A new mode for the diagnosis of angioimmunoblastic T-cell lymphoma.

In order to explore a new mode for the diagnosis of angioimmunoblastic T-cell lymphoma (AITL), 31 cases of AITL and 28 cases of peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) were used as the study subjects. Identifying T follicular helper (TFH) cells with CD4, CD10, Bcl-6, and PD-1, identifying proliferative B cells with CD20 and EZH2, identifying proliferative follicular dendritic cells (FDCs) with CD21 and CD23, and analyzing the value of TFH/B/FDC proliferation and immunolocalization in the diagnosis of AITL. (1) Outside the inherent lymphoid follicles, simultaneous proliferation of TFH/B/FDC (a new diagnostic mode) were observed in AITL [83.87%; 26/31], with their immunolocalizations in the same site [83.87%; 26/31], while this phenomenon was not observed in 28 cases of PTCL-NOS (P<0.05). (2) The sensitivity and specificity of using this new mode to diagnose AITL were both high (83.87%, 100%), which was superior to CD2 (100%, 0%), CD3 (100%, 0%), CD4 (100%, 32.14%), CD5 (100%, 25%), CD10 (61.9%, 100%), Bcl-6 (42.86%, 100%), PD-1 (83.87%, 96.43%), and its Youden Index (0.84) was the highest. The areas under the curve (AUC) of CD10, Bcl-6, PD-1, and new mode to diagnosis AITL were 0.81, 0.71, 0.90, and 0.92, respectively, while the new mode had the highest AUC. The simultaneous proliferation of TFH/B/FDC cells outside the inherent lymphoid follicles can be used to assist in the diagnosis of AITL, and the simultaneous spatiotemporal proliferation of TFH/B/FDC cells is a specific immunomorphology of AITL.

Dual stimulation by autoantigen and CpG fosters the proliferation of exhausted rheumatoid factor-specific CD21low B cells in hepatitis C virus-cured mixed cryoglobulinemia.

Introduction: Hepatitis C virus (HCV) causes mixed cryoglobulinemia (MC) by driving clonal expansion of B cells expressing B cell receptors (BCRs), often encoded by the VH1-69 variable gene, endowed with both rheumatoid factor (RF) and anti-HCV specificity. These cells display an atypical CD21low phenotype and functional exhaustion evidenced by unresponsiveness to BCR and Toll-like receptor 9 (TLR9) stimuli. Although antiviral therapy is effective on MC vasculitis, pathogenic B cell clones persist long thereafter and can cause virus-independent disease relapses. Methods: Clonal B cells from patients with HCV-associated type 2 MC or healthy donors were stimulated with CpG or heath-aggregated IgG (as surrogate immune complexes) alone or in combination; proliferation and differentiation were then evaluated by flow cytometry. Phosphorylation of AKT and of the p65 NF-kB subunit were measured by flow cytometry. TLR9 was quantified by qPCR and by intracellular flow cytometry, and MyD88 isoforms were analyzed using RT-PCR. Discussion: We found that dual triggering with autoantigen and CpG restored the capacity of exhausted VH1-69pos B cells to proliferate. The signaling mechanism for this BCR/TLR9 crosstalk remains elusive, since TLR9 mRNA and protein as well as MyD88 mRNA were normally expressed and CpG-induced phosphorylation of p65 NF-kB was intact in MC clonal B cells, whereas BCR-induced p65 NF-kB phosphorylation was impaired and PI3K/Akt signaling was intact. Our findings indicate that autoantigen and CpG of microbial or cellular origin may unite to foster persistence of pathogenic RF B cells in HCV-cured MC patients. BCR/TLR9 crosstalk might represent a more general mechanism enhancing systemic autoimmunity by the rescue of exhausted autoreactive CD21low B cells.

Autoimmunity in Down's syndrome via cytokines, CD4 T cells and CD11c+ B cells.

