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Abstract: Chronic graft-versus-host disease (cGVHD) is a debilitating, autoimmune-like syndrome that can occur after allogeneic hematopoietic stem cell transplantation. Constitutively activated B cells contribute to ongoing alloreactivity and autoreactivity in patients with cGVHD. Excessive tissue damage that occurs after transplantation exposes B cells to nucleic acids in the extracellular environment. Recognition of endogenous nucleic acids within B cells can promote pathogenic B-cell activation. Therefore, we hypothesized that cGVHD B cells aberrantly signal through RNA and DNA sensors such as Toll-like receptor 7 (TLR7) and TLR9. We found that B cells from patients and mice with cGVHD had higher expression of TLR7 than non-cGVHD B cells. Using ex vivo assays, we found that B cells from patients with cGVHD also demonstrated increased interleukin-6 production after TLR7 stimulation with R848. Low-dose B-cell receptor (BCR) stimulation augmented B-cell responses to TLR7 activation. TLR7 hyperresponsiveness in cGVHD B cells correlated with increased expression and activation of the downstream transcription factor interferon regulatory factor 5. Because RNA-containing immune complexes can activate B cells through TLR7, we used a protein microarray to identify RNA-containing antigen targets of potential pathological relevance in cGVHD. We found that many of the unique targets of active cGVHD immunoglobulin G (IgG) were nucleic acid-binding proteins. This unbiased assay identified the autoantigen and known cGVHD target Ro-52, and we found that RNA was required for IgG binding to Ro-52. Herein, we find that BCR-activated B cells have aberrant TLR7 signaling responses that promote potential effector responses in cGVHD., (Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution.)

Alloreactivity can drive autoimmune syndromes. After allogeneic hematopoietic stem cell transplantation (allo-HCT), chronic graft-versus-host disease (cGVHD), a B cell-associated autoimmune-like syndrome, commonly occurs. Because donor-derived B cells continually develop under selective pressure from host alloantigens, aberrant B cell receptor (BCR) activation and IgG production can emerge and contribute to cGVHD pathobiology. To better understand molecular programing of B cells in allo-HCT, we performed scRNA-Seq analysis on high numbers of purified B cells from patients. An unsupervised analysis revealed 10 clusters, distinguishable by signature genes for maturation, activation, and memory. Within the memory B cell compartment, we found striking transcriptional differences in allo-HCT patients compared with healthy or infected individuals, including potentially pathogenic atypical B cells (ABCs) that were expanded in active cGVHD. To identify intrinsic alterations in potentially pathological B cells, we interrogated all clusters for differentially expressed genes (DEGs) in active cGVHD versus patients who never had signs of immune tolerance loss (no cGVHD). Active cGVHD DEGs occurred in both naive and BCR-activated B cell clusters. Remarkably, some DEGs occurred across most clusters, suggesting common molecular programs that may promote B cell plasticity. Our study of human allo-HCT and cGVHD provides understanding of altered B cell memory during chronic alloantigen stimulation.

Despite the exciting advancement of novel therapies, chronic graft-versus-host disease (cGVHD) remains the most common cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation (HCT). Frontline treatment of cGVHD involves systemic steroids, which are associated with significant morbidities. We previously found that inhibition of spleen tyrosine kinase (SYK) with fostamatinib preferentially eradicated aberrantly activated B cells in both ex vivo studies of cGVHD patient B cells, as well as in vivo mouse studies. These and other preclinical studies implicated hyper-reactive B-cell receptor signaling and increased SYK expression in the pathogenesis of cGVHD and compelled this first in-human allogeneic HCT clinical trial. We investigated the safety and efficacy of the oral SYK inhibitor, fostamatinib, for both the prevention and treatment of cGVHD. The primary objective was to evaluate the safety of fostamatinib and determine its maximum tolerated dose in the post-HCT setting. Secondary objectives included assessing the efficacy of fostamatinib in preventing and treating cGVHD, as well as examining alterations in B-cell compartments with treatment. This was a single-institution phase I clinical trial that evaluated the use of fostamatinib in allogeneic HCT patients before the development of cGVHD or at the time of steroid-refractory cGVHD (SR-cGVHD). Patients received fostamatinib at one of three dose levels using a continual reassessment algorithm to determine the maximum tolerated dose. Multiparameter flow cytometry was used to evaluate changes in B cell subpopulations over the first year of treatment with fostamatinib. Nineteen patients were enrolled in this phase I trial, with 5 in the prophylaxis arm and 14 in the therapeutic arm. One patient (5%) required discontinuation of therapy for a dose-limiting toxicity. At a median follow-up of over 3 years, no patients had cancer relapse while on fostamatinib treatment, and recurrent malignancy was observed in 1 patient 2 years after the end of therapy. In the prophylaxis arm, 1 of 5 patients (20%) developed cGVHD while on fostamatinib. In the therapeutic arm, the overall response rate was 77%, with a complete response rate of 31%. The median duration of response was 19.3 months and the 12-month failure-free survival was 69% (95% confidence interval, 48-100). Patients were able to reduce their steroid dose by a median of 80%, with 73% remaining on a lower dose at 1 year compared to baseline. There was an early reduction in the proportion of IgD - CD38 hi plasmablast-like cells with fostamatinib treatment, particularly in those SR-cGVHD patients who had an eventual response. B-cell reconstitution was not significantly impacted by fostamatinib therapy after allogeneic HCT. Fostamatinib featured a favorable safety profile in the post-HCT setting. Our data suggests an early efficacy signal that was associated with effects on expected cell targets in both the prophylaxis and treatment of cGVHD, providing rationale for a phase II investigation., (Copyright © 2022 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

De novo immune responses to myeloid and other blood-borne tumors are notably limited and ineffective, making our ability to promote immune responses with vaccines a major challenge. While focus has been largely on cytotoxic cell-mediated tumor eradication, B-cells and the antibodies they produce also have roles in anti-tumor responses. Indeed, therapeutic antibody-mediated tumor cell killing is routinely employed in patients with hematolymphoid cancers, but whether endogenous antibody responses can be incited to blood-born tumors remains poorly studied. A major limitation of immunoglobulin therapies is that cell surface expression of tumor-associated antigen (TAA) targets is dynamic and varied, making promotion of polyclonal, endogenous B cell responses appealing. Since many TAAs are self-antigens, developing tumor vaccines that enable production of antibodies to non-polymorphic antigen targets remains a challenge. As B cell responses to RNA vaccines are known to occur, we employed the Viral Replicon Particles (VRP) which was constructed to encode mouse FLT3. The VRP-FLT3 vaccine provoked a rapid IgG B-cell response to this self-antigen in leukemia and lymphoma mouse models. In addition, IgGs to other TAAs were also produced. Our data suggest that vaccination with RNA viral particle vectors incites a loss of B-cell tolerance that enables production of anti-tumor antibodies. This proof of principle work provides impetus to employ such strategies that lead to a break in B-cell tolerance and enable production of broadly reactive anti-TAA antibodies as potential future therapeutic agents for patients with hematolymphoid cancers., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Su, Imai, Jia, Li, DiCioccio, Serody, Poe, Chen, Doan and Sarantopoulos.)

Patients with chronic graft-versus-host disease (cGVHD) have increased B cell-activating factor (BAFF) levels, but whether BAFF promotes disease after allogeneic bone marrow transplantation (allo-BMT) remains unknown. In a major histocompatibility complex-mismatched model with cGVHD-like manifestations, we first examined B-lymphopenic μMT allo-BMT recipients and found that increased BAFF levels in cGVHD mice were not merely a reflection of B-cell number. Mice that later developed cGVHD had significantly increased numbers of recipient fibroblastic reticular cells with higher BAFF transcript levels. Increased BAFF production by donor cells also likely contributed to cGVHD, because BAFF transcript in CD4+ T cells from diseased mice and patients was increased. cGVHD manifestations in mice were associated with high BAFF/B-cell ratios and persistence of B-cell receptor (BCR)-activated B cells in peripheral blood and lesional tissue. By employing BAFF transgenic (Tg) mice donor cells, we addressed whether high BAFF contributed to BCR activation in cGVHD. BAFF increased NOTCH2 expression on B cells, augmenting BCR responsiveness to surrogate antigen and NOTCH ligand. BAFF Tg B cells had significantly increased protein levels of the proximal BCR signaling molecule SYK, and high SYK protein was maintained by BAFF after in vitro BCR activation or when alloantigen was present in vivo. Using T cell-depleted (BM only) BAFF Tg donors, we found that BAFF promoted cGVHD manifestations, circulating GL7+ B cells, and alloantibody production. We demonstrate that pathologic production of BAFF promotes an altered B-cell compartment and augments BCR responsiveness. Our findings compel studies of therapeutic targeting of BAFF and BCR pathways in patients with cGVHD., (© 2021 by The American Society of Hematology.)

