Prevalence of Allelic Variants and Clonality of IGHV1-69 Expressing B-Cells in Patients with Different Severity of COVID 19 Disease

Abstract Introduction: Since the first months of the COVID-19 pandemic, efforts have been made to understand the importance of broadly neutralising natural antibodies in determining the response to SARS-CoV-2. Previous studies have shown that allelic variants of the IGHV1-69 gene play a dominant role in protective natural antibody responses to several other viral pathogens, including influenza virus, hepatitis C virus, human immunodeficiency virus and, most notably, the SARS-CoV-2-related viruses SARS-CoV and MERS-CoV. These allelic variants are commonly known as 51p1-related and differ from the other IGHV1-69 alleles (known as hv1263-related) in the presence of a Phe54 residue in the CDR2 region. Importantly, crystallographic studies have shown that the Phe54 residue is critical for the binding of IGHV1-69 antibodies to the SARS-CoV and MERS-CoV spike proteins. In this study, we evaluated the prevalence of 51p1 and hv1263 alleles and the clonality of 51p1- and hv1263-expressing B cells in a large cohort of healthy individuals and COVID-19 patients and correlated the findings with the severity of the disease. Мaterials and methods: A total of 419 samples were included in the study, of which 78 asymptomatic/mildly symptomatic individuals, 200 hospitalized patients with severe disease, 94 critically ill patients and 47 healthy donors. Peripheral blood was collected 8-20 days after the onset of symptoms and total cellular RNA was extracted from whole blood using an automated procedure. Аllelle-specific Ig-gene fingerprinting of IgM heavy chain transcripts was used to simultaneously analyse the clonality of the IgM+ B-cell population and the clonality of the 51p1- and hv1263-expressing B cell populations. The significance of the differences in the prevalence of clonal B-cell populations between healthy donors and patients and between patients with different severity of the disease was calculated with the Chi-Square test. Results: Analysis of the clonality of the IgM+ B-cell population showed a polyclonal pattern in most of the investigated healthy individuals (33/47, 70%) but in only 20% of all SARS-CoV-2 infected individuals (75/372, p<0.001). A significant difference was also observed between mildly affected and severely/critically ill patients [31/78 (39.7%) vs. 44/294 (15%), respectively) (p<0.001)], but not between severely and critically ill patients [28/200 (14,%) vs. 16/94 (17,1%), (p=n.s.)]. No 51p1 transcripts were detected in 74/372 (19.9%) of SARS-CoV-2 infected individuals and in 14/47 (29,8%) of the control group (p>0,01), while hv1263 transcripts were not detected in 155/289 (53,6%) and in 27/47 (68,6%) tasted patients and controls, respectively (p>0,05). We did not find a statistically significant difference in the prevalence of 51p1 and hv1263 alleles between patients with different disease severity. However, a significantly higher number of patients displayed clonal expansions of 51p1- or hv1263-expressing B cells (219/372(58.9%) and 118/244 (48,4%), respectively in comparison to healthy donors [5/47(10.6%) and 7/47(14.9%), respectively]. There was no statistically significant difference between mildly affected and severely/critically ill patients in the clonallity status of 51p1- 38/61 (62,3%) and 182/237 (76,7%) respectively or between hv1263- expressing B cells in the same two groups of patients [20/25 (80%) and 98/109 (89,9%), p>0.05]. Conclusions: Our results show that SARS-CoV-2 infection stimulates clonal expansions of IGHV1-69 -expressing B-cells, but this is independent of the severity of the disease. In addition, no difference in the prevalence of IGHV1-69 alleles was observed between patients at different stages of the disease, indicating that natural neutralizing antibodies encoded by this gene are not an important determinant of COVID-19 severity and progression. Disclosures No relevant conflicts of interest to declare.

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Deficit of Circulating CD19+CD24hiCD38hi Regulatory B Cells in Severe Aplastic Anemia

Blood ◽

10.1182/blood-2019-127053 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1219-1219

Author(s):

Yoshitaka Zaimoku ◽

Bhavisha A Patel ◽

Sachiko Kajigaya ◽

Xingmin Feng ◽

Lemlem Alemu ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Aplastic Anemia ◽