Down's syndrome (DS) presents with a constellation of cardiac, neurocognitive and growth impairments. Individuals with DS are also prone to severe infections and autoimmunity including thyroiditis, type 1 diabetes, coeliac disease and alopecia areata1,2. Here, to investigate the mechanisms underlying autoimmune susceptibility, we mapped the soluble and cellular immune landscape of individuals with DS. We found a persistent elevation of up to 22 cytokines at steady state (at levels often exceeding those in patients with acute infection) and detected basal cellular activation: chronic IL-6 signalling in CD4 T cells and a high proportion of plasmablasts and CD11c+TbethighCD21low B cells (Tbet is also known as TBX21). This subset is known to be autoimmune-prone and displayed even greater autoreactive features in DS including receptors with fewer non-reference nucleotides and higher IGHV4-34 utilization. In vitro, incubation of naive B cells in the plasma of individuals with DS or with IL-6-activated T cells resulted in increased plasmablast differentiation compared with control plasma or unstimulated T cells, respectively. Finally, we detected 365 auto-antibodies in the plasma of individuals with DS, which targeted the gastrointestinal tract, the pancreas, the thyroid, the central nervous system, and the immune system itself. Together, these data point to an autoimmunity-prone state in DS, in which a steady-state cytokinopathy, hyperactivated CD4 T cells and ongoing B cell activation all contribute to a breach in immune tolerance. Our findings also open therapeutic paths, as we demonstrate that T cell activation is resolved not only with broad immunosuppressants such as Jak inhibitors, but also with the more tailored approach of IL-6 inhibition.

Multiparameter analysis of human B lymphocytes identifies heterogeneous CD19+ CD21lo subsets.

Autoreactive B cell subsets have been described in a variety of settings, using multiple classification schemes and cell surface markers also found on healthy cells. CD19+ CD21lo B cells have been identified as an autoreactive-prone subset of B cells, although the downregulation of CD21 has been observed on a variety of B cell subsets in health and disease. This variation has led to confusion regarding the meaning and applicability of the loss or reduction of CD21 in peripheral B cells. To better understand the relationships between commonly used B cell markers and their associated characteristics, we analyzed human B cells from healthy participants using multiparameter flow cytometry and the visualization algorithm, tSNE. This approach revealed significant phenotypic overlap amongst five previously described autoimmune-prone B cell subsets, including CD19+ CD10- CD27- CD21lo B cells. Interestingly, 12 different subpopulations of CD19+ CD21lo B cells were identified, some of which mapped to previously described autoreactive populations, while others were consistent with healthy B cells. This suggests that CD21 is downregulated in a variety of circumstances involving B cell activation, all of which are present in low numbers even in healthy individuals. These findings describe the utility of unbiased multiparameter analysis using a relatively limited panel of flow cytometry markers to analyze autoreactive-prone and normal activated B cells.

Rapid single-cell identification of Epstein-Barr virus-specific T-cell receptors for cellular therapy.

BACKGROUND AND AIMS: Epstein-Barr virus (EBV) is associated with solid and hematopoietic malignancies. After allogeneic stem cell transplantation, EBV infection or reactivation represents a potentially life-threatening condition with no specific treatment available in clinical routine. In vitro expansion of naturally occurring EBV-specific T cells for adoptive transfer is time-consuming and influenced by the donor's T-cell receptor (TCR) repertoire and requires a specific memory compartment that is non-existent in seronegative individuals. The authors present highly efficient identification of EBV-specific TCRs that can be expressed on human T cells and recognize EBV-infected cells. METHODS AND RESULTS: Mononuclear cells from six stem cell grafts were expanded in vitro with three HLA-B*35:01- or four HLA-A*02:01-presented peptides derived from six EBV proteins expressed during latent and lytic infection. Epitope-specific T cells expanded on average 42-fold and were single-cell-sorted and TCRαß-sequenced. To confirm specificity, 11 HLA-B*35:01- and six HLA-A*02:01-restricted dominant TCRs were expressed on reporter cell lines, and 16 of 17 TCRs recognized their presumed target peptides. To confirm recognition of virus-infected cells and assess their value for adoptive therapy, three selected HLA-B*35:01- and four HLA-A*02:01-restricted TCRs were expressed on human peripheral blood lymphocytes. All TCR-transduced cells recognized EBV-infected lymphoblastoid cell lines. CONCLUSIONS: The authors' approach provides sets of EBV epitope-specific TCRs in two different HLA contexts. Resulting cellular products do not require EBV-seropositive donors, can be adjusted to cell subsets of choice with exactly defined proportions of target-specific T cells, can be tracked in vivo and will help to overcome unmet clinical needs in the treatment and prophylaxis of EBV reactivation and associated malignancies.

How Epstein-Barr virus envelope glycoprotein gp350 tricks the CR2? A molecular dynamics study.

