Xuemei Zhong

Reciprocal generation of Th1/Th17 and Treg cells by B1 and B2 B cells

Regulatory T (Treg) cells are indispensable for maintaining peripheral tolerance, whereas T helper (Th)1 and Th17 cells induce inflammation and tissue destruction. Using Foxp3‐GFP knock‐in mice, we report a novel regulatory role for B cell subsets in influencing the differentiation of Treg versus Th1/Th17 cells. Peritoneal B1 cells strongly promoted T cell proliferation and cytokine secretion when presenting nominal or allogeneic antigens, as compared to conventional follicular B (B2) cells. However, peritoneal B1 cells largely failed to convert naive Foxp3–CD4+ T cells into Foxp3+ Treg cells in the presence of TGF‐β and IL‐2, in marked contrast to conventional B2 cells, which excelled in Treg conversion. Interestingly, under the same Treg conversion conditions, peritoneal B1 cells preferentially promoted Th1 and Th17 cell differentiation. Blockade of CD86 but not CD80 costimulation markedly enhanced Treg cell induction by B1 cells. Thus, B cell antigen presentation function is inversely correlated with de novo Treg cell induction for these B cell subsets. Our findings suggest that B1 and B2 cell subsets play distinct roles in immune regulation by promoting reciprocal differentiation of T cell lineages.

PD-L2 expression extends beyond dendritic cells/macrophages to B1 cells enriched for VH11/VH12 and phosphatidylcholine binding

B1 B cells are the major source of natural antibody that is essential for innate immunity. The B1 repertoire is skewed toward production of phosphatidylcholine (PtC)‐binding VH11 and VH12 immunoglobulin that plays a key role in immune defense against bacterial infection. Programmed death‐ligand 2 (PD‐L2) is a ligand for the immunosuppressive receptor programmed death‐1 (PD‐1). It has been reported that expression of PD‐L2 is restricted to dendritic cells and macrophages in mice. Here we show that 50–70% of resting peritoneal B1 cells express PD‐L2, which is not present or inducible on conventional B2 B cells or PD‐L2– B1 cells. Although PD‐L2+ and PD‐L2– B1 cells are similar in proliferative responses and spontaneous immunoglobulin secretion, PD‐L2+ B1 cells are highly enriched for expression of VH11 and VH12 genes and encompass the bulk of PtC‐binding B1 cells. These findings extend the range of known PD‐L2 expression to B cells and show that B1 cells identified by this marker express a specific repertoire associated with anti‐bacterial immunity.

Fas-induced apoptosis in B cells

Engagement of the cell surface receptor Fas/APO-1 (CD95) initiates a sequence of intracellular events that leads to apoptotic cell death, and this outcome occurs in B cells as it does in other cell types. Fas signaling for B cell death is of particular interest because the expression and function of Fas is altered by engagement of additional cell surface receptors, leading to marked receptor-specific variation in susceptibility to Fas-induced apoptosis. Evidence suggests that the sensitivity of B cells to Fas-mediated apoptosis is intimately connected to homeostasis in the serological arm of the immune system and plays a role in the dysregulation that occurs in certain autoimmune and malignant 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.

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.

An alternatively spliced long form of Fas apoptosis inhibitory molecule (FAIM) with tissue-specific expression in the brain.

The gene encoding Fas apoptosis inhibitory molecule (FAIM) was cloned by differential display using RNA obtained from Fas-resistant and Fas-sensitive primary murine B lymphocytes. FAIM is highly evolutionarily conserved and broadly expressed, suggesting that its gene product plays a key role in cellular physiology. Here we report the identification of a new, longer form of FAIM (FAIM-L) and characterization of the genomic locus that clarifies its origin. The murine FAIM gene is located at chromosome 9f1, a region syntenic to the corresponding location of the human FAIM gene. The gene consists of six exons and contains putative translation initiation sites within exons II and III. The long form of FAIM is generated by all six exons, whereas the originally cloned form of FAIM, now termed FAIM-Short (FAIM-S) is generated from five exons by alternative splicing. FAIM-L is dominantly expressed in the brain whereas FAIM-S is widely expressed in many tissues.