Nk Cells ◽

Cd8 T Cells ◽

Healthy Individuals ◽

Regulatory B Cells ◽

Significant Difference ◽

Cell Phenotypes

Background: Immune aplastic anemia (AA) is caused by cytotoxic T cells (CTLs) that destroy hematopoietic stem and progenitor cells. Regulatory T cells (Tregs) are reduced in AA and increase in response to immunosuppressive therapy (IST; Solomou E et al, Blood 2007). Recent studies suggested an immune regulatory role of regulatory B cells (Bregs). Human CD19+CD24hiCD38hi Bregs suppress Th1 response of CD4+ T cells as well as IFN-γ production by CD8+ CTLs (Mauri C, Menon M, J Clin Invest 2017). The quantity and/or function of Bregs are impaired in autoimmune diseases, malignancies, chronic graft-versus-host disease, and during rejection of transplanted organs. Methods: We investigated B cell phenotypes including CD24hiCD38hi Bregs in previously untreated severe AA (SAA) and very severe AA (VSAA) patients, and healthy individuals aged 18 years and older, and tested their correlation with severity and response to IST. Absolute numbers of lymphocyte subsets, including CD19+ B cells, CD8+ T cells, CD4+ T cells, and NK cell (TBNK), were quantified in fresh blood. Percentages of B cell subsets among total CD19+ B cells, including CD24hiCD38hi Bregs, CD24loCD38lo mature naïve B cells, CD24hiCD38lo memory B cells and CD24loCD38hi plasma cells/plasmablasts, were analyzed using cryopreserved peripheral blood mononuclear cells (PBMCs). Blood samples were obtained from patients close to time of diagnosis and before institution of definitive therapy. All patients were treated with horse anti-thymocyte globulin, cyclosporine, and eltrombopag between 2012 and 2018 at the Hematology Branch, NHLBI (clinicaltrials.gov NCT01623167). Results: TBNK analysis revealed no significant difference in total B cell counts in 104 AA patients compared to 40 healthy individuals (median, 137/μl [IQR, 73-212] vs 163/μl [106-242], P=.11); NK cells were significantly decreased in patients with AA, as previously reported (Gascon P et al, Blood 1986). Total B cell count did not correlate with severity of AA (P=.89) nor with overall response at six months (P=.93). CD8+ T cells and NK cells were lower in VSAA patients compared to SAA patients. None of the TBNK subsets was predictive of overall response in six months after IST. When we assessed the phenotype of B cells among 60 AA patients whose cryopreserved PBMCs were available, CD24hiCD38hi Bregs were markedly decreased as compared to 29 healthy individuals (0.31% [0.14-0.85%] vs 1.9% [1.3-3.6%], P=3×10-7; Figure, Table), while there was no significant difference in other B cell phenotypes. Among these 60 patients, the percentage of CD24hiCD38hi Bregs was especially decreased in VSAA patients compared to SAA (0.18% [0.11-0.34%] vs 0.50% [0.17-1.4%], P=.017). In contrast, CD24loCD38lo mature naïve B cells were higher in VSAA than in SAA (69% [58-86%] vs 60% [42-70%], P=.024). CD24hiCD38hi Breg frequency was positively associated with neutrophil and reticulocyte counts (correlation coefficients [r], 0.34 and 0.26, respectively), while the frequency of CD24loCD38lo mature naïve B cells was negatively correlated (r, -0.34 and -0.40). CD24loCD38lo mature naïve B cells before IST were significantly lower in 47 patients who achieved overall responses at six months compared to 13 non-responders (64% [42-71%), vs 73% [58-88%], P=.014), but CD24hiCD38hi Breg frequency was not correlated with IST responses. At six months after IST, CD24hiCD38hi Bregs in AA patients had recovered to levels present in healthy individuals (2.3% [0.98-4.8%]), in both 34 responders and five non-responders; non-responders showed non-significant increased CD24loCD38lo mature naïve B cells at six months (P=.068). Discussion: A deficit of circulating CD24hiCD38hi Bregs in immune AA with recovery after IST, as occurs with Tregs, suggests Bregs may contribute to the immune pathophysiology in AA. We unexpectedly observed a higher percentage of CD24loCD38lo mature naïve B cells to be associated with more severe disease and a lower probability of responses to IST. B cell phenotype analysis may be beneficial for monitoring of AA and predicting outcomes of therapy. Disclosures No relevant conflicts of interest to declare.

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Circulating Exosomes Inhibit B Cell Proliferation and Activity

Cancers ◽

10.3390/cancers12082110 ◽

2020 ◽

Vol 12 (8) ◽

pp. 2110

Author(s):

Jan C. Schroeder ◽

Lisa Puntigam ◽

Linda Hofmann ◽

Sandra S. Jeske ◽

Inga J. Beccard ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Proliferation ◽

B Cells ◽

B Cell ◽

Surface Expression ◽

Receptor Expression ◽

Healthy Individuals ◽

Inhibitory Effects ◽

Healthy Donors ◽

Checkpoint Receptors

(1) Background: Head and neck squamous cell carcinoma (HNSCC) is characterized by a distinctive suppression of the anti-tumor immunity, both locally in the tumor microenvironment (TME) and the periphery. Tumor-derived exosomes mediate this immune suppression by directly suppressing T effector function and by inducing differentiation of regulatory T cells. However, little is known about the effects of exosomes on B cells. (2) Methods: Peripheral B cells from healthy donors and HNSCC patients were isolated and checkpoint receptor expression was analyzed by flow cytometry. Circulating exosomes were isolated from the plasma of HNSCC patients (n = 21) and healthy individuals (n = 10) by mini size-exclusion chromatography. B cells from healthy individuals were co-cultured with isolated exosomes for up to 4 days. Proliferation, viability, surface expression of checkpoint receptors, and intracellular signaling were analyzed in B cells by flow cytometry. (3) Results: Expression of the checkpoint receptors PD-1 and LAG3 was increased on B cells from HNSCC patients. The protein concentration of circulating exosomes was increased in HNSCC patients as compared to healthy donors. Both exosomes from healthy individuals and HNSCC patients inhibited B cell proliferation and survival, in vitro. Surface expression of inhibitory and stimulatory checkpoint receptors on B cells was modulated in co-culture with exosomes. In addition, an inhibitory effect of exosomes on B cell receptor (BCR) signaling was demonstrated in B cells. (4) Conclusions: Plasma-derived exosomes show inhibitory effects on the function of healthy B cells. Interestingly, these inhibitory effects are similar between exosomes from healthy individuals and HNSCC patients, suggesting a physiological B cell inhibitory role of circulating exosomes.