The connection of Epstein Barr virus (EBV) with diseases such as Burkitt Lymphoma, Hodgkin disease, multiple sclerosis, systemic lupus erythematosus and various B-cell lymphomas made EBV glycoproteins one of the most popular vaccine immunogens. As a protein being encoded by EBV, the viral membrane envelope protein gp350 is studied extensively due to its abundancy on the surface and its interaction with complementary receptor, CR2. The binding of CR2 and gp350 not only leads to the entrance of the virus to the B-cells, but also prevents CR2 and C3d protein interactions that are required for immune response. Thus, understanding the inhibition of gp350 activity is crucial for vaccine development. Although, the active residues on gp350 structure were determined by several mutational studies, the exact mechanism of CR2 binding is still not clear. To this end, we have performed molecular docking followed by molecular dynamics simulations and MM-PBSA on wildtype and several mutated gp350 and CR2 structures. Apart from identifying crucial amino acids, the results of per-residue decomposition energy analysis clarified the individual energy contributions of amino acids and were also found to be accurate in differentiating the active site residues in CR2 binding. Here, we highlight the role of binding region residues (linker-1) but more interestingly, the dynamic relation between the distant sites of gp350 (linker-2 and D3 residues) and CR2. These findings can lead further vaccine development strategies by pointing to the importance of computationally found novel regions that can be potentially used to modulate gp350 activity.

Abacavir inhibits but does not cause self-reactivity to HLA-B*57:01-restricted EBV specific T cell receptors.

Pre-existing pathogen-specific memory T cell responses can contribute to multiple adverse outcomes including autoimmunity and drug hypersensitivity. How the specificity of the T cell receptor (TCR) is subverted or seconded in many of these diseases remains unclear. Here, we apply abacavir hypersensitivity (AHS) as a model to address this question because the disease is linked to memory T cell responses and the HLA risk allele, HLA-B*57:01, and the initiating insult, abacavir, are known. To investigate the role of pathogen-specific TCR specificity in mediating AHS we performed a genome-wide screen for HLA-B*57:01 restricted T cell responses to Epstein-Barr virus (EBV), one of the most prevalent human pathogens. T cell epitope mapping revealed HLA-B*57:01 restricted responses to 17 EBV open reading frames and identified an epitope encoded by EBNA3C. Using these data, we cloned the dominant TCR for EBNA3C and a previously defined epitope within EBNA3B. TCR specificity to each epitope was confirmed, however, cloned TCRs did not cross-react with abacavir plus self-peptide. Nevertheless, abacavir inhibited TCR interactions with their cognate ligands, demonstrating that TCR specificity may be subverted by a drug molecule. These results provide an experimental road map for future studies addressing the heterologous immune responses of TCRs including T cell mediated adverse drug reactions.

Uncontrolled CD21low age-associated and B1 B cell accumulation caused by failure of an EGR2/3 tolerance checkpoint.

CD21low age-associated or atypical memory B cells are autoantibody enriched and poised for plasma cell differentiation. These cells overaccumulate in chronic infections, autoimmune disease, and immunodeficiency, posing the question of what checkpoints normally oppose their accumulation. Here, we reveal a critical role for paralogous calcium-NFAT-regulated transcription factors EGR2 and EGR3 that are induced in self-reactive B cells. CD21low and B1 B cells lacking EGR2 and EGR3 accumulate and circulate in young mice in numbers 10- to 20-fold greater than normal and overexpress a large set of EGR2 ChIP-seq target genes, including known drivers of plasma cell differentiation. Most follicular B cells constitutively express Egr2 proportionally to surface IgM downregulation by self-antigens, and EGR2/3 deficiency abolishes this cardinal feature of B cell anergy. These results explain the cardinal features of B cell anergy, define a key transcriptional checkpoint repressing CD21low B cell formation, and inform how NFATC1 or EGR2 mutations promote B1 cell-derived chronic lymphocytic leukemias.

Uncovering early events in primary Epstein-Barr virus infection using a rabbit model.