Graft-versus-host disease (GVHD) is a major complication of hematopoietic stem cell transplantation (HCT). The tyrosine kinase SYK contributes to both acute and chronic GVHD development, making it an attractive target for GVHD prevention. Entospletinib (ENTO) is a second-generation highly selective SYK inhibitor with a high safety profile. Potential utility of ENTO as GVHD prophylaxis in patients was examined using a preclinical mouse model of eye and skin GVHD and ENTO-compounded chow. We found that early SYK inhibition improved blood immune cell reconstitution in GVHD mice and prolonged survival, with 60% of mice surviving to day +120 compared with 10% of mice treated with placebo. Compared with mice receiving placebo, mice receiving ENTO had dramatic improvements in clinical eye scores, alopecia scores, and skin scores. Infiltrating SYK+ cells expressing B220 or F4/80, resembling SYK+ cells found in lichenoid skin lesions of chronic GVHD patients, were abundant in the skin of placebo mice but were rare in ENTO-treated mice. Thus, ENTO given early after HCT safely prevented GVHD.

B-cell receptor (BCR)-activated B cells contribute to pathogenesis in chronic graft-versus-host disease (cGVHD), a condition manifested by both B-cell autoreactivity and immune deficiency. We hypothesized that constitutive BCR activation precluded functional B-cell maturation in cGVHD. To address this, we examined BCR-NOTCH2 synergy because NOTCH has been shown to increase BCR responsiveness in normal mouse B cells. We conducted ex vivo activation and signaling assays of 30 primary samples from hematopoietic stem cell transplantation patients with and without cGVHD. Consistent with a molecular link between pathways, we found that BCR-NOTCH activation significantly increased the proximal BCR adapter protein BLNK. BCR-NOTCH activation also enabled persistent NOTCH2 surface expression, suggesting a positive feedback loop. Specific NOTCH2 blockade eliminated NOTCH-BCR activation and significantly altered NOTCH downstream targets and B-cell maturation/effector molecules. Examination of the molecular underpinnings of this "NOTCH2-BCR axis" in cGVHD revealed imbalanced expression of the transcription factors IRF4 and IRF8 , each critical to B-cell differentiation and fate. All- trans retinoic acid (ATRA) increased IRF4 expression, restored the IRF4 -to- IRF8 ratio, abrogated BCR-NOTCH hyperactivation, and reduced NOTCH2 expression in cGVHD B cells without compromising viability. ATRA-treated cGVHD B cells had elevated TLR9 and PAX5 , but not BLIMP1 (a gene-expression pattern associated with mature follicular B cells) and also attained increased cytosine guanine dinucleotide responsiveness. Together, we reveal a mechanistic link between NOTCH2 activation and robust BCR responses to otherwise suboptimal amounts of surrogate antigen. Our findings suggest that peripheral B cells in cGVHD patients can be pharmacologically directed from hyperactivation toward maturity.

Objective: Regulatory B cells that inhibit immune responses through interleukin-10 (IL-10) secretion (B10 cells) have been characterized in adult subjects with autoimmune disease. The aim of this study was to characterize B10 cells in individuals across the entire age range of normal human development and changes in their frequency and numbers in children with autoimmunity., Methods: The phenotype and numbers of B10 cells in blood were examined in healthy individuals and children with autoimmunity, using flow cytometry. B10 cell function was assessed by measuring the effect of B cell-derived IL-10 on interferon-γ (IFNγ) expression by CD4+ T cells. Serum cytokine levels were measured by enzyme-linked immunosorbent assay., Results: The frequency of B10 cells transiently increased during childhood, when up to 30% of B cells were competent to produce IL-10, compared with the low frequencies in healthy newborns (3-4%) and adults (7-9%). The surface phenotype of B10 cells in children revealed age-dependent variability. B10 cells from children were distinct from proinflammatory cytokine-producing B cells and down-regulated IFNγ production by CD4+ T cells in vitro. Compared with age-matched healthy controls, children with autoimmunity had lower numbers and frequencies of B10 cells (decreased by 39% and 48%, respectively), higher IFNγ levels, and lower IL-21 levels in serum. IFNγ inhibited, whereas IL-21 promoted, B cell IL-10 competence in vitro., Conclusion: B10 cells, a functionally defined cell subset with a variable surface phenotype reflective of overall B cell development, transiently expand during childhood. B10 cell frequencies and numbers were decreased in children with autoimmunity, which may be explained in part by alterations in serum IFNγ and IL-21 that differentially regulate B10 cell development., Competing Interests: The authors declare no competing financial interests., (© 2016, American College of Rheumatology.)

In cultured cancer cells the E3 ubiquitin ligase Rad18 activates Trans-Lesion Synthesis (TLS) and the Fanconi Anemia (FA) pathway. However, physiological roles of Rad18 in DNA damage tolerance and carcinogenesis are unknown and were investigated here. Primary hematopoietic stem and progenitor cells (HSPC) co-expressed RAD18 and FANCD2 proteins, potentially consistent with a role for Rad18 in FA pathway function during hematopoiesis. However, hematopoietic defects typically associated with fanc-deficiency (decreased HSPC numbers, reduced engraftment potential of HSPC, and Mitomycin C (MMC) -sensitive hematopoiesis), were absent in Rad18(-/-) mice. Moreover, primary Rad18(-/-) mouse embryonic fibroblasts (MEF) retained robust Fancd2 mono-ubiquitination following MMC treatment. Therefore, Rad18 is dispensable for FA pathway activation in untransformed cells and the Rad18 and FA pathways are separable in hematopoietic cells. In contrast with responses to crosslinking agents, Rad18(-/-) HSPC were sensitive to in vivo treatment with the myelosuppressive agent 7,12 Dimethylbenz[a]anthracene (DMBA). Rad18-deficient fibroblasts aberrantly accumulated DNA damage markers after DMBA treatment. Moreover, in vivo DMBA treatment led to increased incidence of B cell malignancy in Rad18(-/-) mice. These results identify novel hematopoietic functions for Rad18 and provide the first demonstration that Rad18 confers DNA damage tolerance and tumor-suppression in a physiological setting., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Non-Hodgkin lymphoma (NHL) is the most commonly diagnosed hematologic cancer of adults in the United States, with the vast majority of NHLs deriving from malignant B lymphocytes that express cell surface CD20. CD20 immunotherapy (rituximab) is widely used to treat NHL, even though the initial effectiveness of rituximab varies widely among patients and typically wanes over time. The mechanisms through which lymphomas initially resist or gain resistance to immunotherapy are not well established. To address this, a preclinical mouse model system was developed to comprehensively identify lymphoma transcriptomic changes that confer resistance to CD20 immunotherapy. The generation of spontaneous primary and familial lymphomas revealed that sensitivity to CD20 immunotherapy was not regulated by differences in CD20 expression, prior exposure to CD20 immunotherapy, or serial in vivo passage. An unbiased forward exome screen of these primary lymphomas was used to validate the utility of this expansive lymphoma cohort, which revealed that increased lymphoma galectin-1 (Gal-1) expression strongly correlated with resistance to immunotherapy. Genetically induced lymphoma Gal-1 expression ablated antibody-dependent lymphoma phagocytosis in vitro and lymphoma sensitivity to CD20 immunotherapy in vivo. Human NHLs also express elevated Gal-1 compared with nonmalignant lymphocytes, demonstrating the ability of this preclinical model system to identify molecular targets that could be relevant to human therapy. This study therefore established a powerful preclinical model system that permits the comprehensive identification of the dynamic lymphoma molecular network that drives resistance to immunotherapy., (© 2016 by The American Society of Hematology.)