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Bone Marrow Derived Mesenchymal Stem Cells from Splenic Marginal Zone Lymphoma Patients Exhibit Altered Proliferative Potential and B Lymphocyte Immunomodulatory Properties

Blood ◽

10.1182/blood.v126.23.2396.2396 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2396-2396

Author(s):

Athanasia Kalyva ◽

Charalampos Pontikoglou ◽

Christina Kalpadakis ◽

Athina Trakaki ◽

Nikitas Zorzos ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Marginal Zone ◽

Ex Vivo ◽

Proliferative Potential ◽

Splenic Marginal Zone Lymphoma ◽

Healthy Individuals ◽

Healthy Donors ◽

Normal Controls

Abstract Splenic marginal zone lymphoma (SMZL) originates from the neoplastic transformation of mature B-lymphocytes. However, there is a concurrent high prevalence of bone marrow (BM) infiltration, suggesting that BM microenvironment dynamics could have a potential involvement in disease pathology. In this regard, we aim to characterise BM derived mesenchymal stem cells (MSCs), since they comprise key components of the BM hematopoietic stroma, in order to investigate if MSCs show altered properties in SMZL patients compared to healthy controls. BM MSCs were isolated from 8 SMZL patients and 10 age- and sex-matched healthy controls. MSCs were in vitro expanded and re-seeded for a total of 5 passages (P). The colony forming unit-fibroblast (CFU-F) assay was used for the estimation of MSC frequency within the BM mononuclear cell (BMMC) fraction. Ex-vivo expanded MSCs were phenotypically characterized by flow cytometry (FC) using appropriate markers. In vitro differentiation to adipocytes and osteoblasts was assessed by cytochemical stains. The proliferative potential of ex vivo expanded MSCs was evaluated by Methyl Triazolyl Tetrazolium (MTT)-based assay and survival characteristics were studied using FC and 7-Aminoactinomycin D (7-AAD) staining. To assess the effect of patient MSCs on B cell growth, B cells were immunomagnetically isolated (Miltenyi Biotec GmbH, Germany) from peripheral blood (PB) of normal individuals, labeled with carboxy fluorescein succinimidyl ester (CFSE; Gibco Invitrogen, Paisley, Scotland) and subsequently cultured in the absence or presence of confluent layers of allogeneic BM-MSCs from SMZL patients or normal controls in the presence of CpG oligonucleotide 2006 (Invivogen, France) and IL-2 (R&D Systems, Minneapolis, MN). In a separate set of experiments, B cell survival was evaluated via FC and 7-AAD staining, after co-culturing with BM-MSCs from patients or healthy donors. Finally, to study BM-MSC capacity to chemotactically attract B-cells, transwell migration assays were set. In the bottom chambers MSCs from patients or healthy individuals were grown until confluency and then isolated B cells from PB of either patients or controls were added into the upper chamber. Twelve hours later migrated cells were enumerated. Grouped data are expressed as means± 1 standard error of the mean (SEM). MSCs were successfully expanded from all participants in the study. Adherent cells from both study groups displayed the typical spindle-shape morphology and immunophenotypic analysis at the end of P2-P3-P4 demonstrated that cultures constituted of a homogeneous cell population, typically expressing CD29, CD44, CD73, CD90 and CD105 while being negative for CD14, CD34 and CD45. SMZL-derived MSCs were similar to their normal counterparts in the capacity to differentiate towards adipocytes and osteocytes as evidenced by Oil Red O and Alizarin Red staining, respectively. The frequency of MSCs within the BMMC compartment was significantly lower in patients as compared to healthy individuals (2.5±0.68/105 ΒΜΜCs and 7.23±0.6/105 ΒΜΜCs, respectively; P=0.0032) apparently due to the predominance of the lymphoma cells within patient BMMCs. SMZL MSCs displayed defective proliferative potential as compared to their normal counterparts at P2, as evidenced by the MTT assay (P<0.0001). To explore the influence of SMZL BM-MSCs in B cells survival we compared the viability of B cells isolated from the PB of healthy individuals cultured in medium alone to that of such cells co-cultured with either BM-MSCs derived from patients or normal controls. 43.85±1.46% of B cells cultured alone were apoptotic, while only 20.8±2.63% and 12±0.77% of B cells co-cultured with either normal MSCs or SMZL MSCs were apoptotic (P<0.0001 and P<0.0001, respectively). Notably patient MSCs confer a survival advantage in B cell viability over their normal counterparts (P=0.0374). Finally SMZL MSCs had a more potent chemotactic activity on B cells from healthy donors, as compared to MSCs from normal controls ( P<0.05). In conclusion we have shown for the first time that SMZL lymphoma MSCs are intrinsically defective in terms of proliferative potential and exert an altered modulation of B cell apoptosis and B cell chemotaxis. These preliminary results concerning the properties of SMZL MSCs merit further investigation and provide the theoretical background for exploring their potential implication in lymphomagenesis. Disclosures No relevant conflicts of interest to declare.