Epstein-Barr virus (EBV) is an oncogenic herpesvirus implicated in the pathogenesis of several malignant and non-malignant conditions. However, a number of fundamental aspects about the biology of EBV and the mechanism(s) by which this virus induces pathology remain unknown. One major obstacle has been the lack of a suitable animal model for EBV infection. In this study, using our recently established rabbit model of EBV infection, we examined the early events following primary EBV infection. We show that, both immunocompetent and immunosuppressed animals were readily susceptible to EBV infection. However, immunosuppressed animals showed marked splenomegaly and widespread infection. Following EBV infection, the virus primarily targeted naïve IgM+, CD20+, CD21+ and CD79a+ B cells. Infected cells expressed varying sets of viral latent/lytic gene products. Notably, co-expression of latent and lytic proteins in the same cell was not observed. Infected cells in type 0/1 latency (EBERs+), were small and proliferating (Ki67+). By contrast, cells in type 2/3 latency (LMP1+), were large, non-proliferating (Ki-67-) and p53+. Although infected B-cells were widely present in splenic follicles, they did not express germinal center marker, BCL-6. Taken together, this study shows for the first time, some of the early events following primary EBV infection.

Serum, but Not Saliva, CXCL13 Levels Associate With Infiltrating CXCL13+ Cells in the Minor Salivary Gland Lesions and Other Histologic Parameters in Patients With Sjögren's Syndrome.

Recent studies suggest that elevated CXCL13 serum levels in patients with primary Sjögren's syndrome (pSS) associate with minor salivary gland (MSG) histologic features, disease severity, as well as high-risk status for non-Hodgkin lymphoma (NHL) development and NHL itself. In contrast, limited discriminative value of CXCL13 saliva levels has been reported. Prompt by these reports, we sought to validate the clinical utility of CXCL13 by investigating potential correlations of serum and saliva levels with MSG histopathologic [including CXCL13+-cell number, severity of infiltrates and germinal center (GC) formation], serologic and clinical parameters, as well as NHL. CXCL13 levels were evaluated in paired serum and saliva specimens of 45 pSS patients (15 with NHL; pSS-associated NHL: SSL), 11 sicca-controls (sicca-complaining individuals with negative MSG biopsy and negative autoantibody profile), 10 healthy individuals (healthy-controls) and 6 non-SS-NHLs. CXCL13+-cells were measured in paired MSG-tissues of 22 of pSS patients studied (including 7 SSLs) and all sicca-controls. CXCL13 serum levels were significantly increased in pSS and SSL patients compared to sicca- and healthy-controls and were positively correlated with the CXCL13+-cell number and biopsy focus-score. Serum CXCL13 was significantly higher in pSS patients with GCs, rheumatoid factor, hypocomplementemia, high disease activity, NHL and in high-risk patients for NHL development. CXCL13 saliva levels were significantly increased in SSL patients (compared to non-SS-NHLs), patients with GCs and in high-risk for NHL patients. Univariate analysis revealed that CXCL13 serum, but not saliva, levels were associated with lymphoma, an association that did not survive multivariate analysis. Conclusively, our findings confirm that serum, but not saliva, levels of CXCL13 are associated with histologic, serologic and clinical features indicative of more severe pSS.

Gastric epithelial attachment of Helicobacter pylori induces EphA2 and NMHC-IIA receptors for Epstein-Barr virus.

Epstein-Barr virus (EBV)-associated gastric cancer belongs to 1 of the 4 subtypes of gastric cancer and accounts for 10% of total gastric cancers. However, most cases of gastric cancer have a history of Helicobacter pylori infection. Therefore, we investigated the possibility that H. pylori infection promotes the development of EBV-associated gastric cancer. H. pylori was exposed to principal EBV receptor, CD21, negative gastric epithelial cells, and then infected with EBV recombinant expressing enhanced green fluorescent protein. Changes in EBV infectivity due to prior H. pylori exposure were analyzed using flow cytometry. The treatment of gastric epithelial cells with H. pylori increased the efficiency of EBV infection. An increase was also observed when CagA-deficient, VacA-deficient, and FlaA-deficient H. pylori strains were used, but not when cag pathogenicity island-deficient H. pylori was used. The treatment of epithelial cells with H. pylori induced the expression of accessory EBV receptors, EphA2 and NMHC-IIA, and increased the efficiency of EBV infection depending on their expression levels. When gastric epithelial cells were treated with EPHA2 or NMHC-IIA siRNA, EBV infection via H. pylori attachment was decreased. The adhesion of H. pylori induced the expression of accessory EBV receptors in gastric epithelial cells and increased the efficiency of EBV infection.

CD27-CD38lowCD21low B-Cells Are Increased in Axial Spondyloarthritis.

