Nichol E. Holodick

Human B1 cells in umbilical cord and adult peripheral blood express the novel phenotype CD20+CD27+CD43+CD70−

Human B1 cells consist of CD20+CD27+CD43+CD70− cells bearing a skewed B cell receptor repertoire, and are present in umbilical cord and adult peripheral blood.

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.

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.

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.

CD21/CD19 Coreceptor Signaling Promotes B Cell Survival during Primary Immune Responses

The adaptive immune response is tightly regulated to limit responding cells in an Ag-specific manner. On B cells, coreceptors CD21/CD19 modulate the strength of BCR signals, potentially influencing cell fate. The importance of the CD95 pathway was examined in response of B cells to moderate affinity Ag using an adoptive transfer model of lysozyme-specific Ig transgenic (HEL immunoglobulin transgene (MD4) strain) B cells. Although adoptively transferred Cr2+/+ MD4 B cells are activated and persist within splenic follicles of duck egg lysozyme-immunized mice, Cr2−/− MD4 B cells do not. In contrast, Cr2−/− MD4 lpr B cells persist after transfer, suggesting that lack of CD21/CD35 signaling results in CD95-mediated elimination. Cr2 deficiency did not affect CD95 levels, but cellular FLIP (c-FLIP) protein and mRNA levels were reduced 2-fold compared with levels in Cr2+/+ MD4 B cells. In vitro culture with Cr2+/+ MD4 B cells demonstrated that equimolar amounts of rHEL-C3d3 were more effective than hen egg lysozyme alone in up-regulating c-FLIP levels and for protection against CD95-mediated apoptosis. Collectively, this study implies a mechanism for regulating B cell survival in vivo whereby the strength of BCR signaling (including coreceptor) determines c-FLIP levels and protection from CD95-induced death.

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.

Human B1 cells are CD3-: A reply to "A human equivalent of mouse B-1 cells?" and "The nature of circulating CD27+CD43+ B cells".

Griffin et al. respond: In the January 2011 issue of the JEM , we identified a population of B cells in human umbilical cord and adult peripheral blood that manifests three key functional features of mouse B1 cells (spontaneous immunoglobulin secretion, tonic intracellular signaling, and efficient

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.

B-1a Cell Diversity: Nontemplated Addition in B-1a Cell Ig Is Determined by Progenitor Population and Developmental Location

Natural Abs produced by B-1a cells are required for immediate protection against infection. The protective capacity of natural Abs is attributed to germline-like structure, which includes the relative absence of N-region addition. Previous studies have shown B-1a cell Ig from aged mice contains abundant nontemplated (N)-additions. B-1a cells have been shown to derive from a specific lineage-negative (Lin−)CD45Rlow/−CD19+ progenitor found both in fetal liver and adult bone marrow. In this study, we report identification of a fetal liver population characterized phenotypically as Lin−CD45R−CD19−, which gives rise to IgM+IgDlowCD45RlowCD5+Mac-1+CD19highCD43+CD23low B-1a cells upon adoptive transfer to SCID recipients. These B-1a cells derived from the Lin−CD45R−CD19− fetal liver population produce natural Ab that binds pneumococcal Ags, but this Ig contains substantial N-addition despite initial absence of TdT. Furthermore, we show extensive N-addition is also present in B-1a cells derived from the Lin−CD45Rlow/−CD19+ B-1 progenitor found in the bone marrow. Together these results demonstrate B-1a cell N-addition depends on the type of progenitor and the location of the progenitor during its development. These findings have implications for how regulation of different progenitors from fetal liver and bone marrow may play a role in the age-related increase in N-region addition by B-1a cells in normal animals.

Splenic B-1a Cells Expressing CD138 Spontaneously Secrete Large Amounts of Immunoglobulin in Naïve Mice

B-1a cells constitutively secrete natural antibody that provides immediate protection against microbial pathogens and functions homeostatically to speed removal of apoptotic cell debris. Although B-1a cells are especially prominent in the peritoneal and pleural cavities, some B-1a cells reside in the spleen. A small subset of splenic B-1a cells in naïve, unimmunized mice express CD138, a recognized plasma cell antigen, whereas the bulk of splenic B-1a cells are CD138 negative. Splenic B-1a cells in toto have been shown to generate much more antibody per cell than peritoneal B-1a cells; however, specific functional information regarding CD138+ splenic B-1a cells has been lacking. Here, we find a higher proportion of CD138+ splenic B-1a cells spontaneously secrete more IgM as compared to CD138− B-1a cells. Moreover, IgM secreted by CD138+ splenic B-1a cells is skewed with respect to N-region addition, and some aspects of VH and JH utilization, as compared to CD138− splenic B-1a cells and peritoneal B-1a cells. The small population of CD138+ splenic B-1a cells is likely responsible for a substantial portion of natural IgM and differs from IgM produced by other B-1a cell subsets.