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Hematopoietic Stem and Progenitor Cells in Patients with Mature B-Cell Malignancies Are Quantitatively and Qualitatively Deregulated Compared to Healthy Controls

Blood ◽

10.1182/blood.v124.21.4307.4307 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4307-4307

Author(s):

Bokang Maswabi ◽

Dana Prukova ◽

Jan Molinsky ◽

Magdalena Klanova ◽

Lucie Lateckova ◽

...

Keyword(s):

Gene Expression ◽

B Cells ◽

B Cell ◽

Cell Lymphoma ◽

Cell Populations ◽

B Cell Malignancies ◽

Hematopoietic Stem ◽

Healthy Donors ◽

Stem And Progenitor Cells ◽

Expression Frequency

Abstract Mature B-cell lymphomas represent a heteregenous group of malignancies, which are considered to arise along different steps of B-cell development, particularly from germinal or post-germinal center B-cells. Recent studies, however, indicate that hematopoietic stem and progenitor cells (HSPC) could be involved in the pathogenesis of these diseases. We analyzed HSPC populations from 131 patients with mature B-cell malignancies, including chronic lymphocytic leukemia (CLL, n=20), mantle cell lymphoma (MCL, n=26), diffuse large B-cell lymphoma (DLBCL, n=35), follicular lymphoma (FL, n=25), and multiple myeloma (MM, n=25). For comparison, HSPC populations obtained from 22 healthy donors were used. Hematopoietic stem cells (HSC, Lin-CD34+CD38-CD90+CD45RA-), multipotent progenitors (MPP, Lin-CD34+CD38-CD90-CD45RA-), multi-lymphoid progenitors (MLP, Lin-CD34+CD38-CD90-CD45RA+), and pro-B cells (CD34+CD38+CD10+CD19+) were analyzed by flow cytometry. Proportions of HSPC and pro-B cells were related to CD34+CD38- and CD34+CD38+ cells, respectively. HSC and pro-B cell populations were sorted directly into tubes containing lysis solution for subsequent gene expression analyses. Preamplified cDNA was used for quantitative PCR of selected gene targets (n=28 in HSC, and n=27 in pro-B). The expression of targets was normalized to GAPDH expression. Corresponding cell populations isolated from healthy donors were used as controls. The aforementioned flow cytometry analyses revealed significantly decreased MLP population in all 5 tested diagnoses: CLL (p<0.0001), MCL (p=0.0004), DLBCL (p=0.002), FL (p=0.004), and MM (p=0.004) compared to CTRL (Figure 1). Pro-B cell populations were also decreased in all tested diagnoses, but statistical significance was reached only in MCL (p<0.0001), DLBCL (p=0.0003), and MM (p=0.0025), but not in CLL (p=0.078) or FL (p=0.308). HSC populations were significantly increased in DLBCL (p=0.0023), FL (p=0.015), and MCL (p=0.036), but not in MM (p=0.207) or CLL (p=0.875). The only significant change in MPP population was an increase in CLL (p=0.031). To gain more insight into biology of the stem and progenitor cell populations we analyzed gene expression changes of the key transcriptional regulators, HSC and pro-B cell specific surface markers, or genes that are overexpressed in particular lymphoma subtypes. We observed: 1) Significant upregulation of BCL11A, RUNX1, IKAROS, GATA2, PROM1, and CD44 mRNA within HSC populations obtained from all tested diagnoses compared to CTRL-HSC. 2) None of the targets in pro-B cells were significantly deregulated across all 5 diagnoses, the only significantly upregulated genes were IKAROS and EBF1 in CLL compared to CTRL pro-B cells. 3) NOTCH1 and CCND1 mRNA were not detected in CTRL-HSC, but NOTCH1 was detectable in more than 50% of DLBCL and MM-HSC samples, while CCND1 was detectable in more than 50% of CLL-HSC samples. 4) More than 50% difference in gene expression frequency between CTRL and patient HSC samples was observed in the case of BMI1 (CLL, DLBCL, FL and MM), FOXO1 (MM), FOXP1 (DLBCL), and IRF4 (CLL). 5) Pro-B cells obtained from CTRL samples did not express BCL2, BMI1, MYC, PAX5, or ZAP70, but these genes were detected in more than 50% of patient pro-B cell samples as follows: BCL2 in FL, BMI1 and ZAP70 in CLL and DLBCL, MYC in CLL, DLBCL and FL, PAX5 in CLL. 6) More than 50% difference in gene expression frequency between CTRL and patient pro-B cells was observed in case of BCL11A (CLL, DLBCL, FL, MM, and MCL), BCL2L1 (CLL, DLBCL, FL, MM), CD38 (CLL, DLBCL, FL), CD44 (in CLL, DLBCL), IRF4 (CLL, DLBCL, FL), IRF8 and LEF1 (CLL), PU.1 (CLL, FL), and RUNX1 (CLL, DLBCL). In this study, we showed that bone marrow HSPC in patients with mature B-cell malignancies are quantitatively and qualitatively different compared to HSPC obtained from healthy controls. We currently study whether this deregulation is caused by the influence of malignant cells on HSPC or reflects HSPC intrinsic changes that are involved in the pathogenesis of these malignancies. Grant support: IGA-MZ NT13201-4/2012, GACR14-19590S, UNCE 204021, SVV-2013-266509, GA-UK 595912, PRVOUK-27/LF1/1 Figure 1: Percentage of HSPC in patients with diverse mature B-cell malignancies and healthy donors (controls). Symbols are plotted at means and standard deviations are omitted for clarity. Asterisk denotes statistically significant changes. Figure 1:. Percentage of HSPC in patients with diverse mature B-cell malignancies and healthy donors (controls). Symbols are plotted at means and standard deviations are omitted for clarity. Asterisk denotes statistically significant changes. Disclosures No relevant conflicts of interest to declare.