B-cells have received little attention in axial spondyloarthritis (axSpA) and for this reason their role in pathogenesis remains unclear. However, there are indications that B-cells may be involved in the disease process. Our objective was to obtain insights into the composition of the peripheral B-cell compartment of axSpA patients compared to healthy donors (HD) and patients with primary Sjögren's syndrome (pSS), a typical B-cell-associated autoimmune disease. Special emphasis was given to CD27-negative B-cells expressing low levels of CD21 (CD21low B-cells), since this subset is implicated in autoimmune diseases with strong involvement of B-cells. Transitional B-cells (CD38hi) were excluded from the analysis of the CD27-CD21low B-cell compartment. This study included 45 axSpA patients, 20 pSS patients and 30 HDs. Intriguingly, compared to HDs the frequency of CD27-CD38lowCD21low B-cells was significantly elevated in both axSpA and pSS patients (P<0.0001 for both comparisons). The frequency of CD27-CD38lowCD21low B-cells expressing the activation-induced immune markers T-bet and CD11c was decreased in axSpA patients compared to HDs. A higher proportion of CD27-CD38lowCD21low B-cells expressed the chemokine receptor CXCR3 in axSpA compared to HDs, suggestive for active involvement of these cells in an inflammatory process. The frequency of CD27-CD38lowCD21low B-cells in axSpA patients correlated positively with age and erythrocyte sedimentation rate. Furthermore, axSpA patients with extra-skeletal manifestations (ESM) showed increased frequencies of CD27-CD38lowCD21low B-cells compared to patients without ESM. In conclusion, our findings are suggestive of active B-cell involvement in the pathogenesis of axSpA, against prevailing dogma.

T-bet+CD27+CD21- B cells poised for plasma cell differentiation during antibody-mediated rejection of kidney transplants.

Alloimmune responses driven by donor-specific antibodies (DSAs) can lead to antibody-mediated rejection (ABMR) in organ transplantation. Yet, the cellular states underlying alloreactive B cell responses and the molecular components controlling them remain unclear. Using high-dimensional profiling of B cells in a cohort of 96 kidney transplant recipients, we identified expanded numbers of CD27+CD21- activated memory (AM) B cells that expressed the transcription factor T-bet in patients who developed DSAs and progressed to ABMR. Notably, AM cells were less frequent in DSA+ABMR- patients and at baseline levels in DSA- patients. RNA-Seq analysis of AM cells in patients undergoing ABMR revealed these cells to be poised for plasma cell differentiation and to express restricted IGHV sequences reflective of clonal expansion. In addition to T-bet, AM cells manifested elevated expression of interferon regulatory factor 4 and Blimp1, and upon coculture with autologous T follicular helper cells, differentiated into DSA-producing plasma cells in an IL-21-dependent manner. The frequency of AM cells was correlated with the timing and severity of ABMR manifestations. Importantly, T-bet+ AM cells were detected within kidney allografts along with their restricted IGHV sequences. This study delineates a pivotal role for AM cells in promoting humoral responses and ABMR in organ transplantation and highlights them as important therapeutic targets.

Generation of Epstein-Barr Virus Antigen-Specific T Cell Receptors Recognizing Immunodominant Epitopes of LMP1, LMP2A, and EBNA3C for Immunotherapy.

Epstein-Barr virus (EBV) infections in healthy individuals are usually cleared by immune cells, wherein CD8+ T lymphocytes play the most important role. However, in some immunocompromised individuals, EBV infections can lead to the development of cancer in B, T, natural killer (NK) cells and epithelial cells. Most EBV-associated cancers express a limited number of virus-specific antigens such as latent membrane proteins (LMP1 and LMP2) and nuclear proteins (EBNA1, -2, EBNA3A, -B, -C, and EBNA-LP). These antigens represent true tumor-specific antigens and can be considered useful targets for T cell receptor (TCR) gene therapy to treat EBV-associated diseases. We used a TCR isolation platform based on a single major histocompatibility complex class I (MHC I) K562 cell library for the detection, isolation, and re-expression of TCRs targeting immunodominant peptide MHC (pMHC). Mature dendritic cells (mDCs) were pulsed with in vitro-transcribed (ivt) RNA encoding for the selected antigen to stimulate autologous T cells. The procedure allowed the mDCs to select an immunogenic epitope of the antigen for processing and presentation on the cell surface in combination with the most suitable MHC I molecule. We isolated eight EBV-specific TCRs. They recognize various pMHCs of EBV antigens LMP1, LMP2A, and EBNA3C, some of them described previously and some newly identified in this study. The TCR genes were molecularly cloned into retroviral vectors and the resultant TCR-engineered T cells secreted interferon-γ after antigen contact and were able to lyse tumor cells. The EBV-specific TCRs can be used as a basis for the generation of a TCR library, which provides a valuable source of TCRs for the production of EBV-specific T cells to treat EBV-associated diseases in patients with different MHC I types.