Novel therapies for chronic graft-versus-host disease (cGVHD) are needed. Aberrant B-cell activation has been demonstrated in mice and humans with cGVHD. Having previously found that human cGVHD B cells are activated and primed for survival, we sought to further evaluate the role of the spleen tyrosine kinase (Syk) in cGVHD in multiple murine models and human peripheral blood cells. In a murine model of multiorgan system, nonsclerodermatous disease with bronchiolitis obliterans where cGVHD is dependent on antibody and germinal center (GC) B cells, we found that activation of Syk was necessary in donor B cells, but not T cells, for disease progression. Bone marrow-specific Syk deletion in vivo was effective in treating established cGVHD, as was a small-molecule inhibitor of Syk, fostamatinib, which normalized GC formation and decreased activated CD80/86(+) dendritic cells. In multiple distinct models of sclerodermatous cGVHD, clinical and pathological disease manifestations were not eliminated when mice were therapeutically treated with fostamatinib, though both clinical and immunologic effects could be observed in one of these scleroderma models. We further demonstrated that Syk inhibition was effective at inducing apoptosis of human cGVHD B cells. Together, these data demonstrate a therapeutic potential of targeting B-cell Syk signaling in cGVHD., (© 2015 by The American Society of Hematology.)

Desmoglein 3 is a transmembrane component of desmosome complexes that mediate epidermal cell-to-cell adhesion and tissue integrity. Antibody blockade of desmoglein 3 function in pemphigus vulgaris patients leads to skin blistering (acantholysis) and oral mucosa lesions. Desmoglein 3 deficiency in mice leads to a phenotype characterized by cyclic alopecia in addition to the dramatic skin and mucocutaneous acantholysis observed in pemphigus patients. In this study, mice that developed an overt squeaky (sqk) phenotype were identified with obstructed airways, cyclic hair loss, and severe immunodeficiency subsequent to the development of oral lesions and malnutrition. Single-nucleotide polymorphism-based quantitative trait loci mapping revealed a genetic deletion that resulted in expression of a hypomorphic desmoglein 3 protein with a truncation of an extracellular cadherin domain. Because hypomorphic expression of a truncated desmoglein 3 protein led to a spectrum of severe pathology not observed in mice deficient in desmoglein 3, similar human genetic alterations may also disrupt desmosome function and induce a disease course distinct from pathogenesis of pemphigus vulgaris., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Balanced transmembrane signals maintain a competent peripheral B cell pool limited in self-reactive B cells that may produce pathogenic autoantibodies. To identify molecules regulating peripheral B cell survival and tolerance to self-antigens (Ags), a gene modifier screen was performed with B cells from CD22-deficient C57BL/6 (CD22(-/-[B6])) mice that undergo activation-induced cell death (AICD) and fail to up-regulate c-Myc expression after B cell Ag receptor ligation. Likewise, lysozyme auto-Ag-specific B cells in Ig(Tg) hen egg lysozyme (HEL) transgenic mice inhabit the spleen but undergo AICD after auto-Ag encounter. This gene modifier screen identified EndoU, a single-stranded RNA-binding protein of ancient origin, as a major regulator of B cell survival in both models. EndoU gene disruption prevents AICD and normalizes c-Myc expression. These findings reveal that EndoU is a critical regulator of an unexpected and novel RNA-dependent pathway controlling peripheral B cell survival and Ag responsiveness that may contribute to peripheral B cell tolerance.

CD22 is a transmembrane glycoprotein expressed by mature B cells. It inhibits signal transduction by the B-cell receptor and its coreceptor CD19. Recent reports indicate that most human lung cancer cells and cell lines express CD22, making it an important new therapeutic target for lung cancer. The objective of our studies was to independently validate these results with the goal of testing the efficacy of our CD22 immunotoxins on lung cancer cell lines. As determined by quantitative real-time PCR analysis, we found that levels of CD22 mRNA in a panel of human lung cancer cell lines were 200 to 60,000-fold lower than those observed in the human CD22(+) Burkitt lymphoma cells, Daudi. Using flow cytometry with a panel of CD22 monoclonal antibodies and Western blot analyses, we could not detect surface or intracellular expression of CD22 protein in a panel of lung cancer cell lines. In addition, the in vitro proliferation of the lung tumor cell lines was not affected by either CD22 antibodies or our highly potent anti-CD22 immunotoxin. In contrast, CD22(+) Daudi cells expressed high levels of CD22 mRNA and protein, and were sensitive to our CD22 immunotoxin. Importantly, primary non-small cell lung cancers from more than 250 patient specimens did not express detectable levels of CD22 protein as assessed by immunohistochemistry. We conclude that CD22 is not expressed at measurable levels on the surface of lung cancer cells, and that these cells cannot be killed by anti-CD22 immunotoxins.

The spleen regulatory B cell subset with the functional capacity to express IL-10 (B10 cells) modulates both immune responses and autoimmune disease severity. However, the peritoneal cavity also contains relatively high frequencies of functionally defined IL-10-competent B10 cells. In this study, peritoneal cavity B10 cells shared similar cell surface phenotypes with their spleen counterparts. However, peritoneal cavity B10 cells were 10-fold more frequent among B cells than occurred within the spleen, intestinal tract, or mesenteric lymph nodes and were present at higher proportions among the phenotypically defined peritoneal B1a > B1b > B2 cell subpopulations. The development or localization of B10 cells within the peritoneal cavity was not dependent on the presence of commensal microbiota, T cells, IL-10 or B10 cell IL-10 production, or differences between their fetal liver or adult bone marrow progenitor cell origins. The BCR repertoire of peritoneal cavity B10 cells was diverse, as occurs in the spleen, and predominantly included germline-encoded VH and VL regions commonly found in either the conventional or B1 B cell compartments. Thereby, the capacity to produce IL-10 appears to be an intrinsic functional property acquired by clonally diverse B cells. Importantly, IL-10 production by peritoneal cavity B cells significantly reduced disease severity in spontaneous and induced models of colitis by regulating neutrophil infiltration, colitogenic CD4(+) T cell activation, and proinflammatory cytokine production during colitis onset. Thus, the numerically small B10 cell subset within the peritoneal cavity has regulatory function and is important for maintaining homeostasis within gastrointestinal tissues and the immune system.

B cells regulate immune responses by producing antigen-specific antibodies. However, specific B-cell subsets can also negatively regulate T-cell immune responses, and have been termed regulatory B cells. Human and mouse regulatory B cells (B10 cells) with the ability to express the inhibitory cytokine interleukin-10 (IL-10) have been identified. Although rare, B10 cells are potent negative regulators of antigen-specific inflammation and T-cell-dependent autoimmune diseases in mice. How B10-cell IL-10 production and regulation of antigen-specific immune responses are controlled in vivo without inducing systemic immunosuppression is unknown. Using a mouse model for multiple sclerosis, here we show that B10-cell maturation into functional IL-10-secreting effector cells that inhibit in vivo autoimmune disease requires IL-21 and CD40-dependent cognate interactions with T cells. Moreover, the ex vivo provision of CD40 and IL-21 receptor signals can drive B10-cell development and expansion by four-million-fold, and generate B10 effector cells producing IL-10 that markedly inhibit disease symptoms when transferred into mice with established autoimmune disease. The ex vivo expansion and reinfusion of autologous B10 cells may provide a novel and effective in vivo treatment for severe autoimmune diseases that are resistant to current therapies.

