Transitional cells represent a crucial step in the differentiation and selection of the mature B cell compartment. Human transitional B cells have previously been variably identified based on the high level of expression of CD10, CD24, and CD38 relative to mature B cell populations and are expanded in the peripheral blood following rituximab-induced B cell-depletion at reconstitution. In this study, we take advantage of the gradual acquisition of the ABCB1 transporter during B cell maturation to delineate refined subsets of transitional B cells, including a late transitional B cell subset with a phenotype intermediate between T2 and mature naive. This late transitional subset appears temporally following the T1 and T2 populations in the peripheral compartment after rituximab-induced B cell reconstitution (and is thus termed T3) and is more abundant in normal peripheral blood than T1 and T2 cells. The identity of this subset as a developmental intermediate between early transitional and mature naive B cells was further supported by its ability to differentiate to naive during in vitro culture. Later transitional B cells, including T2 and T3, are found at comparatively increased frequencies in cord blood and spleen but were relatively rare in bone marrow. Additional studies demonstrate that transitional B cells mature across a developmental continuum with gradual up-regulation of mature markers, concomitant loss of immature markers, and increased responsiveness to BCR cross-linking in terms of proliferation, calcium flux, and survival. The characterization of multiple transitional B cell subpopulations provides important insights into human B cell development.

B-1cells play critical roles in defending against microbial invasion and in housekeeping removal of cellular debris. B-1cells 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-2cells, but their identity in the human system remained poorly defined for many years. Recently, functional criteria for human B-1cells were established on the basis of murine findings, and reverse engineering resulted in identification of the phenotypic profile, CD20+CD27+CD43+CD70−, for B-1cells found in both umbilical cord blood and adult peripheral blood. Human B-1cells may contribute to multiple disease states through production of autoantibody and stimulation/modulation of T cell activity. Human B-1cells 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-1cell numbers and/or activity may be a new avenue for altering T cell function and treating immune dyscrasias.

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. While 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 naïve 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 to naïve B cells with higher levels of sIgM. Phenotypically, in healthy subjects, IgMlo cells are characterized by the absence of activation markers, reduction of co-stimulatory molecules (CD19 and CD21) and increased levels of inhibitory CD22. Functionally, IgMlo cells display significantly weaker proliferation, impaired differentiation, and poor antibody production. In aggregate, the data indicates that hypo-responsiveness to BCR cross-linking associated with sIgM down-regulation is present in a much larger fraction of all human naïve 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 SLE patients, naïve IgMlo cells display increased levels of CD95 and decreased levels of CD22, a phenotype consistent with enhanced activation of autoreactive naïve B cells in this autoimmune 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.

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.

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.

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.

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.