Thomas L. Rothstein

Human B‐1 cells take the stage

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.

Pleural innate response activator B cells protect against pneumonia via a GM-CSF-IgM axis

In response to lung infection, pleural innate response activator B cells produce GM-CSF–dependent IgM and ensure a frontline defense against bacterial invasion.

Adult BM generates CD5+ B1 cells containing abundant N-region additions

The self‐renewing capacity of B1 cells infers homeostatic regulation; however, previous work suggests the low level of N‐region addition characterizing B1 cells early in life increases with age, which implies that the B1‐cell population is not a closed system. To explore this, we evaluated N‐region addition in CD5+ B1 cells generated from adult BM. Adult BM cells were marked with GFP introduced by mouse stem cell virus transduction, and were then adoptively transferred into lethally irradiated recipients. Within 2–3 months, we found GFP‐marked CD5+ B cells in the peritoneal cavities of recipients, which we demonstrate here meet a variety of criteria for B1‐cell traits including Mac‐1 surface expression; annexin, elfin, and Pax‐5 gene expression; mitogenic responsiveness to phorbol ester; and spontaneous immunoglobulin secretion. Notably, we found by single‐cell PCR that this population of BM‐derived CD5+ B1 cells expressed immunoglobulin with abundant N‐region addition (and little VH11/VH12 skewing), unlike CD5+ B1 cells obtained from unmanipulated animals but reminiscent of B2 cells. Further, we confirmed that native CD5+ B1 cells from older mice contain more N‐region additions than native CD5+ B1 cells from younger mice. These results suggest that adult BM progenitors contribute to the peritoneal CD5+ B1‐cell pool over time.

Functional Outcome of B Cell Activation by Chromatin Immune Complex Engagement of the B Cell Receptor and TLR9

We have previously shown that rheumatoid factors produced by Fas-deficient autoimmune-prone mice typically bind autologous IgG2a with remarkably low affinity. Nevertheless, B cells representative of this rheumatoid factor population proliferate vigorously in response to IgG2a/chromatin immune complexes through a mechanism dependent on the sequential engagement of the BCR and TLR9. To more precisely address the role of both receptors in this response, we analyzed the signaling pathways activated in AM14 B cells stimulated with these complexes. We found that the BCR not only serves to direct the chromatin complex to an internal compartment where it can engage TLR9 but also transmits a suboptimal signal that in combination with the signals emanating from TLR9 leads to NF-κB activation and proliferation. Importantly, engagement of both receptors leads to the up-regulation of a group of gene products, not induced by the BCR or TLR9 alone, that include IL-2. These data indicate that autoreactive B cells, stimulated by a combination of BCR and TLR9 ligands, acquire functional properties that may contribute to the activation of additional cells involved in the autoimmune disease process.

CXCL13 is elevated in Sjögren's syndrome in mice and humans and is implicated in disease pathogenesis

SS is an autoimmune disease. pSS affects exocrine glands predominantly, whereas sSS occurs with other autoimmune connective tissue disorders. Currently, care for patients with SS is palliative, as no established therapeutics target the disease directly, and its pathogenetic mechanisms remain uncertain. B‐cell abnormalities have been identified in SS. CXCL13 directs B‐cell chemotaxis and is elevated in several autoimmune diseases. In this study, we tested the hypothesis that CXCL13 is elevated in SS in mice and humans and that neutralization of the chemokine ameliorates disease in a murine model. We assayed CXCL13 in mouse models and human subjects with SS to determine whether CXCL13 is elevated both locally and systemically during SS progression and whether CXCL13 may play a role in and be a biomarker for the disease. Cxcl13 expression in salivary tissue increases with disease progression, and its blockade resulted in a modest reduction in glandular inflammation in an SS model. We demonstrate that in humans CXCL13 is elevated in serum and saliva, and an elevated salivary CXCL13 level distinguishes patients with xerostomia. These data suggest a role for CXCL13 as a valuable biomarker in SS, as 74% of patients with SS displayed elevated CXCL13 in sera, saliva, or both. Thus, CXCL13 may be pathogenically involved in SS and may serve as a new marker and a potential therapeutic target.

Human B1 Cell Frequency: Isolation and Analysis of Human B1 Cells

Controversy over the frequency of human B1 cells in normal individuals has arisen as different labs have begun to employ non-uniform techniques to study this population. The phenotypic profile and relative paucity of circulating human B1 cells place constraints on methodology to identify and isolate this population. Multiple steps must be optimized to insure accurate enumeration and optimal purification. In the course of working with human B1 cells we have developed a successful strategy that provides consistent analysis of B1 cells for frequency determination and efficient isolation of B1 cells for functional studies. Here we discuss issues attendant to identifying human B1 cells and outline a carefully optimized approach that leads to uniform and reproducible data.

Immunoglobulin secretion by B1 cells: Differential intensity and IRF4-dependence of spontaneous IgM secretion by peritoneal and splenic B1 cells

Peritoneal B1 cells are typified by spontaneous, constitutive secretion of IgM natural antibody, detected by ELISPOT assay, among other means. Recently, this key characteristic has been called into question, a reason for which we evaluated the integrity of IgM+ ELISPOT spots. We found that fixed B1 cells fail to produce ELISPOT spots, that interference with Golgi function inhibits ELISPOT spot formation, and that B1 cell‐derived immunoglobulin in supernatant samples is EndoH‐resistant. These findings indicate that spots produced by B1 cells on ELISPOT assay reflect secretory IgM actively exported by viable B1 cells. Current paradigms propose that interferon response factor 4 (IRF4) is required for plasma cell differentiation and immunoglobulin secretion. However, we found that IgM secretion by peritoneal B1 cells is not altered in IRF4‐null mice. In contrast, spontaneous IgM secretion by splenic B1 cells, which amounts to much more IgM secreted per cell, is dramatically reduced in the absence of IRF4. These results indicate that peritoneal B1 cells spontaneously secrete low levels of IgM via an IRF4‐independent non‐classical pathway, and, considering the low level of serum IgM in IRF‐null mice, further suggest that accumulation of serum immunoglobulin depends on IRF4‐dependent secretion by splenic B1 cells.