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Can the cytokine adsorber CytoSorb® help to mitigate cytokine storm and reduce mortality in critically ill patients? A propensity score matching analysis

Annals of Intensive Care ◽

10.1186/s13613-021-00905-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Christina Scharf ◽

Ines Schroeder ◽

Michael Paal ◽

Martin Winkels ◽

Michael Irlbeck ◽

...

Keyword(s):

Propensity Score ◽

Critically Ill ◽

Critically Ill Patients ◽

Wilcoxon Test ◽

Relative Reduction ◽

Cytokine Storm ◽

Saps Ii ◽

Significant Difference ◽

Group 2 ◽

Group 1

Abstract Background A cytokine storm is life threatening for critically ill patients and is mainly caused by sepsis or severe trauma. In combination with supportive therapy, the cytokine adsorber Cytosorb® (CS) is increasingly used for the treatment of cytokine storm. However, it is questionable whether its use is actually beneficial in these patients. Methods Patients with an interleukin-6 (IL-6) > 10,000 pg/ml were retrospectively included between October 2014 and May 2020 and were divided into two groups (group 1: CS therapy; group 2: no CS therapy). Inclusion criteria were a regularly measured IL-6 and, for patients allocated to group 1, CS therapy for at least 90 min. A propensity score (PS) matching analysis with significant baseline differences as predictors (Simplified Acute Physiology Score (SAPS) II, extracorporeal membrane oxygenation, renal replacement therapy, IL-6, lactate and norepinephrine demand) was performed to compare both groups (adjustment tolerance: < 0.05; standardization tolerance: < 10%). U-test and Fisher’s-test were used for independent variables and the Wilcoxon test was used for dependent variables. Results In total, 143 patients were included in the initial evaluation (group 1: 38; group 2: 105). Nineteen comparable pairings could be formed (mean initial IL-6: 58,385 vs. 59,812 pg/ml; mean SAPS II: 77 vs. 75). There was a significant reduction in IL-6 in patients with (p < 0.001) and without CS treatment (p = 0.005). However, there was no significant difference (p = 0.708) in the median relative reduction in both groups (89% vs. 80%). Furthermore, there was no significant difference in the relative change in C-reactive protein, lactate, or norepinephrine demand in either group and the in-hospital mortality was similar between groups (73.7%). Conclusion Our study showed no difference in IL-6 reduction, hemodynamic stabilization, or mortality in patients with Cytosorb® treatment compared to a matched patient population.

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AB0144 STRATIFICATION OF PATIENTS WITH PRIMARY SJÖGREN’S SYNDROME BY MEASURING B-CELL HEALTH

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.3507 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 1372-1373

Author(s):

G. M. Verstappen ◽

J. C. Tempany ◽

H. Cheon ◽

A. Farchione ◽

S. Downie-Doyle ◽

...

Keyword(s):

