodeled immune cell metabolism enhance loss of tolerance in lupus-prone mice, our findings help to explain the restoration of tolerance observed after short-term combination costimulatory blockade.

Age-related deficits in the immune system have been associated with an increased incidence of infections, autoimmune diseases, and cancer. Human B cell populations change quantitatively and qualitatively in the elderly. However, the function of human B-1 cells, which play critical anti-microbial and housekeeping roles, have not been studied in the older age population. In the present work, we analyzed how the frequency, function and repertoire of human peripheral blood B-1 cells (CD19+CD20+CD27+CD38low/intCD43+) change with age. Our results show that not only the percentage of B-1 cells but also their ability to spontaneously secrete IgM decreased with age. Further, expression levels of the transcription factors XBP-1 and Blimp-1 were significantly lower, while PAX-5, characteristic of non-secreting B cells, was significantly higher, in healthy donors over 65 years (old) as compared to healthy donors between 20 and 45 years (young). To further characterize the B-1 cell population in older individuals, we performed single cell sequencing analysis of IgM heavy chains from healthy young and old donors. We found reduced repertoire diversity of IgM antibodies in B-1 cells from older donors as well as differences in usage of certain VH and DH specific genes, as compared to younger. Overall, our results show impairment of the human B-1 cell population with advancing age, which might impact the quality of life and onset of disease within the elderly population.

Human antibody secreting cell (ASC) populations in circulation are not well studied. In addition to B-1 (CD20+CD27+CD38lo/intCD43+) cell and the conventional plasmablast (CD20-CD27hiCD38hi) cell populations, here we identified a novel B cell population termed 20+38hi B cells (CD20+CD27hiCD38hi) that spontaneously secretes antibody. At steady state, 20+38hi B cells are distinct from plasmablasts on the basis of CD20 expression, amount of antibody production, frequency of mutation, and diversity of B cell receptor repertoire. However, cytokine treatment of 20+38hi B cells induces loss of CD20 and acquisition of CD138, suggesting that 20+38hi B cells are precursors to plasmablasts, or pre-plasmablasts. We then evaluated similarities and differences between CD20+CD27+CD38lo/intCD43+ B-1 cells, CD20+CD27hiCD38hi 20+38hi B cells, CD20-CD27hiCD38hi plasmablasts, and CD20+CD27+CD38lo/intCD43- memory B cells. We found that B-1 cells differ from 20+38hi B cells and plasmablasts in numbers of ways, including antigen expression, morphological appearance, transcriptional profiling, antibody skewing, antibody repertoire, and secretory response to stimulation. In terms of gene expression, B-1 cells align more closely with memory B cells than with 20+38hi B cells or plasmablasts, but differ in that memory B cells do not express antibody secretion related genes. We found that, B-1 cell antibodies utilize Vh4-34, which is often associated with autoreactivity, 3 to 6-fold more often than other B cell populations. Along with selective production of IgM anti-PC, this data suggests that human B-1 cells might be preferentially selected for autoreactivity/natural-specificity. In sum, our results indicate that human healthy adult peripheral blood at steady state consists of 3 distinct ASC populations.

The induction of autoantibodies and autoimmunity in patients treated with TNF inhibitors (TNFi) is well-known, but the mechanism by which TNFi induce breach of B cell tolerance is not known. TNF deficiency induces TFH and TH17 cell expansions and disrupts germinal centers (GC), that regulate high affinity antigen specific antibody production and autoreactive B cell selection. We found that TNF deficiency in Sle1 mice (Sle1.TNF−/−) induced high titers of germline encoded, T-dependent IgG anti-cardiolipin. Upon treated with TLR9 ligand (DNA/curli amyloid protein), Sle1.TNF−/− mice developed high IgG anti-dsDNA; although CD95+PNA+/GL7lowGC phenotype B cells were induced, they did not form GC nor acquire dark zone phenotype, and their Ig heavy chains accumulated low numbers of mutations. Similarly, without proper GC, GC phenotype B cells were found in the TNFR1/2−/−NZM2328 mice, who developed accelerated lupus, suggesting disturbed GC by TNF deficiency dysregulated GC phenotype B cell selection. Using an antibody to Neu5Gc (N-Glycolylneuraminic acid, a CD22 high affinity ligand which is the hydrolyzed product of GL7 ligand, N-acetylneuraminic acid, Neu5Ac), we found that GC phenotype B cells from TNF deficient mice failed to stop hydrolyzing Neu5Ac to Neu5Gc, suggesting a failure in recruitment of CD22 to the BCR complex upon activation. Furthermore, activated B cells from Sle1.TNF−/− mice constitutively expressed high level of pS6 and SHIP-1; whereas, additional stimulants, anti-Ig and/or anti-CD40, were required to induce pS6 expression in those of the Sle1 mice. Here, we report a possible mechanism by which TNF deficiency alters the homeostasis of regulatory molecules such as CD22 and FcgRIIB, and thus activated B cell selection.

