Recovery of the Normal B-Cell Compartment in Children Treated for B-Cell Precursor Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3792-3792
Author(s):  
Prisca Theunissen ◽  
Ester Mejstrikova ◽  
Tomasz Szczepanski ◽  
Lukasz Sedek ◽  
Alita van der Sluijs ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Healthy Controls ◽  
Cell Precursor ◽  
Cell Compartment ◽  
Cell Numbers ◽  
Time Points ◽  
Compensatory Proliferation ◽  
B Cell Subsets

Abstract BACKGROUND Cytotoxic treatment in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients induces a dramatic decrease in B-cell precursor (BCP) and mature B-cell numbers, followed by regeneration of BCPs in the bone marrow (BM) and subsequent replenishment of mature B-cells in the peripheral blood (PB) in between treatment blocks and after stop of treatment. To understand the degree of B-cell recovery after such dramatic changes, we first evaluated the composition of the B-cell population in the BM and PB of pediatric BCP-ALL patients during and after therapy. Secondly, we investigated whether the immunophenotypic maturation of BCPs in regenerating BM is similar to normal BCP development or whether such regeneration induces immunophenotypic aberrancies, which could potentially hamper minimal residual disease (MRD) detection. Finally, we assessed whether compensatory proliferation plays a role during B-cell regeneration, since enhanced proliferation might limit the B-cell receptor diversity and consequently may affect susceptibility to infections during and after therapy. METHODS For immunophenotypic characterization of different B-cell subsets, 8-color flow cytometry was performed on fresh BM and PB samples at different time points after start of therapy (DCOG ALL11-protocol). To study BCP maturation, a multidimensional maturation pathway based on 5 backbone markers was designed and the expression pattern of several differentiation markers during this maturation pathway was evaluated. To assess proliferation in BCP subsets, BM samples were stained with subset-specific antibodies and DRAQ5 for cell cycle analysis. The proliferation history of sorted pre-B-II-small and immature subsets in BM and sorted mature B-cell subsets in PB was assessed by performing a kappa-deleting recombination excision circle (KREC)-assay. RESULTS BCP regeneration occurred mainly at day 78, month 5 and after stop of therapy. The BCP compartment in regenerating BM at time points during therapy showed a shift towards the most immature stages. In PB, mature B-cell numbers decreased after start of therapy and newly generated mature B-cells subsets reappeared at month 5 and after stop of therapy. Importantly, the BCP maturation pathway with its expression patterns of CD10, CD34, CD58, CD66c, CD38, CD123, CD9, CD81, CD24, TdT, Igκ and Igλ was comparable between regenerating BM and BM of healthy individuals, albeit that a shift in the relative BCP subset distribution was observed in regenerating BM. As expected, most proliferation in BM of healthy controls occurred in the pre-B-II-large subset (68% ±11% (mean ±SD) proliferating cells). Comparable percentages of proliferating pre-B-II-large cells were found in regenerating BM: 74%±10% at day 78, 72%±10% at month 5 and 63% (preliminary data, n=1) at one year after stop of therapy (month 36). Also pre-B-I cells showed some proliferation, with no significant differences between normal and regenerating BM (Figure 1). If present, the pre-B-II-small and immature BCP subsets showed no proliferation in regenerating and normal BM. KREC-analysis of sorted pre-B-II-small and immature subsets confirmed that no cell divisions had occurred after IGK-rearrangement in normal BM as well as regenerating BM at month 5 and month 36. Low numbers of pre-B-II-small and immature cells precluded KREC-analysis at day 78. KREC-analysis of the various mature B-cell subsets in PB showed no significant difference in proliferation history between PB of patients at different time points during or after therapy and PB of healthy controls. CONCLUSIONS In BCP-ALL patients, the B-cell compartment is drastically affected during treatment. Subsequent regeneration of BCPs and mature B-cells occurs at different time points during therapy and after stop of therapy. Immunophenotypically, BCP maturation in regenerating BM is similar to normal B-cell differentiation, indicating that MRD detection will not be hampered by aberrant immunophenotypes of regenerating BCPs. Importantly, no enhanced proliferation is observed in BCP subsets in BM and mature B-cells subsets in PB of patients during and after therapy. The lack of compensatory proliferation suggests that B-cell regeneration is due to a larger influx of non-committed stem cells into the B-cell lineage and indicates that a diverse immune repertoire will most likely be restored during recovery of the B-cell compartment. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Abnormal B Cell Compartment Associated With Tfh Cells in Children With Henoch-schonlein Purpura