Malignant B cells responding to external stimuli are likely to gain a growth advantage in vivo. These cells may therefore maintain surface CD19 expression to amplify transmembrane signals and promote their expansion and survival. To determine whether CD19 expression influences this process, Eμ-Myc transgenic (c-Myc(Tg)) mice that develop aggressive and lethal B cell lymphomas were made CD19 deficient (c-Myc(Tg)CD19⁻/⁻). Compared with c-Myc(Tg) and c-Myc(Tg)CD19⁺/⁻ littermates, the median life span of c-Myc(Tg)CD19⁻/⁻ mice was prolonged by 81-83% (p < 0.0001). c-Myc(Tg)CD19⁻/⁻ mice also lived 42% longer than c-Myc(Tg) littermates following lymphoma detection (p < 0.01). Tumor cells in c-Myc(Tg) and c-Myc(Tg)CD19⁻/⁻ mice were B lineage derived, had a similar phenotype with a large blastlike appearance, invaded multiple lymphoid tissues, and were lethal when adoptively transferred into normal recipient mice. Importantly, reduced lymphomagenesis in c-Myc(Tg)CD19⁻/⁻ mice was not due to reductions in early B cell numbers prior to disease onset. In mechanistic studies, constitutive c-Myc expression enhanced CD19 expression and phosphorylation on active sites. Reciprocally, CD19 expression in c-Myc(Tg) B cells enhanced c-Myc phosphorylation at regulatory sites, sustained higher c-Myc protein levels, and maintained a balance of cyclin D2 expression over that of cyclin D3. These findings define a new and novel c-Myc:CD19 regulatory loop that positively influences B cell transformation and lymphoma progression.

The immune system has evolved into two main arms: the primitive innate arm that is the first line of defense but relatively short-lived and broad acting; and the advanced adaptive arm that generates immunological memory, allowing rapid, specific recall responses. T cell-independent type-2 (TI-2) antigens (Ags) invoke innate immune responses. However, due to its 'at the ready' nature, how the innate arm of the immune system maintains tolerance to potentially abundant host TI-2 Ags remains elusive. Therefore, it is important to define the mechanisms that establish innate immune tolerance. This review highlights recent insights into B cell tolerance to theoretical self TI-2 Ags, and examines how the B cell-restricted sialic acid binding Ig-like lectins (Siglecs), CD22 and Siglec-G, might contribute to this process., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Susac syndrome (SS) is the triad of encephalopathy, branch retinal artery occlusions (BRAOs), and hearing loss. Migraines may herald and accompany encephalopathy. Little is known about pathogenesis. Based on light microscopic findings in brain biopsy material analogous to anti-endothelial cell antibody (AECA)-mediated microvascular injury, we postulated that SS microangiopathy was attributable to AECAs. We examined serum samples from 11 patients with SS for AECAs; 9 were positive by indirect immunofluorescence and Western blot studies. A highly distinctive band on Western blots corresponding to a 50-kDa protein was observed in 8 positive SS samples; the other positive case exhibited specific reactivity with a protein band at 40 kDa. Of the 2 negative cases, 1 had been inactive since 1988; the other was an abortive variant characterized solely by BRAOs. There was enhanced surface binding of SS serum using live endothelial cell substrates compared with samples from healthy subjects. Additional serum samples from apparently healthy patients, 2 with atypical migraines, and patients with other forms of autoinflammatory disease did not show the distinctive band of immunoreactivity. SS is a distinct autoimmune endotheliopathy syndrome associated with AECAs; the antibody target seems specific in many cases and may be a disease biomarker. The exact role of AECAs in disease propagation remains unanswered.

Degos disease is a lethal small vessel angiopathy targeting the skin, gastrointestinal tract, and central nervous system, potentially developing in the setting of known autoimmune disease, although forme fruste primary variants exist. Its pathogenetic basis is unknown. Four cases of Degos disease were encountered in archival material, representing 2 men, ages 38 and 43 years, and 2 females, ages 48 and 2 years; 3 patients died of disease. All had characteristic skin lesions with gastrointestinal involvement; other affected organs included brain in one and pericardium and pleura in another. Skin biopsies showed pauci-inflammatory thrombogenic microangiopathy with endothelial cell injury. Extracutaneous organs demonstrated fibromucinous occlusive arteriopathy. Prominent vascular C5b-9 was seen in the skin, gastrointestinal tract, and brain. All cases had evidence of high expression of interferon-α (based on tissue expression of MXA, a type I interferon-inducible protein), endothelial tubuloreticular inclusions, and an interferon gene signature in peripheral blood mononuclear cells. The MXA expression paralleled the pattern of C5b-9 deposition. Degos disease is a distinct vascular injury syndrome whereby a dysregulated interferon-α response in concert with membranolytic attack complex deposition may contribute to the unique vascular changes. Understanding the pathophysiology of the disease process could lead to more directed therapies, including terminal complement inhibition with agents such as eculizumab.

Background: Aberrant CD40 ligand (CD154) expression occurs on both T cells and B cells in human lupus patients, which is suggested to enhance B cell CD40 signaling and play a role in disease pathogenesis. Transgenic mice expressing CD154 by their B cells (CD154(TG)) have an expanded spleen B cell pool and produce autoantibodies (autoAbs). CD22 deficient (CD22(-/-)) mice also produce autoAbs, and importantly, their B cells are hyper-proliferative following CD40 stimulation ex vivo. Combining these 2 genetic alterations in CD154(TG)CD22(-/-) mice was thereby predicted to intensify CD40 signaling and autoimmune disease due to autoreactive B cell expansion and/or activation., Methodology/principal Findings: CD154(TG)CD22(-/-) mice were assessed for their humoral immune responses and for changes in their endogenous lymphocyte subsets. Remarkably, CD154(TG)CD22(-/-) mice were not autoimmune, but instead generated minimal IgG responses against both self and foreign antigens. This paucity in IgG isotype switching occurred despite an expanded spleen B cell pool, higher serum IgM levels, and augmented ex vivo B cell proliferation. Impaired IgG responses in CD154(TG)CD22(-/-) mice were explained by a 16-fold expansion of functional, mature IL-10-competent regulatory spleen B cells (B10 cells: 26.7×10(6)±6 in CD154(TG)CD22(-/-) mice; 1.7×10(6)±0.4 in wild type mice, p<0.01), and an 11-fold expansion of B10 cells combined with their ex vivo-matured progenitors (B10+B10pro cells: 66×10(6)±3 in CD154(TG)CD22(-/-) mice; 6.1×10(6)±2 in wild type mice, p<0.01) that represented 39% of all spleen B cells., Conclusions/significance: These results demonstrate for the first time that the IL-10-producing B10 B cell subset has the capacity to suppress IgG humoral immune responses against both foreign and self antigens. Thereby, therapeutic agents that drive regulatory B10 cell expansion in vivo may inhibit pathogenic IgG autoAb production in humans.

Peripheral B-cell numbers are tightly regulated by homeostatic mechanisms that influence the transitional and mature B-cell compartments and dictate the size and clonotypic diversity of the B-cell repertoire. B-lymphocyte stimulator (BLyS, a trademark of Human Genome Sciences, Inc.) plays a key role in regulating peripheral B-cell homeostasis. CD22 also promotes peripheral B-cell survival through ligand-dependent mechanisms. The B-cell subsets affected by the absence of BLyS and CD22 signals overlap, suggesting that BLyS- and CD22-mediated survival are intertwined. To examine this, the effects of BLyS insufficiency following neutralizing BLyS mAb treatment in mice also treated with CD22 ligand-blocking mAb were examined. Combined targeting of the BLyS and CD22 survival pathways led to significantly greater clearance of recirculating bone marrow, blood, marginal zone and follicular B cells than either treatment alone. Likewise, BLyS blockade further reduced bone marrow, blood and spleen B-cell numbers in CD22(-/-) mice. Notably, BLyS receptor expression and downstream signaling were normal in CD22(-/-) B cells, suggesting that CD22 does not directly alter BLyS responsiveness. CD22 survival signals were likewise intact in the absence of BLyS, as CD22 mAb treatment depleted blood B cells from mice with impaired BLyS receptor 3 (BR3) signaling. Finally, enforced BclxL expression, which rescues BR3 impairment, did not affect B-cell depletion following CD22 mAb treatment. Thus, the current studies support a model whereby CD22 and BLyS promote the survival of overlapping B-cell subsets but contribute to their maintenance through independent and complementary signaling pathways.