Belimumab has therapeutic benefit in active systemic lupus erythematosus (SLE), especially in patients with high-titer anti-dsDNA antibodies. We asked whether the profound B cell loss in belimumab-treated SLE patients is accompanied by shifts in the immunoglobulin repertoire. We enrolled 15 patients who had been continuously treated with belimumab for more than 7 years, 17 matched controls, and 5 patients who were studied before and after drug initiation. VH genes of sort-purified mature B cells and plasmablasts were subjected to next-generation sequencing. We found that B cell-activating factor (BAFF) regulates the transitional B cell checkpoint, with conservation of transitional 1 (T1) cells and approximately 90% loss of T3 and naive B cells after chronic belimumab treatment. Class-switched memory B cells, B1 B cells, and plasmablasts were also substantially depleted. Next-generation sequencing revealed no redistribution of VH, DH, or JH family usage and no effect of belimumab on representation of the autoreactive VH4-34 gene or CDR3 composition in unmutated IgM sequences, suggesting a minimal effect on selection of the naive B cell repertoire. Interestingly, a significantly greater loss of VH4-34 was observed among mutated IgM and plasmablast sequences in chronic belimumab-treated subjects than in controls, suggesting that belimumab promotes negative selection of activated autoreactive B cells.

B-1 cells represent a subpopulation of B cells that has been extensively studied in mice and shown to spontaneously generate natural antibody that provides antimicrobial protection and helps dispose of cellular debris. Mouse B-1 cells originate from distinct progenitors and express additional immune properties that include phagocytosis, antigen presentation, immune suppression, and polarization of T cell differentiation. Confusion regarding the existence of human B-1 cells with mouse B-1-like properties has recently been addressed by identification of a new phenotypic profile. Human B-1 cells spontaneously secrete antibody and are distinct from other circulating B cell populations by multiple criteria. A number of laboratories have recently reported on changes to the human B-1 cell population in human disease.

In response to lung infection, pleural innate response activator B cells produce GM-CSF–dependent IgM and ensure a frontline defense against bacterial invasion., Pneumonia is a major cause of mortality worldwide and a serious problem in critical care medicine, but the immunophysiological processes that confer either protection or morbidity are not completely understood. We show that in response to lung infection, B1a B cells migrate from the pleural space to the lung parenchyma to secrete polyreactive emergency immunoglobulin M (IgM). The process requires innate response activator (IRA) B cells, a transitional B1a-derived inflammatory subset which controls IgM production via autocrine granulocyte/macrophage colony-stimulating factor (GM-CSF) signaling. The strategic location of these cells, coupled with the capacity to produce GM-CSF–dependent IgM, ensures effective early frontline defense against bacteria invading the lungs. The study describes a previously unrecognized GM-CSF-IgM axis and positions IRA B cells as orchestrators of protective IgM immunity.

B-1 cells play critical roles in defending against microbial invasion and in housekeeping removal of cellular debris. B-1 cells secrete natural antibody and manifest functions that influence T cell expansion and differentiation and in these and other ways differ from conventional B-2 cells. B-1 cells were originally studied in mice where they are easily distinguished from B-2 cells, but their identity in the human system remained poorly defined for many years. Recently, functional criteria for human B-1 cells were established on the basis of murine findings, and reverse engineering resulted in identification of the phenotypic profile, CD20 + CD27 + CD43 + CD70 − , for B-1 cells found in both umbilical cord blood and adult peripheral blood. Human B-1 cells may contribute to multiple disease states through production of autoantibody and stimulation/modulation of T cell activity. Human B-1 cells could be a rich source of antibodies useful in treating diseases present in elderly populations where natural antibody protection may have eroded. Manipulation of human B-1 cell numbers and/or activity may be a new avenue for altering T cell function and treating immune dyscrasias.