2020 ◽  
Author(s):  
Ning Zhang ◽  
Ge Tian ◽  
Yuanyuan Sun ◽  
Jing Pan ◽  
Wei Xu ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Mononuclear Cells ◽  
Healthy Controls ◽  
Henoch Schonlein Purpura ◽  
Cell Compartment ◽  
Tfh Cells ◽  
B Cell Subsets ◽  
Schonlein Purpura ◽  
Henoch Schönlein Purpura

Abstract Aim IgA-producing B cells were found to be associated with children diagnosed with Henoch-Schonlein purpura (HSP). The present study aimed to determine whether children with HSP possess abnormal B cell subsets. Methods A total of 14 children diagnosed with HSP, and age- and gender-matched healthy controls were enrolled in our study. Peripheral blood mononuclear cells were isolated, and the percentage of B cells subsets and Tfh cells were determined by flow cytometry. Finally, Spearman’s correlation coefficient was used to analyze the correlation between the percentage of Tfh cells and B cell subsets. Results We found that the frequency of total B cells was significantly increased in children with HSP; however, the percentage of plasma cells was significantly lower in HSP children. A significant reduction in the count of naïve B cells and an increase in class-switched B cells were found in children with HSP compared with healthy controls. We observed that the expression of C-X-C chemokine receptor type 5 (CXCR5) on total CD4+ T cells and the percentage of CD4+CXCR5+ cells were significantly increased within HSP patients. Moreover, significant correlations between Tfh cells and various B cells subsets were observed. Conclusion Our study showed a Tfh cell-associated abnormal B cell compartment in HSP children.

scholarly journals SARS-CoV-2 infection causes immunodeficiency in recovered patients by downregulating CD19 expression in B cells via enhancing B-cell metabolism

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Yukai Jing ◽  
Li Luo ◽  
Ying Chen ◽  
Lisa S. Westerberg ◽  
Peng Zhou ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Immune Regulation ◽  
Cell Metabolism ◽  
Healthy Controls ◽  
Bcr Signaling ◽  
Before And After ◽  
B Cell Subsets ◽  
Almost All ◽  
Cd19 Expression

AbstractThe SARS-CoV-2 infection causes severe immune disruption. However, it is unclear if disrupted immune regulation still exists and pertains in recovered COVID-19 patients. In our study, we have characterized the immune phenotype of B cells from 15 recovered COVID-19 patients, and found that healthy controls and recovered patients had similar B-cell populations before and after BCR stimulation, but the frequencies of PBC in patients were significantly increased when compared to healthy controls before stimulation. However, the percentage of unswitched memory B cells was decreased in recovered patients but not changed in healthy controls upon BCR stimulation. Interestingly, we found that CD19 expression was significantly reduced in almost all the B-cell subsets in recovered patients. Moreover, the BCR signaling and early B-cell response were disrupted upon BCR stimulation. Mechanistically, we found that the reduced CD19 expression was caused by the dysregulation of cell metabolism. In conclusion, we found that SARS-CoV-2 infection causes immunodeficiency in recovered patients by downregulating CD19 expression in B cells via enhancing B-cell metabolism, which may provide a new intervention target to cure COVID-19.

scholarly journals Proportions of B-cell subsets are altered in incomplete systemic lupus erythematosus and correlate with interferon score and IgG levels

Rheumatology ◽  
2020 ◽  
Vol 59 (9) ◽  
pp. 2616-2624
Author(s):  
Svenja Henning ◽  
Wietske M Lambers ◽  
Berber Doornbos-van der Meer ◽  
Wayel H Abdulahad ◽  
Frans G M Kroese ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Lupus Erythematosus ◽  
Cell Subset ◽  
Cross Sectional Study ◽  
Type I ◽  
Healthy Controls ◽  
Sectional Study ◽  
Cross Sectional ◽  
B Cell Subsets