Cell Division ◽

Cell Differentiation ◽

T Cell ◽

B Cells ◽

B Cell ◽

B Cell Differentiation ◽

Research Support ◽

Differentiation Capacity ◽

Cell Responses ◽

Healthy Donors

Background:Primary Sjögren’s syndrome (pSS) is a heterogeneous immune disorder with broad clinical phenotypes that can arise from a large number of genetic, hormonal, and environmental causes. B-cell hyperactivity is considered to be a pathogenic hallmark of pSS. However, whether B-cell hyperactivity in pSS patients is a result of polygenic, B cell-intrinsic factors, extrinsic factors, or both, is unclear. Despite controversies about the efficacy of rituximab, new B-cell targeting therapies are under investigation with promising early results. However, for such therapies to be successful, the etiology of B-cell hyperactivity in pSS needs to be clarified at the individual patient level.Objectives:To measure naïve B-cell function in pSS patients and healthy donors using quantitative immunology.Methods:We have developed standardised, quantitative functional assays of B-cell responses that measure division, death, differentiation and isotype switching, to reveal the innate programming of B cells in response to T-independent and dependent stimuli. This novel pipeline to measure B-cell health was developed to reveal the sum total of polygenic defects and underlying B-cell dysfunction at an individual level. For the current study, 25 pSS patients, fulfilling 2016 ACR-EULAR criteria, and 15 age-and gender-matched healthy donors were recruited. Standardized quantitative assays were used to directly measure B cell division, death and differentiation in response to T cell-independent (anti-Ig + CpG) and T-cell dependent (CD40L + IL-21) stimuli. Naïve B cells (IgD+CD27-) were sorted from peripheral blood mononuclear cells and were labeled with Cell Trace Violet at day 0 to track cell division until day 6. B cell differentiation was measured at day 5.Results:Application of our standardized assays, and accompanying parametric models, allowed us to study B cell-intrinsic defects in pSS patients to a range of stimuli. Strikingly, we demonstrated a hyperresponse of naïve B cells to combined B cell receptor (BCR) and Toll-like receptor (TLR)-9 stimulation in pSS patients. This hyperresponse was revealed by an increased mean division number (MDN) at day 5 in pSS patients compared with healthy donors (p=0.021). A higher MDN in pSS patients was observed at the cohort level and was likely attributed to an increased division burst (division destiny) time. The MDN upon BCR/TLR-9 stimulation correlated with serum IgG levels (rs=0.52; p=0.011). No difference in MDN of naïve B cells after T cell-dependent stimulation was observed between pSS patients and healthy donors. B cell differentiation capacity (e.g., plasmablast formation and isotype switching) after T cell-dependent stimulation was also assessed. At the cohort level, no difference in differentiation capacity between groups was observed, although some pSS patients showed higher plasmablast frequencies than healthy donors.Conclusion:Here, we demonstrate defects in B-cell responses both at the cohort level, as well as individual signatures of defective responses. Personalized profiles of B cell health in pSS patients reveal a group of hyperresponsive patients, specifically to combined BCR/TLR stimulation. These patients may benefit most from B-cell targeted therapies. Future studies will address whether profiles of B cell health might serve additional roles, such as prediction of disease trajectories, and thus accelerate early intervention and access to precision therapies.Disclosure of Interests:Gwenny M. Verstappen: None declared, Jessica Catherine Tempany: None declared, HoChan Cheon: None declared, Anthony Farchione: None declared, Sarah Downie-Doyle: None declared, Maureen Rischmueller Consultant of: Abbvie, Bristol-Meyer-Squibb, Celgene, Glaxo Smith Kline, Hospira, Janssen Cilag, MSD, Novartis, Pfizer, Roche, Sanofi, UCB, Ken R. Duffy: None declared, Frans G.M. Kroese Grant/research support from: Unrestricted grant from Bristol-Myers Squibb, Consultant of: Consultant for Bristol-Myers Squibb, Speakers bureau: Speaker for Bristol-Myers Squibb, Roche and Janssen-Cilag, Hendrika Bootsma Grant/research support from: Unrestricted grants from Bristol-Myers Squibb and Roche, Consultant of: Consultant for Bristol-Myers Squibb, Roche, Novartis, Medimmune, Union Chimique Belge, Speakers bureau: Speaker for Bristol-Myers Squibb and Novartis., Philip D. Hodgkin Grant/research support from: Medimmune, Vanessa L. Bryant Grant/research support from: CSL

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YY1 plays an essential role at all stages of B-cell differentiation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1606297113 ◽

2016 ◽

Vol 113 (27) ◽

pp. E3911-E3920 ◽

Cited By ~ 43

Author(s):

Eden Kleiman ◽

Haiqun Jia ◽

Salvatore Loguercio ◽

Andrew I. Su ◽

Ann J. Feeney

Keyword(s):

Transcription Factor ◽

Cell Differentiation ◽

B Cells ◽

B Cell ◽

Signaling Pathways ◽

Mrna Splicing ◽

Cell Populations ◽

Sequencing Analysis ◽

B Cell Differentiation ◽

Chip Sequencing

Ying Yang 1 (YY1) is a ubiquitously expressed transcription factor shown to be essential for pro–B-cell development. However, the role of YY1 in other B-cell populations has never been investigated. Recent bioinformatics analysis data have implicated YY1 in the germinal center (GC) B-cell transcriptional program. In accord with this prediction, we demonstrated that deletion of YY1 by Cγ1-Cre completely prevented differentiation of GC B cells and plasma cells. To determine if YY1 was also required for the differentiation of other B-cell populations, we deleted YY1 with CD19-Cre and found that all peripheral B-cell subsets, including B1 B cells, require YY1 for their differentiation. Transitional 1 (T1) B cells were the most dependent upon YY1, being sensitive to even a half-dosage of YY1 and also to short-term YY1 deletion by tamoxifen-induced Cre. We show that YY1 exerts its effects, in part, by promoting B-cell survival and proliferation. ChIP-sequencing shows that YY1 predominantly binds to promoters, and pathway analysis of the genes that bind YY1 show enrichment in ribosomal functions, mitochondrial functions such as bioenergetics, and functions related to transcription such as mRNA splicing. By RNA-sequencing analysis of differentially expressed genes, we demonstrated that YY1 normally activates genes involved in mitochondrial bioenergetics, whereas it normally down-regulates genes involved in transcription, mRNA splicing, NF-κB signaling pathways, the AP-1 transcription factor network, chromatin remodeling, cytokine signaling pathways, cell adhesion, and cell proliferation. Our results show the crucial role that YY1 plays in regulating broad general processes throughout all stages of B-cell differentiation.