The B-1 B cell population is an important bridge between innate and adaptive immunity primarily because B-1 cells produce natural Ab. Murine B-1 and B-2 cells arise from distinct progenitors; however, in humans, in part because it has been difficult to discriminate between them phenotypically, efforts to pinpoint the developmental origins of human B-1 and B-2 cells have lagged. To characterize progenitors of human B-1 and B-2 cells, we separated cord blood and bone marrow Lin−CD34+ hematopoietic stem cells into Lin−CD34+CD38lo and Lin−CD34+CD38hi populations. We found that transplanted Lin−CD34+CD38lo cells, but not Lin−CD34+CD38hi cells, generated a CD19+ B cell population after transfer into immunodeficient NOD.Cg-Prkdcscid Il2rgtm1wjl/SxJ neonates. The emergent CD19+ B cell population was found in spleen, bone marrow, and peritoneal cavity of humanized mice and included distinct populations displaying the B-1 or the B-2 cell phenotype. Engrafted splenic B-1 cells exhibited a mature phenotype, as evidenced by low-to-intermediate expression levels of CD24 and CD38. The engrafted B-1 cell population expressed a VH-DH-JH composition similar to cord blood B-1 cells, including frequent use of VH4-34 (8 versus 10%, respectively). Among patients with hematologic malignancies who underwent hematopoietic stem cell transplantation, B-1 cells were found in the circulation as early as 8 wk posttransplantation. Altogether, our data demonstrate that human B-1 and B-2 cells develop from a Lin−CD34+CD38lo stem cell population, and engrafted B-1 cells in humanized mice exhibit an Ig-usage pattern comparable to B-1 cells in cord blood.

B cell anergy represents an important mechanism of peripheral immunological tolerance for mature autoreactive B cells that escape central tolerance enforced by receptor editing and clonal deletion. Although well documented in mice, the extent of its participation in human B cell tolerance remains to be fully established. In this study, we characterize the functional behavior of strictly defined human naive B cells separated on the basis of their surface IgM (sIgM) expression levels. We demonstrate that cells with lower sIgM levels (IgMlo) are impaired in their ability to flux calcium in response to either anti-IgM or anti-IgD cross-linking and contain a significantly increased frequency of autoreactive cells compared with naive B cells with higher levels of sIgM. Phenotypically, in healthy subjects, IgMlo cells are characterized by the absence of activation markers, reduction of costimulatory molecules (CD19 and CD21), and increased levels of inhibitory CD22. Functionally, IgMlo cells display significantly weaker proliferation, impaired differentiation, and poor Ab production. In aggregate, the data indicate that hyporesponsiveness to BCR cross-linking associated with sIgM downregulation is present in a much larger fraction of all human naive B cells than previously reported and is likely to reflect a state of anergy induced by chronic autoantigen stimulation. Finally, our results indicate that in systemic lupus erythematosus patients, naive IgMlo cells display increased levels of CD95 and decreased levels of CD22, a phenotype consistent with enhanced activation of autoreactive naive B cells in this autoimmune disease.

N-glycolylated (NeuGc) gangliosides are not naturally expressed in human tissues but are overexpressed in several tumors and have immunosuppressive capacity. The present work shows the existence of natural cytotoxic antibodies against NeuGcGM3, whose levels decrease with age. Furthermore, this reactivity was not detected in the sera of non-small cell lung cancer patients that matched in sex and age the assessed healthy donors. Aging decreases the efficiency of immune functions, which could be associated with an increased incidence of cancer. In order to identify and study the effect of aging on the B cell population involved in the response against NeuGcGM3 naïve, memory, plasma and B-1 cells were purified from healthy human donors PBMC and stimulated in vitro to produce immunoglobulins. Only B-1 cell supernatants recognized tumor cells expressing this ganglioside. Interestingly, B-1 cells from NSCLC patients had a reduced activation capacity. The percentage of B-1 cells and their capacity to spontaneously secrete IgM decreased with age in healthy donors. In fact, expression levels of Xbp1 and Blimp1 were significantly lower, while those of Pax5 were significantly higher, in elderly donors. Elderly donors showed a B-1 antibody repertoire with higher number of repeated clones and therefore a reduced variability in comparison with young individuals. It was interesting to observe in aged individuals an increased frequency of VH3-23 and VH4-34 usage, genes which have been linked to autoimmune diseases and hematopoietic malignancies, while there is a significant reduction of clones expressing the sequence VH3-33, frequently found on antibodies against PC, oxidized LDL, apoptotic cells and induced after vaccination with Pneumavax23.