Abstract Objectives Incomplete SLE (iSLE) patients display symptoms typical for SLE but have insufficient criteria to fulfil the diagnosis. Biomarkers are needed to identify iSLE patients that will progress to SLE. IFN type I activation, B-cell-activating factor (BAFF) and B-cell subset distortions play an important role in the pathogenesis of SLE. The aim of this cross-sectional study was to investigate whether B-cell subsets are altered in iSLE patients, and whether these alterations correlate with IFN scores and BAFF levels. Methods iSLE patients (n = 34), SLE patients (n = 41) with quiescent disease (SLEDAI ≤4) and healthy controls (n = 22) were included. Proportions of B-cell subsets were measured with flow cytometry, IFN scores with RT-PCR and BAFF levels with ELISA. Results Proportions of age-associated B-cells were elevated in iSLE patients compared with healthy controls and correlated with IgG levels. In iSLE patients, IFN scores and BAFF levels were significantly increased compared with healthy controls. Also, IFN scores correlated with proportions of switched memory B-cells, plasma cells and IgG levels, and correlated negatively with complement levels in iSLE patients. Conclusion In this cross-sectional study, distortions in B-cell subsets were observed in iSLE patients and were correlated with IFN scores and IgG levels. Since these factors play an important role in the pathogenesis of SLE, iSLE patients with these distortions, high IFN scores, and high levels of IgG and BAFF may be at risk for progression to SLE.

scholarly journals Complexity of the human memory B-cell compartment is determined by the versatility of clonal diversification in germinal centers

2015 ◽  
Vol 112 (38) ◽  
pp. E5281-E5289 ◽  
Author(s):  
Bettina Budeus ◽  
Stefanie Schweigle de Reynoso ◽  
Martina Przekopowitz ◽  
Daniel Hoffmann ◽  
Marc Seifert ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Clone ◽  
Human Memory ◽  
Memory B Cells ◽  
Sequencing Analysis ◽  
Cell Compartment ◽  
Memory B Cell ◽  
Cell Clones ◽  
B Cell Subsets

Our knowledge about the clonal composition and intraclonal diversity of the human memory B-cell compartment and the relationship between memory B-cell subsets is still limited, although these are central issues for our understanding of adaptive immunity. We performed a deep sequencing analysis of rearranged immunoglobulin (Ig) heavy chain genes from biological replicates, covering more than 100,000 memory B lymphocytes from two healthy adults. We reveal a highly similar B-cell receptor repertoire among the four main human IgM+ and IgG+ memory B-cell subsets. Strikingly, in both donors, 45% of sequences could be assigned to expanded clones, demonstrating that the human memory B-cell compartment is characterized by many, often very large, B-cell clones. Twenty percent of the clones consisted of class switched and IgM+(IgD+) members, a feature that correlated significantly with clone size. Hence, we provide strong evidence that the vast majority of Ig mutated B cells—including IgM+IgD+CD27+ B cells—are post-germinal center (GC) memory B cells. Clone members showed high intraclonal sequence diversity and high intraclonal versatility in Ig class and IgG subclass composition, with particular patterns of memory B-cell clone generation in GC reactions. In conclusion, GC produce amazingly large, complex, and diverse memory B-cell clones, equipping the human immune system with a versatile and highly diverse compartment of IgM+(IgD+) and class-switched memory B cells.

scholarly journals Metabolic phenotype of B cells from young and elderly HIV individuals

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Daniela Frasca ◽  
Suresh Pallikkuth ◽  
Savita Pahwa
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Function ◽  
Metabolic Phenotype ◽  
Healthy Controls ◽  
Metabolic Abnormalities ◽  
Metabolic Markers ◽  
Extracellular Flux ◽  
Cell Pool ◽  
B Cell Subsets