Humoral immunity to T cell-independent type 2 Ags (TI-2 Ag) is critical for protection against encapsulated bacteria such as Streptococcus pneumoniae. The CD21/35 receptor is thought to promote protective humoral immunity to encapsulated bacteria by enabling complement-decorated capsular polysaccharides to coligate the CD21/35-CD19 signaling complex with the B cell Ag receptor (BCR), thereby enhancing Ag-specific B cell activation. However, Ab responses to S. pneumoniae type 3 capsular polysaccharide (PPS-3) and other strong TI-2 Ags were significantly impaired in CD21/35(-/-) but not C3(-/-) or C4(-/-) mice. B cells from CD21/35(-/-) mice expressed significantly higher levels of cell surface CD19. CD21/35(-/-) B cells exhibited enhanced BCR-induced calcium responses and significantly higher expression of the inhibitory programmed death-1 (PD-1) receptor following immunization with a TI-2 Ag or BCR crosslinking. Reducing CD19 expression in CD21/35(-/-) mice normalized BCR-induced calcium responses, PD-1 induction, and PPS-3-specific IgG3 responses and restored protection during S. pneumoniae infection. PD-1 blockade also selectively rescued PPS-3-specific IgG3 responses in CD21/35(-/-) mice. Thereby, CD21/35 promotes protective humoral immunity to S. pneumoniae and other strong TI-2 Ags through a complement-independent pathway by negatively regulating CD19 expression and PD-1 induction.

Despite the demonstrated clinical efficacy of CD20 monoclonal antibody (mAb) for lymphoma therapy, the in vivo mechanisms of tumor depletion remain controversial and variable. To identify the molecular mechanisms responsible for lymphoma killing by CD20 mAb in a homologous system amenable to mechanistic studies and genetic manipulation, a mouse lymphoma model was developed using primary tumor cells from a C57BL/6 Emicro-cMyc transgenic mouse and mouse antimouse CD20 mAbs. CD20 mAb treatment of syngeneic mice with adoptively transferred lymphomas prevented tumor development or significantly prolonged mouse survival depending on tumor volume, mAb dose, and treatment timing. Cooperative FcgammaRIV, FcgammaRIII, and FcgammaRI interactions mediated optimal lymphoma depletion by CD20 mAb in vivo, whereas clodronate-mediated depletion of macrophages eliminated the therapeutic benefit of CD20 mAb. Although CD20 mAbs activated complement in vitro and in vivo, normal and malignant B-cell depletion was induced through C1q- and C3-independent mechanisms. Thus, the ability of CD20 mAbs to deplete malignant B cells in vivo required FcgammaR-dependent use of the innate mononuclear cell immune system. These findings allow for mechanism-based predictions of the biologic outcome of CD20 mAb therapy and treatment optimization.

B cells mediate multiple functions that influence immune and inflammatory responses. In this study, T cell-mediated inflammation was exaggerated in CD19-deficient (Cd19(-/-)) mice and wild-type mice depleted of CD20(+) B cells, whereas inflammation was substantially reduced in mice with hyperactive B cells as a result of CD19 overexpression (hCD19Tg). These inflammatory responses were negatively regulated by a unique CD1d(hi)CD5(+) B cell subset that was absent in Cd19(-/-) mice, represented only 1%-2% of spleen B220(+) cells in wild-type mice, but was expanded to approximately 10% of spleen B220(+) cells in hCD19Tg mice. Adoptive transfer of these CD1d(hi)CD5(+) B cells normalized inflammation in wild-type mice depleted of CD20(+) B cells and in Cd19(-/-) mice. Remarkably, IL-10 production was restricted to this CD1d(hi)CD5(+) B cell subset, with IL-10 production diminished in Cd19(-/-) mice, yet increased in hCD19Tg mice. Thereby, CD1d(hi)CD5(+) B cells represent a unique subset of potent regulatory B cells.

Because pathogens induce infectious symptoms in a time-dependent manner, a rapid immune response is beneficial for defending hosts from pathogens, especially those inducing acute infectious diseases. However, it is largely unknown how the time course of immune responses is regulated. In this study, we demonstrate that B cells deficient in the inhibitory coreceptor CD22 undergo accelerated cell division after Ag stimulation, resulting in rapid generation of plasma cells and Ab production. This finding indicates that CD22 regulates the time course of B cell responses and suggests that CD22 is a good target to shorten the time required for Ab production, thereby augmenting host defense against acute infectious diseases as "universal vaccination."

CD20 antibody depletion of B lymphocytes effectively ameliorates multiple T cell-mediated autoimmune diseases through mechanisms that remain unclear. To address this, a mouse CD20 antibody that depletes >95% of mature B cells in mice with otherwise intact immune systems was used to assess the role of B cells in CD4(+) and CD8(+) T cell activation and expansion in vivo. B cell depletion had no direct effect on T cell subsets or the activation status of CD4(+) and CD8(+) T cells in naive mice. However, B cell depletion impaired CD4(+) T cell activation and clonal expansion in response to protein antigens and pathogen challenge, whereas CD8(+) T cell activation was not affected. In vivo dendritic cell ablation, along with CD20 immunotherapy, revealed that optimal antigen-specific CD4(+) T cell priming required both B cells and dendritic cells. Most importantly, B cell depletion inhibited antigen-specific CD4(+) T cell expansion in both collagen-induced arthritis and autoimmune diabetes mouse models. These results provide direct evidence that B cells contribute to T cell activation and expansion in vivo and offer insights into the mechanism of action for B cell depletion therapy in the treatment of autoimmunity.

The CD22 extracellular domain regulates B lymphocyte function by interacting with alpha2,6-linked sialic acid-bearing ligands. To understand how CD22 ligand interactions affect B cell function in vivo, mouse anti-mouse CD22 mAbs were generated that inhibit CD22 ligand binding to varying degrees. Remarkably, mAbs which blocked CD22 ligand binding accelerated mature B cell turnover by 2- to 4-fold in blood, spleen, and lymph nodes. CD22 ligand-blocking mAbs also inhibited the survival of adoptively transferred normal (73-88%) and malignant (90%) B cells in vivo. Moreover, mAbs that bound CD22 ligand binding domains induced significant CD22 internalization, depleted marginal zone B cells (82-99%), and reduced mature recirculating B cell numbers by 75-85%. The CD22 mAb effects were independent of complement and FcRs, and the CD22 mAbs had minimal effects in CD22AA mice that express mutated CD22 that is not capable of ligand binding. These data demonstrate that inhibition of CD22 ligand binding can disrupt normal and malignant B cell survival in vivo and suggest a novel mechanism of action for therapeutics targeting CD22 ligand binding domains.

Cell surface molecules on lymphocytes positively or negatively modulate the Ag receptor signaling, and thus regulate the fate of the cell. CD22 is a B cell-specific cell surface protein that contains multiple ITIMs in the cytoplasmic tail, and critically regulates B cell activation and survival. CD22 regulation on B cell signaling is complex because CD22 can have both positive and negative roles in various contexts. We generated phosphospecific polyclonal Abs reacting four major CD22 tyrosine motifs (Y762, Y807, Y822, and Y842) and analyzed the pattern and intensity of phosphorylation of these tyrosine residues. The tyrosine motifs, Y762, Y822, and Y842, are considered as ITIM, whereas the other, Y807, is suggested to be important for Grb2 recruitment. Approximately 10% of the four tyrosine residues were constitutively phosphorylated. Upon anti-IgM ligation, CD22 Y762 underwent most rapid phosphorylation, whereas all four tyrosine residues were eventually phosphorylated equally at approximately 35% of all CD22 molecules in the cell. By contrast, anti-CD40 stimulation specifically up-regulated anti-IgM-induced phosphorylation of tyrosines within two ITIM motifs, Y762 and Y842, which was consistent with in vivo finding of the negative role of CD22 in CD40 signaling. Thus, CD22 phosphorylation is not only quantitatively but also qualitatively regulated by different stimulations, which may determine the outcome of B cell signaling.