Increased glucose levels are associated with the generation of advanced glycation endproduct (AGE) modifications. Interaction between AGE-modified plaque components and immune cells is believed to have an important role in the development of vascular complications in diabetes. Methylglyoxal (MGO) is one type of reactive aldehyde that gives rise to AGE modification. The present study analyzed whether autoantibodies against MGO-modified epitopes of the low-density lipoprotein apolipoprotein B (apoB) 100 predict cardiovascular events. A library consisting of 302 peptides comprising the complete apoB100 molecule was screened to identify peptides targeted by MGO-specific autoantibodies. Peptide (p) 220 (apoB amino acids 3286-3305) was identified as a major target. Baseline IgM and IgG against MGO–peptide 220 (p220) were measured in 700 individuals from the Malmö Diet and Cancer Cohort. A total of 139 cardiovascular events were registered during the 15-y follow-up period. Controlling for major cardiovascular risk factors demonstrated that subjects in the lowest tertile of MGO-p220 IgM had an increased risk for cardiovascular events (hazard ratio [95% confidence interval]: 2.07 [1.22–3.50]; ptrend = 0.004). Interestingly, the association between MGO-p220 IgM and cardiovascular events remained and even tended to become stronger when subjects with prevalent diabetes were excluded from the analysis (2.51 [1.37-4.61]; ptrend = 0.002). MGO-p220 IgM was inversely associated with blood glucose, but not with oxidized low-density lipoprotein. Finally, we demonstrate that anti-MGO-p220 IgM is produced by B1 cells. These data show that subjects with low levels of IgM recognizing MGO-modified p220 in apoB have an increased risk to develop cardiovascular events and that this association is present in nondiabetic subjects.

It is well known that B-1 cells are the main B cell subset responsible for the production of natural antibodies. Several studies suggest that natural IgM antibodies play an important role in immunosurveillance against transformed cells in humans, preferentially binding glycolipids and carbohydrate structures. NeuGcGM3 ganglioside is a sialic acid containing glycosphingolipid that has been considered an attractive target for cancer immunotherapy since is not naturally expressed in healthy human tissues but is overexpressed in several tumors. We previously described the existence of cytotoxic anti-tumor antibodies against NeuGcGM3 ganglioside in healthy young subjects. However, whether this anti-tumor antibody response has a “natural origin” is unknown. With the present work we aimed to elucidate whether B-1 cells have the potential to secrete anti-NeuGcGM3 antibodies. First we generated four mouse monoclonal antibodies from Balb/c mouse peritoneal B cells. All hibridomas secreted IgM which bound NeuGcGM3 but no other gangliosides and recognized and killed NeuGcGM3 positive tumor cells. All the anti-NeuGcGM3 IgM-producing hybridomas were CD5+ B-1 cells. Then naïve, memory, plasma and B-1 cells were purified from healthy human donors PBMC and stimulated in vitro to produce immunoglobulins. All the B-1 cell supernatants reacted with NeuGcGM3 and most of them also recognized tumor cells expressing this ganglioside. Only 40% of memory samples, and none from naïve or plasma cells, recognized this antigen. These data suggest a protective role for B-1 cells against malignant cells through the production of natural antibodies reactive to tumor associated antigens such as NeuGcGM3 ganglioside.

Work from multiple groups continues to provide additional evidence for the powerful and highly diverse roles, both protective and pathogenic, that B cells play in autoimmune diseases. Similarly, it has become abundantly clear that antibody-independent functions may account for the opposing influences that B cells exercise over other arms of the immune response and ultimately over autoimmunity itself. Finally, it is becoming apparent that the clinical impact of B cell depletion therapy may be to a large extent determined by the functional balance between different B cell subsets that may be generated by this therapeutic intervention. In this review, we postulate that our perspective of B cell tolerance and our experimental approach to its understanding are fundamentally changed by this view of B cells. Accordingly, we shall first discuss current knowledge of B cell tolerance conventionally defined as the censoring of autoantibody-producing B cells (with an emphasis on human B cells). Therefore, we shall discuss a different model that contemplates B cells not only as targets of tolerance but also as mediators of tolerance. This model is based on the notion that the onset of clinical autoimmune disease may require a B cell gain-of-pathogenic function (or a B cell loss-of-regulatory-function) and that accordingly, disease remission may depend on the restoration of the physiological balance between B cell pathogenic and protective functions.