Deep Phenotyping of CD11c+ B Cells in Systemic Autoimmunity and Controls.

Circulating CD11c+ B cells are a key phenomenon in certain types of autoimmunity but have also been described in the context of regular immune responses (i.e., infections, vaccination). Using mass cytometry to profile 46 different markers on individual immune cells, we systematically initially confirmed the presence of increased CD11c+ B cells in the blood of systemic lupus erythematosus (SLE) patients. Notably, significant differences in the expression of CD21, CD27, and CD38 became apparent between CD11c- and CD11c+ B cells. We observed direct correlation of the frequency of CD21-CD27- B cells and CD21-CD38- B cells with CD11c+ B cells, which were most pronounced in SLE compared to primary Sjögren's syndrome patients (pSS) and healthy donors (HD). Thus, CD11c+ B cells resided mainly within memory subsets and were enriched in CD27-IgD-, CD21-CD27-, and CD21-CD38- B cell phenotypes. CD11c+ B cells from all donor groups (SLE, pSS, and HD) showed enhanced CD69, Ki-67, CD45RO, CD45RA, and CD19 expression, whereas the membrane expression of CXCR5 and CD21 were diminished. Notably, SLE CD11c+ B cells showed enhanced expression of the checkpoint molecules CD86, PD1, PDL1, CD137, VISTA, and CTLA-4 compared to HD. The substantial increase of CD11c+ B cells with a CD21- phenotype co-expressing distinct activation and checkpoint markers, points to a quantitative increased alternate (extrafollicular) B cell activation route possibly related to abnormal immune regulation as seen under the striking inflammatory conditions of SLE which shows a characteristic PD-1/PD-L1 upregulation.

Systematic memory B cell archiving and random display shape the human splenic marginal zone throughout life.

Human memory B cells (MBCs) are generated and diversified in secondary lymphoid tissues throughout the organism. A paired immunoglobulin (Ig)-gene repertoire analysis of peripheral blood (PB) and splenic MBCs from infant, adult, and elderly humans revealed that throughout life, circulating MBCs are comprehensively archived in the spleen. Archive MBC clones are systematically preserved and uncoupled from class-switching. Clonality in the spleen increases steadily, but boosts at midlife, thereby outcompeting small clones. The splenic marginal zone (sMZ) represents a primed MBC compartment, generated from a stochastic exchange within the archive memory pool. This is supported by functional assays, showing that PB and splenic CD21+ MBCs acquire transient CD21high expression upon NOTCH2-stimulation. Our study provides insight that the human MBC system in PB and spleen is composed of three interwoven compartments: the dynamic relationship of circulating, archive, and its subset of primed (sMZ) memory changes with age, thereby contributing to immune aging.

High proportion of anergic B cells in the bone marrow defined phenotypically by CD21(-/low)/CD38- expression predicts poor survival in diffuse large B cell lymphoma.

BACKGROUND: Diffuse large B cell lymphoma (DLBCL) is the commonest lymphoma that is highly aggressive where one-third of the patients relapse despite effective treatment. Interaction between the lymphoma cells and the non-clonal immune cells within the bone marrow microenvironment is thought to play a critical role in the pathogenesis of DLBCL. METHODS: We used flow cytometry to characterize the proportion of B cell subpopulations in the bone marrow (N = 47) and peripheral blood (N = 54) of 75 DLBCL patients at diagnosis and study their impact on survival. RESULTS: Anergic B cells in the bone marrow (BM), characterized as having CD21(-/low)/CD38- expression, influenced survival with high numbers (defined as > 13.9%) being associated with significantly shorter overall survival (59.7 months vs 113.6 months, p = 0.0038). Interestingly, low numbers of anergic B cells in the BM (defined as ≤13.9%) was associated with germinal center B cell type of DLBCL (p = 0.0354) that is known to have superior rates of survival when compared to activated B cell type. Finally, Cox regression analysis in our cohort of patients established that the inferior prognosis of having high numbers of anergic B cells in the bone marrow was independent of the established Revised International Prognostic Index (R-IPI) score. CONCLUSIONS: High proportion of anergic B cells in the BM characterized by CD21(-/low)/CD38- expression predicts poor survival outcomes in DLBCL.