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The autoantigen-binding B cell repertoires of normal and of chronically graft-versus-host-diseased mice.

Journal of Experimental Medicine ◽

10.1084/jem.165.6.1675 ◽

1987 ◽

Vol 165 (6) ◽

pp. 1675-1687 ◽

Cited By ~ 54

Author(s):

A G Rolink ◽

T Radaszkiewicz ◽

F Melchers

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

The Other ◽

Cell Populations ◽

Foreign Antigen ◽

Alloreactive T Cells ◽

Polyclonal Activator ◽

Activated B Cells

A quantitative analysis of the frequencies of autoantibody-producing B cells in GVHD and in normal mice has been undertaken by generating collections of hybridomas of activated B cells. These hybridomas secreted sufficient quantities of Ig to allow binding analyses on a panel of autoantigens. B cells have been activated in a variety of ways. In vivo they were activated by injection of alloreactive T cells of one parent, leading to GVHD by a foreign antigen, sheep erythrocytes, in a secondary response, or by the polyclonal activator LPS. B cells from an experimentally unstimulated animal were used for an analysis of the normal background. In vitro B cells were activated by alloreactive T cells or by LPS. The frequencies of hybridomas and, therefore, of activated B cells producing autoantibodies to DNA or to kidney were not significantly different in mice activated by a graft-vs.-host T cell response as compared with B cell populations activated by any of the other procedures. They were found to compose 7.1-17.1% of the total repertoire of activated B cells. Moreover, the frequencies of autoantibody-producing activated B cells does not change with time after induction of the graft-vs.-host reaction. The pattern and frequencies of autoantigen-binding specificities to cytoskeleton, smooth muscle, nuclei, mitochondria, and DNA were not found to be different in any of the groups of hybridomas. The single notable exception, found in GVHD mice, were hybridomas producing autoantibodies to kidney proximal tubular brush border. These results allow the conclusion that autoantigen-binding B cells exist in an activated state in GVHD mice, as well as in mice activated by a foreign antigen or by a polyclonal activator, in B cell populations activated in vitro either by alloreactive T cells or by a polyclonal activator, and even in the background of experimentally unstimulated animals. T cell-mediated graft-vs.-host activation, in large part, does not lead to a selective expansion of autoantigen-binding B cells. The main difference between the graft-vs.-host-activated B cell repertoire and all others is that approximately 90% of teh autoantibodies were of the IgG class, whereas al autoantibodies found in the other groups were IgM.

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Characterization of B-cell Receptor Heavy Chain Complementarity-determining Region 3 Repertoire Based on the Differences of Symbiotic Microorganisms in the Gut of Mice

10.21203/rs.3.rs-203289/v1 ◽

2021 ◽

Author(s):

Jun Li ◽

Yurong Pan ◽

Qingqing Ma ◽

Long Ma ◽

Bin Shi ◽

...

Keyword(s):

Small Intestine ◽

B Cells ◽

B Cell ◽

Heavy Chain ◽

Intestinal Microflora ◽

Cell Receptor ◽

Memory B Cells ◽

B Cell Receptor ◽

Positive Effects ◽

Significant Difference

Abstract Background Colonization of gut microorganism is related to maturation of B cells in peripheral immune organs. This study aims to investigate the effect of intestinal microflora in Germ-free (GF), Specific Pathogen-free (SPF) and Clean (CL) BALB/C mice to small intestine total B-cell and memory B-cell receptor (BCR) complementary-determining region 3 (CDR3) repertoire. Results The composition and characteristics of intestinal microflora were analyzed by 16S rDNA sequencing. Genomic DNA extracted from small intestine tissue and memory B-cells of GF, SPF and CL mice were conducted via high-throughput DNA sequencing methods. As expected, significant differences of gut microflora diversity were observed in the three mice groups. CL group showed the most diversity, followed by SPF group, and GF group had the lowest diversity. Moreover, anormogenesis of intestinal lymphoid tissue were observed in GF mice. Diversity of the BCR heavy chain CDR3 repertoire in memory B cells were significant difference among three groups, but not in total B cells. The nucleotide polymorphism, usage frequency of gene segments (V, D, J, V–J gene segments) and amino acid of total B cells and memory B cells CDR3 were comparable among three mice groups, and there was significant difference between CL and GF mice groups. Conclusions The results of this study advocate that the colonization of intestinal microorganisms affect the diversity of B cells CDR3 repertoire. Elucidating mechanism of microbiome participated in the function of intestinal mucosal immune system may have positive effects on human health, and it requires further investigation.