C3d can function as a molecular adjuvant by binding CD21 and thereby enhancing B cell activation and humoral immune responses. However, recent studies suggest both positive and negative roles for C3d and the CD19/CD21 signaling complex in regulating humoral immunity. To address whether signaling through the CD19/CD21 complex can negatively regulate B cell function when engaged by physiological ligands, diphtheria toxin (DT)-C3d fusion protein and C3dg-streptavidin (SA) complexes were used to assess the role of CD21 during BCR-induced activation and in vivo immune responses. Immunization of mice with DT-C3d3 significantly reduced DT-specific Ab responses independently of CD21 expression or signaling. By contrast, SA-C3dg tetramers dramatically enhanced anti-SA responses when used at low doses, whereas 10-fold higher doses did not augment immune responses, except in CD21/35-deficient mice. Likewise, SA-C3dg (1 microg/ml) dramatically enhanced BCR-induced intracellular calcium concentration ([Ca2+]i) responses in vitro, but had no effect or inhibited [Ca2+]i responses when used at 10- to 50-fold higher concentrations. SA-C3dg enhancement of BCR-induced [Ca2+]i responses required CD21 and CD19 expression and resulted in significantly enhanced CD19 and Lyn phosphorylation, with enhanced Lyn/CD19 associations. BCR-induced CD22 phosphorylation and Src homology 2 domain-containing protein tyrosine phosphatase-1/CD22 associations were also reduced, suggesting abrogation of negative regulatory signaling. By contrast, CD19/CD21 ligation using higher concentrations of SA-C3dg significantly inhibited BCR-induced [Ca2+]i responses and inhibited CD19, Lyn, CD22, and Syk phosphorylation. Therefore, C3d may enhance or inhibit Ag-specific humoral immune responses through both CD21-dependent and -independent mechanisms depending on the concentration and nature of the Ag-C3d complexes.

Immunotherapy with unconjugated CD20 monoclonal antibodies has proven effective for treating non-Hodgkin's lymphoma and autoimmune disease. CD20 immunotherapy depletes mature B cells but does not effectively deplete pre-B or immature B cells, some B cell subpopulations, antibody-producing cells, or their malignant counterparts. Because CD19 is expressed earlier during B cell development, a therapeutic strategy for the treatment of early lymphoblastic leukemias/lymphomas was developed by using CD19-specific monoclonal antibodies in a transgenic mouse expressing human CD19. Pre-B cells and their malignant counterparts were depleted as well as antibody- and autoantibody-producing cells. These results demonstrate clinical utility for the treatment of diverse B cell malignancies, autoimmune disease, and humoral transplant rejection.

B-1a and B-1b lymphocytes were found to exhibit specialized roles in providing immunity to Streptococcus pneumoniae and differ dramatically in their developmental requirements. Transgenic mice overexpressing CD19 (hCD19Tg) generated B-1a cells and natural antibodies that provided protection during infection, while CD19-deficient (CD19(-/-)) mice lacked B-1a cells, lacked natural antibodies, and were more susceptible to infection. By contrast, pneumococcal polysaccharide (PPS) immunization protected CD19(-/-) mice during lethal challenge, whereas hCD19Tg mice remained unprotected. This resulted from differences in the B-1b subset: the key population found to produce protective PPS-specific antibody in both wild-type and CD19(-/-) mice. Thus, CD19(-/-) mice generated B-1b cells and protective adaptive PPS-specific antibody responses, whereas hCD19Tg mice lacked B-1b cells and adaptive PPS-specific antibody responses. This reciprocal contribution of B-1a and B-1b subsets to innate and acquired immunity reveals an unexpected division of labor within the B-1 compartment that is normally balanced by their coordinated development.

Although anti-CD20 immunotherapy effectively treats human lymphoma and autoimmune disease, the in vivo effect of immunotherapy on tissue B cells and their subsets is generally unknown. To address this, anti-mouse CD20 mAbs were used in a mouse model in which the extent and kinetics of tissue B cell depletion could be assessed in vivo. CD20 mAb treatment depleted most mature B cells within 2 days, with 95-98% of B cells in the bone marrow, blood, spleen, lymph nodes, and gut-associated lymphoid tissues depleted by day 7, including marginal zone and follicular B cells. The few spleen B cells remaining after CD20 mAb treatment included pre-B, immature, transitional, and some B1 B cells that expressed CD20 at low levels. By contrast, peritoneal cavity B cells expressed normal CD20 densities and were coated with CD20 mAb, but only 30-43% of B1 cells and 43-78% of B2 cells were depleted by day 7. Spleen B cells adoptively transferred into the peritoneal cavity were similarly resistant to mAb-induced depletion, while transferred B cells that had migrated to the spleen were depleted. However, peritoneal B1 and B2 cells were effectively depleted in mAb-treated wild-type and C3-deficient mice by thioglycolate-induced monocyte migration into this otherwise privileged niche. Inflammation-elicited effector cells did not promote peritoneal cavity B cell depletion in FcR-deficient mice treated with CD20 mAb. Thus, the majority of CD20(+) cells and B cell subsets within lymphoid tissues and the peritoneum could be depleted efficiently in vivo through Fc-dependent, but C-independent pathways during anti-CD20 immunotherapy.

Cell-surface CD19 functions as a general rheostat for defining intrinsic and antigen receptor-induced signaling thresholds critical for clonal expansion of the B cell pool and humoral immunity. CD19 also governs B cell responses initiated through the CD21 receptor, where complement C3d binding to CD21 links humoral immune responses with the innate immune system. Alterations in this signaling pathway can predispose mice and humans to autoantibody production and systemic autoimmunity. Transgenic mice that overexpress CD19 by 20-170% lose tolerance and generate autoantibodies. Likewise, B cells from CD21-deficient mice overexpress CD19 by approximately 50%, which leads to autoantibody production. Autoimmune patients with systemic sclerosis also overexpress CD19 by approximately 20%, which may contribute to their intrinsic B cell abnormalities and autoantibody production. Thus, chronic B cell activation resulting from augmented CD19 expression or signaling through the CD19 pathway may reveal a prototype autoimmune disease susceptibility pathway in mice and humans.

Recent advances in the study of CD22 indicate a complex role for this transmembrane glycoprotein member of the immunoglobulin superfamily in the regulation of B lymphocyte survival and proliferation. CD22 has been previously recognized as a potential lectin-like adhesion molecule that binds alpha2,6-linked sialic acid-bearing ligands and as an important regulator of B-cell antigen receptor (BCR) signaling. However, genetic studies in mice reveal that some CD22 functions are regulated by ligand binding, whereas other functions are ligand-independent and may only require expression of an intact CD22 cytoplasmic domain at the B-cell surface. Until recently, most of the functional activity of CD22 has been widely attributed to CD22's ability to recruit potent intracellular phosphatases and limit the intensity of BCR-generated signals. However, a more complex role for CD22 has recently emerged, including a central role in a novel regulatory loop controlling the CD19/CD21-Src-family protein tyrosine kinase (PTK) amplification pathway that regulates basal signaling thresholds and intensifies Src-family kinase activation after BCR ligation. CD22 is also central to the regulation of peripheral B-cell homeostasis and survival, the promotion of BCR-induced cell cycle progression, and is a potent regulator of CD40 signaling. Herein we discuss our current understanding of how CD22 governs these complex and overlapping processes, how alterations in these tightly controlled regulatory activities may influence autoimmune disease, and the current and future applications of CD22-directed therapies in oncology and autoimmunity.

The interaction of CD22 with alpha2,6-linked sialic acid ligands has been widely proposed to regulate B lymphocyte function and migration. Here, we generated gene-targeted mice that express mutant CD22 molecules that do not interact with these ligands. CD22 ligand binding regulated the expression of cell surface CD22, immunoglobulin M and major histocompatibility complex class II on mature B cells, maintenance of the marginal zone B cell population, optimal B cell antigen receptor-induced proliferation, and B cell turnover rates. However, CD22 negative regulation of calcium mobilization after B cell antigen receptor ligation, CD22 phosphorylation, recruitment of SHP-1 to CD22 and B cell migration did not require CD22 ligand engagement. These observations resolve longstanding questions regarding the physiological importance of CD22 ligand binding in the regulation of B cell function in vivo.