Notch signaling induces a transcriptionally permissive state at the Complement C3d Receptor 2 (CR2) promoter in a pre-B cell model.

During mammalian lymphoid development, Notch signaling is necessary at multiple stages of T lymphopoiesis, including lineage commitment, and later stages of T cell effector differentiation. In contrast, outside of a defined role in the development of splenic marginal zone B cells, there is conflicting evidence regarding whether Notch signaling plays functional roles in other B cell sub-populations. Complement receptor 2 (CR2) modulates BCR-signaling and is tightly regulated throughout differentiation. During B lymphopoiesis, CR2 is detected on immature and mature B cells with high surface expression on marginal zone B cells. Here, we have explored the possibility that Notch regulates human CR2 transcriptional activity using in vitro models including a co-culture system, co-transfection gene reporters and chromatin accessibility assays. We provide evidence that Notch signaling regulates CR2 promoter activity in a mature B cell line, as well as the induction of endogenous CR2 mRNA in a non-expressing pre-B cell line. The dynamics of endogenous gene activation suggests additional unidentified factors are required to mediate surface CR2 expression on immature and mature B lineage cells.

A novel mouse model expressing human forms for complement receptors CR1 and CR2.

BACKGROUND: The complement cascade is increasingly implicated in development of a variety of diseases with strong immune contributions such as Alzheimer's disease and Systemic Lupus Erythematosus. Mouse models have been used to determine function of central components of the complement cascade such as C1q and C3. However, species differences in their gene structures mean that mice do not adequately replicate human complement regulators, including CR1 and CR2. Genetic variation in CR1 and CR2 have been implicated in modifying disease states but the mechanisms are not known. RESULTS: To decipher the roles of human CR1 and CR2 in health and disease, we engineered C57BL/6J (B6) mice to replace endogenous murine Cr2 with human complement receptors, CR1 and CR2 (B6.CR2CR1). CR1 has an array of allotypes in human populations and using traditional recombination methods (Flp-frt and Cre-loxP) two of the most common alleles (referred to here as CR1long and CR1short) can be replicated within this mouse model, along with a CR1 knockout allele (CR1KO). Transcriptional profiling of spleens and brains identified genes and pathways differentially expressed between mice homozygous for either CR1long, CR1short or CR1KO. Gene set enrichment analysis predicts hematopoietic cell number and cell infiltration are modulated by CR1long, but not CR1short or CR1KO. CONCLUSION: The B6.CR2CR1 mouse model provides a novel tool for determining the relationship between human-relevant CR1 alleles and disease.

Naive- and Memory-like CD21low B Cell Subsets Share Core Phenotypic and Signaling Characteristics in Systemic Autoimmune Disorders.

An expansion of CD21low B cells has been described in a variety of diseases associated with persistent immune stimulation as in chronic infection, immunodeficiency, or autoimmunity. Different developmental stages of CD21low B cells have been highlighted in specific diseases; however, a systematic comparison of distribution, phenotype, and signaling capacity of these populations has not yet been performed to delineate the pivotal character of this unusual B cell population. Screening of more than 200 patients with autoimmune disease demonstrated that the prevalence of patients with expanded CD21low B cells varies between diseases. The expansion was frequent in patients with systemic lupus erythematosus, in which it correlated to relative B cell lymphopenia and duration of disease. Different proportions of distinct developmental stages of CD21low B cells co-occur in nearly all patients with autoimmune disease. Although in most patients, naive-like and CD27- switched memory B cells were the most prominent CD21low subpopulations, there was no detectable association of the pattern with the underlying disease. Despite their distinct developmental stage, all CD21low B cells share a common core phenotype including the increased expression of inhibitory receptors, associated with an elevated constitutive phosphorylation of proximal signaling molecules downstream of the BCR but impaired Ca2+ mobilization and NF-κB activation after BCR stimulation. Further, this was accompanied by impaired upregulation of CD69, although CD86 upregulation was preserved. Beyond maturation-associated differences, the common core characteristics of all CD21low B cell populations suggests either a common ancestry or a shared sustained imprint by the environment they originated in.