A Novel Mechanism of B Cell–Mediated Immune Suppression through CD73 Expression and Adenosine Production

Immune suppression by regulatory T cells and regulatory B cells is a critical mechanism to limit excess inflammation and autoimmunity. IL-10 is considered the major mediator of B cell–induced immune suppression. We report a novel mechanism for immune suppression through adenosine generation by B cells. We identified a novel population of B cells that expresses CD73 as well as CD39, two ectoenzymes that together catalyze the extracellular dephosphorylation of adenine nucleotides to adenosine. Whereas CD39 expression is common among B cells, CD73 expression is not. Approximately 30–50% of B-1 cells (B220+CD23−) and IL-10–producing B (B10) cells (B220+CD5+CD1dhi) are CD73hi, depending on mouse strain, whereas few conventional B-2 cells (B220+CD23+AA4.1−) express CD73. In keeping with expression of both CD73 and CD39, we found that CD73+ B cells produce adenosine in the presence of substrate, whereas B-2 cells do not. CD73−/− mice were more susceptible to dextran sulfate sodium salt (DSS)-induced colitis than wild type (WT) mice were, and transfer of CD73+ B cells ameliorated the severity of colitis, suggesting that B cell CD73/CD39/adenosine can modulate DSS-induced colitis. IL-10 production by B cells is not affected by CD73 deficiency. Interestingly, adenosine generation by IL-10−/− B cells is impaired because of reduced expression of CD73, indicating an unexpected connection between IL-10 and adenosine and suggesting caution in interpreting the results of studies with IL-10−/− cells. Our findings demonstrate a novel regulatory role of B cells on colitis through adenosine generation in an IL-10–independent manner.

A Novel Subpopulation of B-1 Cells Is Enriched With Autoreactivity in Normal and Lupus-Prone Mice

OBJECTIVE B-1 cells have long been suggested to play an important role in lupus. However, reports to date have been controversial regarding their pathogenic or protective roles in different animal models. We undertook this study to investigate a novel subpopulation of B-1 cells and its roles in murine lupus. METHODS Lymphocyte phenotypes were assessed by flow cytometry. Autoantibody secretion was analyzed by enzyme-linked immunosorbent assay, autoantigen proteome array, and antinuclear antibody assay. Cell proliferation was measured by thymidine incorporation and 5,6-carboxyfluorescein succinimidyl ester dilution. B cell Ig isotype switching was measured by enzyme-linked immunospot assay. RESULTS Anti-double-stranded DNA (anti-dsDNA) autoantibodies were preferentially secreted by a subpopulation of CD5+ B-1 cells that expressed programmed death ligand 2 (termed L2pB1 cells). A substantial proportion of hybridoma clones generated from L2pB1 cells reacted to dsDNA. Moreover, these clones were highly cross-reactive with other lupus-related autoantigens. L2pB1 cells were potent antigen-presenting cells and promoted Th17 cell differentiation in vitro. A dramatic increase of circulating L2pB1 cells in lupus-prone BXSB mice was correlated with elevated serum titers of anti-dsDNA antibodies. A significant number of L2pB1 cells preferentially switched to IgG1 and IgG2b when stimulated with interleukin-21. CONCLUSION Our findings identify a novel subpopulation of B-1 cells that is enriched for autoreactive specificities, undergoes isotype switch, manifests enhanced antigen presentation, promotes Th17 cell differentiation, and is preferentially associated with the development of lupus in a murine model. Together, these findings suggest that L2pB1 cells have the potential to initiate autoimmunity through serologic and T cell-mediated mechanisms.

Autoantigen can promote progression to a more aggressive TCL1 leukemia by selecting variants with enhanced B-cell receptor signaling

Significance These studies indicate that autoantigen-reactivity plays a role in the progression of a murine leukemia that models human chronic lymphocytic leukemia. This indication is consistent with the notion that chronic lymphocytic leukemia evolves by selection of normal B cells that bind autoantigen via the B-cell antigen receptor. (Auto)antigen engagement by the B-cell receptor (BCR) and possibly the sites where this occurs influence the outcome of chronic lymphocytic leukemia (CLL). To test if selection for autoreactivity leads to increased aggressiveness and if this selection plays out equally in primary and secondary tissues, we used T-cell leukemia (TCL)1 cells reactive with the autoantigen phosphatidylcholine (PtC). After repeated transfers of splenic lymphocytes from a single mouse with oligoclonal PtC-reactive cells, outgrowth of cells expressing a single IGHV-D-J rearrangement and superior PtC-binding and disease virulence occurred. In secondary tissues, increased PtC-binding correlated with enhanced BCR signaling and cell proliferation, whereas reduced signaling and division of cells from the same clone was documented in cells residing in the bone marrow, blood, and peritoneum, even though cells from the last site had highest surface membrane IgM density. Gene-expression analyses revealed reciprocal changes of genes involved in BCR-, CD40-, and PI3K-signaling between splenic and peritoneal cells. Our results suggest autoantigen-stimulated BCR signaling in secondary tissues promotes selection, expansion, and disease progression by activating pro-oncogenic signaling pathways, and that—outside secondary lymphoid tissues—clonal evolution is retarded by diminished BCR-signaling. This transferrable, antigenic-specific murine B-cell clone (TCL1-192) provides a platform to study the types and sites of antigen-BCR interactions and genetic alterations that result and may have relevance to patients.