Anti-CD20 antibody immunotherapy effectively treats non-Hodgkin's lymphoma and autoimmune disease. However, the cellular and molecular pathways for B cell depletion remain undefined because human mechanistic studies are limited. Proposed mechanisms include antibody-, effector cell-, and complement-dependent cytotoxicity, the disruption of CD20 signaling pathways, and the induction of apoptosis. To identify the mechanisms for B cell depletion in vivo, a new mouse model for anti-CD20 immunotherapy was developed using a panel of twelve mouse anti-mouse CD20 monoclonal antibodies representing all four immunoglobulin G isotypes. Anti-CD20 antibodies rapidly depleted the vast majority of circulating and tissue B cells in an isotype-restricted manner that was completely dependent on effector cell Fc receptor expression. B cell depletion used both FcgammaRI- and FcgammaRIII-dependent pathways, whereas B cells were not eliminated in FcR common gamma chain-deficient mice. Monocytes were the dominant effector cells for B cell depletion, with no demonstrable role for T or natural killer cells. Although most anti-CD20 antibodies activated complement in vitro, B cell depletion was completely effective in mice with genetic deficiencies in C3, C4, or C1q complement components. That the innate monocyte network depletes B cells through FcgammaR-dependent pathways during anti-CD20 immunotherapy has important clinical implications for anti-CD20 and other antibody-based therapies.

Complement component C3 covalently attaches to Ags following activation, where the C3d cleavage fragment can function as a molecular adjuvant to augment humoral immune responses. C3d is proposed to exert its adjuvant-like activities by targeting Ags to the C3d receptor (CD21/35) expressed by B cells and follicular dendritic cells. To directly assess the importance of CD21/35 in mediating the immunostimulatory effects of C3d, CD21/35-deficient (CD21/35(-/-)) mice were immunized with streptavidin (SA), SA-C3dg tetramers, recombinant HIV gp120 (gp120), or gp120 fused with linear multimers of C3d. Remarkably, SA- and gp120-specific Ab responses were significantly augmented in CD21/35(-/-) mice when these Ags were complexed with C3d in comparison to Ag alone. In fact, primary and secondary Ab responses and Ab-forming cell responses of CD21/35(-/-) mice approached those of wild-type mice immunized with SA-C3dg and gp120-C3d. Thus, C3d can function as a molecular adjuvant in the absence of CD21/35 expression.

Understanding the molecular mechanisms through which CD22 regulates B lymphocyte homeostasis, signal transduction, and tolerance is critical to defining normal B cell function and understanding the role of CD22 in autoimmunity. Therefore, CD22 function was examined in vivo and in vitro using B cells from CD22-deficient (CD22(-/-)) mice. Backcrossing of founder CD22(-/-) mice onto the C57BL/6 (B6) genetic background from a B6/129 mixed background resulted in a dramatically reduced B cell proliferative response following IgM ligation, characterized by a paucity of lymphoblasts and augmented apoptosis. Also, the phenotype of splenic B6 CD22(-/-) B cells was uniquely HSA(high) and IgD(low)/CD21(low) with intermediate levels of CD5 expression, although the percentages of mature and transitional B cells were normal. That B6 CD22(-/-) B cells predominantly underwent apoptosis following IgM ligation correlated with this unique tolerant phenotype, as well as defective induction of the c-Myc:Cullin 1 (CUL1) ubiquitin ligase pathway that is necessary for progression to the S phase of cell cycle. CD40 ligation compensated for CD22 deficiency by restoring lymphoblast development, proliferation, c-Myc and CUL1 expression, and protein ubiquitination/degradation in IgM-stimulated B6 CD22(-/-) B cell cultures. Thereby, this study expands our current understanding of the complex role of CD22 during B cell homeostasis and Ag responsiveness, and reveals that the impact of CD22 deficiency is dictated by the genetic background on which it is rendered. Moreover, this study defines CD22 and CD40 as the first examples of lymphocyte coreceptors that influence induction of the c-Myc:CUL1 ubiquitin ligase pathway.

CD20 plays a role in human B cell proliferation and is an effective target for immunotherapy. In this study, mouse CD20 expression and biochemistry were assessed for the first time using a new panel of CD20-specific mAb, with CD20 function assessed using CD20-deficient (CD20(-/-)) mice. CD20 expression was B cell restricted and was initiated during late pre-B cell development. The frequency and density of CD20 expression increased during B cell maturation in the bone marrow, with a subpopulation of transitional IgM(hi) B cells expressing higher CD20 levels than the majority of mature recirculating B cells. Transitional T1 B cells in the spleen also expressed high CD20 levels, providing a useful new marker for this B cell subset. In CD20(-/-) mice, immature and mature B cell IgM expression was approximately 20-30% lower relative to B cells from wild-type littermates. In addition, CD19-induced intracellular calcium responses were significantly reduced in CD20(-/-) B cells, with a less dramatic effect on IgM-induced responses. These results reveal a role for CD20 in transmembrane Ca(2+) movement in mouse primary B cells that complements previous results obtained using human CD20 cDNA-transfected cell lines. Otherwise, B cell development, tissue localization, signal transduction, proliferation, T cell-dependent antibody responses and affinity maturation were normal in CD20(-/-) mice. Thus, mouse and human CD20 share similar patterns of expression and function. These studies thereby provide an animal model for studying CD20 function in vivo and the molecular mechanisms that influence anti-CD20 immunotherapy.

CD81 is a widely expressed tetraspanin that associates in B cells with CD19 in the CD19-CD21-CD81 signaling complex. CD81 is necessary for normal CD19 expression; cd81(-/-) B cells express lower levels of CD19, especially cd81(-/-) small pre-BII cells, which are almost devoid of surface CD19. The dependence of CD19 expression on CD81 is specific to this particular tetraspanin since cd9(-/-) B cells express normal levels of CD19. Furthermore, expression of human CD81 in mouse cd81(-/-) B cells restored surface CD19 to normal levels. Quantitative analysis of CD19 mRNA demonstrated normal levels, even in cd81(-/-) pre-BII cells. Analysis of CD19 at the protein level identified two CD19 glycoforms in both wild-type and cd81(-/-) B cells. The higher M(r) glycoform is significantly reduced in cd81(-/-) B cells and is endoglycosidase H (endo-H) resistant. In contrast, the low M(r) glycoform is comparably expressed in cd81(-/-) and in wild-type B cells and is endo-H sensitive. Because endo-H sensitivity is tightly correlated with endoplasmic reticulum localization, we suggest that the dependency of CD19 expression on CD81 occurs in a postendoplasmic reticulum compartment where CD81 is necessary for normal trafficking or for surface membrane stability of CD19.

The CD21/35 receptor provides an important link between innate and adaptive immunity. Its importance during protective immune responses to encapsulated extracellular bacteria was assessed using a new line of mice completely deficient in CD21/35 expression (CD21/35(-/-)). CD21/35 expression was essential for the rapid trapping of C3dg-antigen complexes by B cells in vivo, especially in splenic marginal zones. Despite normal B cell development in CD21/35(-/-) mice, T cell-independent and -dependent antibody responses to low-dose antigens were significantly decreased, with a striking impairment in IgG3 responses. Accordingly, CD21/35(-/-) mice were more susceptible to acute lethal Streptococcus pneumoniae infection. Thus, CD21/35 expression is critical for early protective antibody responses to lethal pathogens that rapidly multiply and quickly overwhelm the immune system.

L-selectin mediates the initial capture and subsequent rolling of leukocytes along inflamed vascular endothelium and mediates lymphocyte migration to peripheral lymphoid tissues. Leukocyte activation induces rapid endoproteolytic cleavage of L-selectin from the cell surface, generating soluble L-selectin (sL-selectin). Because human sL-selectin retains ligand-binding activity in vitro, mouse sL-selectin and its in vivo relevance were characterized. Comparable with humans, sL-selectin was present in adult C57BL/6 mouse sera at approximately 1.7 micro g/ml. Similar levels of sL-selectin were present in sera from multiple mouse strains, despite their pronounced differences in cell surface L-selectin expression levels. Adhesion molecule-deficient mice prone to spontaneous chronic inflammation and mice suffering from leukemia/lymphoma had 2.5- and 20-fold increased serum sL-selectin levels, respectively. By contrast, serum sL-selectin levels were reduced by 70% in Rag-deficient mice lacking mature lymphocytes. The majority of serum sL-selectin had a molecular mass of 65-75 kDa, consistent with its lymphocyte origin. Slow turnover may explain the relatively high levels of sL-selectin in vivo. The t(1/2) of sL-selectin, assessed by transferring sera from wild-type mice into L-selectin-deficient mice and monitoring serum sL-selectin levels by ELISA, was >20 h, and it remained detectable for longer than 1 wk. Short-term in vivo lymphocyte migration assays demonstrated that near physiologic levels ( approximately 0.9 micro g/ml) of sL-selectin decreased lymphocyte migration to peripheral lymph nodes by >30%, with dose-dependent inhibition occurring with increasing sL-selectin concentrations. These results suggest that sL-selectin influences lymphocyte migration in vivo and that the increased sL-selectin levels present in certain pathologic conditions may adversely affect leukocyte migration.