Cellometer Vision as an alternative to flow cytometry for cell cycle analysis, mitochondrial potential, and immunophenotyping

Cell phenotyping and cell cycle analysis are two commonly used assays in both clinical diagnosis and biomedical research. Cell phenotyping by identifying different biomarkers is essential for the diagnosis of hematologic malignancy, sub‐classifying diseases, monitoring response to treatment, predicting prognosis, detecting rare cell populations and residual malignant cells. Cell cycle analysis distinguishes cells in different phases of cell cycle and is often used to determine the cellular response to drugs and biological stimulations. These assays have been traditionally carried out by sensitive fluorescence detection methods such as flow cytometry and laser scanning cytometry for fluorescence‐based cell population analysis. However, these instruments remain relatively expensive, large in size, and require a considerable amount of maintenance, which may not be feasible for smaller research groups that do not have access to these equipments or field clinics that require quick diagnostic results on site. Recently, a small portable imaging cytometry system (Cellometer Vision) has been developed by Nexcelom Bioscience LLC (Lawrence, MA) for automated cell concentration and viability measurement using bright‐field and fluorescent imaging methods. Here we report new applications of the Cellometer imaging cytometry for fluorescence‐based cell population analysis and compared them with conventional flow cytometry. Cell population analysis assays such as immunophenotyping, cell cycle, and mitochondrial membrane potential detection methods have not yet been reported for the Cellometer Vision system. Using this imaging cytometry method for fluorescence‐based assays that are typically done by flow cytometry offers a quick, simple, and inexpensive alternative method for biomedical research, which may be beneficial for smaller research laboratories and clinics.

Blockade of B-cell-activating factor suppresses lupus-like syndrome in autoimmune BXSB mice

B‐cell‐activating factor (BAFF), a member of the tumour necrosis factor superfamily, plays a critical role in the maturation, homeostasis and function of B cells. In this study, we demonstrated the biological outcome of BAFF blockade in BXSB murine lupus model, using a soluble fusion protein consisting of human BAFF‐R and human mutant IgG4 Fc. Mutation of Leu235 to Glu in IgG4 Fc eliminated antibody‐dependent cell cytotoxicity (ADCC) and complement lysis activity, and generated a protein devoid of immune effector functions. Treatment of BXSB mice with BAFF‐R‐IgG4mut fusion protein for 5 weeks resulted in significant B‐cell reduction in both the peripheral blood and spleen. Treated mice developed lower proteinuria, reduced glomerulonephritis and much delayed host death than untreated animals. Thus, BAFF blockade with BAFF‐R‐IgG4mut protein is an effective strategy to treat B‐cell‐mediated lupus‐like pathology. Moreover, compared with other IgG isotypes with undesired effector functions, mutant IgG4 Fc should prove useful in constructing novel therapeutic reagents to block immune molecule signalling in various diseases.

The immunomodulatory parasitic worm product ES-62 reduces lupus-associated accelerated atherosclerosis in a mouse model.

ES-62 is an anti-inflammatory phosphorylcholine-containing glycoprotein secreted by the filarial nematode Acanthocheilonema viteae. Accelerated atherosclerosis frequently occurs in systemic lupus erythematosus, resulting in substantial cardiovascular morbidity and mortality. We examined the effects of ES-62 in the gld.apoE(-/-) mouse model of this condition. Treatment with ES-62 did not substantially modulate renal pathology but caused decreased anti-nuclear autoantibody levels. Moreover, a striking 60% reduction in aortic atherosclerotic lesions was observed, with an associated decrease in macrophages and fibrosis. We believe that these latter findings constitute the first example of a defined parasitic worm product with therapeutic potential in atherosclerosis: ES-62-based drugs may represent a novel approach to control accelerated atherosclerosis in systemic lupus erythematosus.