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Antibody Cloning Identifies Pathogenic and Non-Pathogenic Antibodies in Heparin-Induced Thrombocytopenia and Defines the Molecular Signatures That Differentiate the Two Types of Antibodies

Blood ◽

10.1182/blood-2019-126097 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 439-439

Author(s):

Wen Zhu ◽

Yongwei Zheng ◽

Mei Yu ◽

Yaling Wu ◽

Jianhui Wei ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Platelet Activation ◽

Solid Phase ◽

Variable Regions ◽

Heparin Induced Thrombocytopenia ◽

Tyrosine Residues ◽

Healthy Donors ◽

Activation Assay ◽

Positively Charged

Heparin-induced thrombocytopenia (HIT) is a common adverse drug reaction associated with frequent life-threatening thrombotic complications. The hallmark of HIT is polyclonal antibodies (Abs) that recognize platelet alpha granule chemokine PF4 when it binds to heparin (PF4/H). These Abs can be detected in solid phase assays that use PF4/H as a target (PF4 ELISA), but only a minority of patients testing positive actually have HIT, i.e., most heparin-induced Abs are non-pathogenic. In patients who have clinical HIT, Abs that activate platelets can be detected using a platelet-activation assay such as the serotonin release assay, or the PF4-dependent p-selectin expression assay (PEA) (Chest 2016; 150:506). Thus, there are at least two distinct types of heparin-induced Abs - those that react only in PF4 ELISA and are seemingly "non-platelet-activating" and "non-pathogenic" and those that are "platelet-activating" and "pathogenic". To date, the molecular basis for the differing clinical and serologic behaviors of pathogenic and non-pathogenic Abs is uncertain. To address this issue, we performed single cell cloning to clone B cell receptors from IgG1+ B cells from HIT patients. We deposited single B cells (CD19+IgG1+) from 6 patients with "classical" and 2 patients with "spontaneous" HIT into 96 well plates containing feeder cells (from G Kelsoe, Duke U) that support B cell proliferation and Ab secretion (Immunity 2018;48:174). Clones secreting IgG were first screened in PF4 ELISA and positive results were obtained with 55 clones from 6 patients. Further screening showed that 7 of these clones (from 4 patients) were also PEA-positive (platelet-activating). Clones positive only in PF4 ELISA, positive in both PF4 ELISA and PEA, or negative in PF4 ELISA were designated NP (non-pathogenic), PA (platelet-activating) and NB (non-binding), respectively. H and L chain variable regions were defined in 7 PA, 42 NP and 34 NB clones. The following findings were made when sequences in the 3 clonal groups were compared: PA clones preferentially used JH6 (p=0.002) and the VH3/JH6 combination (p=0.0003)The PA and NP Abs all employed κ chains, whereas κ chain usage for NB clones was 61% (p<0.0001).No preferred signatures were identified in κ chain complementarity determining regions (LCDR3) of PA clones that differentiate them from NP and NB Abs.PA Abs had longer heavy chain CDR3s (HCDR3) than NP (p<0.001) or NB (p=0.0001) AbsPA Abs contained more positively charged amino acid residues compared to NP (p=0.058) or NB (p=0.002) Abs.PA Abs contained more tyrosine residues compared to NP (p=0.067) or NB (p<0.0001) AbsFive of 7 PA clones contained an RX1-2K/RX1-2R/H (RKH) motif in HCDR3; the remaining 2 PA clones contained a string of at least 5 tyrosines (Y5 motif) in HCDR3. The RKH and Y5 motifs were not found in any of the 76 NP and NB clones. Substitution of alanine for positively charged residues of the RKH motif or of tyrosine residues in the Y5 motif in PA clones reduced PF4/H binding and platelet activation, arguing for functional significance of both motifs. Utilization of nearly identical H and L chains within 3 groups of clones and of shared H chains within 3 groups of clones (both PA and NP) was observed in multiple patients. Moreover, utilization of a shared H chain was observed within 3 NP clones from two unrelated patients. These findings indicate clonal amplification and convergence of the B cell (both PA and NP) response, likely in response to a common antigen. High throughput sequencing of IgG H chains were performed on peripheral blood mononuclear cells (PBMC) from 7 HIT patients and 3 healthy donors. Eleven of 1585 H chain sequences (0.69%) from HIT patients contained the RKH and 18 (1.1%) contained the Y5 motif. In 3 healthy donors, 4 of 1418 H chain sequences (0.28%) contained RKH and none (0%) contained Y5. The findings reflect amplification of B cells with receptors containing RKH and Y5 motifs in HIT patients (p=0.1 for excess RKH and p<0.0001 for Y5 in HIT). These observations provide the first characterization of Ig structural motifs that are favored for selection in the humoral immune response leading to HIT and suggest that the RKH and Y5 CDR3 motifs in particular may contribute importantly to Ab pathogenicity. Findings made are expected to facilitate further work to define features specific to "pathogenic" HIT Abs and, possibly, to identify genetic variants that predispose individuals to experience HIT. Disclosures Padmanabhan: Terumo BCT: Consultancy; Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees; Versiti Wisconsin: Patents & Royalties: Related to HIT patents; Retham Technologies: Equity Ownership; Janssen R&D: Consultancy.

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