CD19 is a B-lymphocyte cell surface molecule that functions as a general response regulator or rheostat, which defines intrinsic and B-cell antigen receptor-induced signalling thresholds that are critical for humoral immunity and expansion of the peripheral B-cell pool. In addition, B-cell responses are influenced by signals transduced through a CD19-CD21 cell surface receptor complex, where the binding of complement C3d to CD21 links humoral immune responses with the innate immune system. This review outlines recent biochemical and genetic studies that characterize the signal transduction pathways utilized by this receptor complex to regulate B-cell intracellular calcium responses.

CD19 and Bruton's tyrosine kinase (Btk) may function along common signaling pathways in regulating intrinsic and B cell Ag receptor (BCR)-induced signals. To identify physical and functional interactions between CD19 and Btk, a CD19-negative variant of the A20 B cell line was isolated, and CD19-deficient (CD19(-/-)) and CD19-overexpressing mice with the X-linked immunodeficient (Xid; Btk) mutation were generated. In A20 cells, Btk physically associated with CD19 following BCR engagement. CD19 and Btk interactions were not required for initial Btk phosphorylation, but CD19 expression maintained Btk in an activated state following BCR engagement. In primary B cells, CD19 signaling also required downstream Btk function since CD19-induced intracellular Ca(2+) ([Ca(2+)](i)) responses were modest in Xid B cells. In addition, CD19 overexpression did not normalize the Xid phenotype and most phenotypic and functional hallmarks of CD19 overexpression were not evident in these mice. However, CD19 and Btk also regulate independent signaling pathways since their combined loss had additive inhibitory effects on BCR-induced [Ca(2+)](i) responses and CD19 deficiency induced a severe immunodeficiency in Xid mice. Thus, CD19 expression amplifies or prolongs Btk-mediated signaling, rather than serving as a required agent for Btk activation. Consistent with this, phosphatidylinositol 3-monophosphate kinase and Akt activation were normal in CD19(-/-) B cells following IgM engagement, although their kinetics of activation was altered. Thus, these biochemical and compound gene dosage studies indicate that Btk activation and [Ca(2+)](i) responses following BCR engagement are regulated through multiple pathways, including a CD19/Src family kinase-dependent pathway that promotes the longevity of Btk signaling.

B lymphocyte antigen receptor (BCR) signals are regulated by CD19, with BCR-induced intracellular calcium ([Ca(2+)](i)) responses enhanced by CD19 co-ligation. In this study, CD19 engagement using a dimeric anti-CD19 antibody induced [Ca(2+)](i) mobilization and significantly enhanced BCR-induced [Ca(2+)](i) responses without a requirement for CD19/BCR co-ligation. Although simultaneous CD19 and BCR engagement significantly enhanced CD19/Lyn complex formation and [Ca(2+)](i) responses, downstream tyrosine phosphorylation of CD22 and multiple other cellular proteins was inhibited, as was SHP1 recruitment to phosphorylated CD22. CD19 overexpression also enhanced BCR-induced [Ca(2+)](i) responses, but down-regulated tyrosine phosphorylation of CD22 and multiple other cellular proteins following BCR ligation. Because CD19 and Lyn expression are genetically titrated in B cells, CD19 engagement may augment BCR-induced [Ca(2+)](i) responses by sequestering the available pool of functional Lyn away from downstream negative regulatory proteins such as CD22. Consistent with this, simultaneous CD19 engagement did not further enhance the BCR-induced [Ca(2+)](i) responses of Lyn- or CD22-deficient B cells. Thus, CD19 recruitment of Lyn may preferentially activate selective signaling pathways downstream of the CD19/Lyn complex to the exclusion of other downstream regulatory and effector pathways. Other receptors may also utilize a similar strategy to regulate kinase availability and downstream intermolecular signaling.

B lymphocytes are critically regulated by signals transduced through the CD19-CD21 cell surface receptor complex, where complement C3d binding to CD21 supplies an already characterized ligand. To determine the extent that CD19 function is controlled by complement activation, CD19-deficient mice (that are hyporesponsive to transmembrane signals) and mice overexpressing CD19 (that are hyperresponsive) were crossed with CD21- and C3-deficient mice. Cell surface CD19 and CD21 expression were significantly affected by the loss of CD21 and C3 expression, respectively. Mature B cells from CD21-deficient littermates had approximately 36% higher cell surface CD19 expression, whereas CD21/35 expression was increased by approximately 45% on B cells from C3-deficient mice. Negative regulation of CD19 and CD21 expression by CD21 and C3, respectively, may be functionally significant because small increases in cell surface CD19 overexpression can predispose to autoimmunity. Otherwise, B cell development and function in CD19-deficient and -overexpressing mice were not significantly affected by a simultaneous loss of CD21 expression. Although CD21-deficient mice were found to express a hypomorphic cell surface CD21 protein at low levels that associated with mouse CD19, C3 deficiency did not significantly affect B cell development and function in CD19-deficient or -overexpressing mice. These results, and the severe phenotype exhibited by CD19-deficient mice compared with CD21- or C3-deficient mice, collectively demonstrate that CD19 can regulate B cell signaling thresholds independent of CD21 engagement and complement activation.

CD19 and the Src family protein tyrosine kinases (PTKs) are important regulators of intrinsic signaling thresholds in B cells. Regulation is achieved by cross-talk between Src family PTKs and CD19; Lyn is essential for CD19 phosphorylation, while CD19 establishes an Src family PTK activation loop that amplifies kinase activity. However, CD19-deficient (CD19(-/-)) B cells are hyporesponsive to transmembrane signals, while Lyn-deficient (Lyn(-/-)) B cells exhibit a hyper-responsive phenotype resulting in autoimmunity. To identify the outcome of interactions between CD19 and Src family PTKs in vivo, B cell function was examined in mice deficient for CD19 and Lyn (CD19/Lyn(-/-)). Remarkably, CD19 deficiency suppressed the hyper-responsive phenotype of Lyn(-/-) B cells and autoimmunity characterized by serum autoantibodies and immune complex-mediated glomerulonephritis in Lyn(-/-) mice. Consistent with Lyn and CD19 each regulating conventional B cell development, B1 cell development was markedly reduced by Lyn deficiency, with further reductions in the absence of CD19 expression. Tyrosine phosphorylation of Fyn and other cellular proteins induced following B cell Ag receptor ligation was dramatically reduced in CD19/Lyn(-/-) B cells relative to Lyn(-/-) B cells, while Syk phosphorylation was normal. In addition, the enhanced intracellular Ca(2+) responses following B cell Ag receptor ligation that typify Lyn deficiency were delayed by the loss of CD19 expression. BCR-induced proliferation and humoral immune responses were also markedly inhibited by CD19/Lyn deficiency. These findings demonstrate that while the CD19/Lyn amplification loop is a major regulator of signal transduction thresholds in B lymphocytes, CD19 regulation of other Src family PTKs also influences B cell function and the development of autoimmunity.

B lymphocyte development and function depend upon the activity of intrinsic and B cell antigen receptor (BCR)-induced signals. These signals are interpreted, amplified, fine-tuned, or suppressed through the precise actions of specialized cell surface coreceptors, or "response regulators," that inform B cells of their extracellular environment. Important cell surface response regulators include the CD19/CD21 complex, CD22, and CD72. CD19 establishes a novel Src-family protein tyrosine kinase (PTK) amplification loop that regulates basal signaling thresholds and intensifies Src-family PTK activation following BCR ligation. In turn, CD22 limits the intensity of CD19-dependent, BCR-generated signals through the recruitment of potent phosphotyrosine and phosphoinositide phosphatases. Herein we discuss our current understanding of how CD19/CD21 and CD22 govern the emergence and intensity of BCR-mediated signals, and how alterations in these tightly controlled regulatory activities contribute to autoimmunity in mice and humans.