Abstract Background HIV infection induces inflammaging and chronic immune activation (IA), which are negatively associated with protective humoral immunity. Similar to HIV, aging is also associated with increased inflammaging and IA. The metabolic requirements of B cell responses in HIV infected (HIV+) individuals are not known, although metabolic abnormalities have been reported in these individuals. How these metabolic abnormalities are exacerbated by aging is also not known. Methods B cells were isolated by magnetic sorting from the blood of young and elderly HIV + individuals, as well as from the blood of age-matched healthy controls. We evaluated the composition of the B cell pool by flow cytometry, the expression of RNA for pro-inflammatory and metabolic markers by qPCR and their metabolic status using a Seahorse XFp extracellular flux analyzer. Results In this study we have evaluated for the first time the metabolic phenotype of B cells from young and elderly HIV + individuals as compared to those obtained from age-matched healthy controls. Results show that the B cell pool of HIV + individuals is enriched in pro-inflammatory B cell subsets, expresses higher levels of RNA for pro-inflammatory markers and is hyper-metabolic, as compared to healthy controls, and more in elderly versus young HIV + individuals, suggesting that this higher metabolic phenotype of B cells is needed to support B cell IA. We have identified the subset of Double Negative (DN) B cells as the subset mainly responsible for this hyper-inflammatory and hyper-metabolic profile. Conclusions Our results identify a relationship between intrinsic B cell inflammation and metabolism in HIV + individuals and suggest that metabolic pathways in B cells from HIV + individuals may be targeted to reduce inflammaging and IA and improve B cell function and antibody responses.

Reduced Circulating Memory B-Cells Account for Humoral Immune Defects in Multiple Myeloma, Associated with Infective Risk and Poor Vaccination Responses

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3393-3393
Author(s):  
Jonathan Carmichael ◽  
Clive R Carter ◽  
Christopher Parrish ◽  
Charlotte Kallmeyer ◽  
Sylvia Feyler ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Bacterial Infections ◽  
Memory B Cells ◽  
Antibody Responses ◽  
Progressive Reduction ◽  
Cell Numbers ◽  
Humoral Immune ◽  
Transitional B Cells ◽  
B Cell Subsets

Abstract Multiple myeloma (MM) is characterized by an increased risk of infection due to the immunosuppressive effect of the disease and conjointly of therapy. Furthermore, there is impaired responses to vaccination to counter the infection risk. The factors that underpin defective B-cell homeostasis and effective humoral immunity are not clear, nor are the extent of the defects. Also, the level of impaired humoral immunity in MGUS is not fully understood. The aim of this study was to delineate the circulating B-cell populations and recall antibody responses in patients with MGUS & MM, compared to age-matched controls, correlating with the responsiveness to vaccinations, incidence of infective complications and concomitant therapy. We performed comprehensive B-cell immunophenotyping by multi-parameter flow cytometry of peripheral blood samples from patients with MGUS (n=16), asymptomatic MM (n=18) and MM (n=108) with a median age of 63 years (range 38-94) comparing them to age-matched controls (n=9). B-cell subsets included naïve (CD19+CD27-), memory (CD19+CD27+; non-switch CD19+IgD+CD27+, switch CD19+IgD-CD27+), transitional (CD19+CD27-CD24hiCD38hi) & regulatory (CD19+CD27+CD24hi) B-cells. Serum uninvolved total IgG, IgM & IgA levels along with vaccine-specific antibody responses were analysed. There is a progressive decrease in the uninvolved immunoglobulin classes with significant reduction in total IgA (p=0.006) and IgM levels (p=0.007) in aMM/MM compared to MGUS & control (Figure 1). When anti-pneumococcal antibodies were measured, only 30% of aMM/MM patients had adequate protective levels compared to 79% of age-matched controls, with 40% of aMM/MM patients with inadequate levels experiencing recurrent respiratory tract infections compared to 25% of aMM/MM patients with adequate proactive antibodies. Patients with MGUS, aMM and MM have lower total B-cell numbers compared to controls (1-way ANOVA p=0.004; Figure 1). The reduction in B-cell numbers were primarily the consequence of reduced memory B-cells (percentage and absolute 1-way ANOVA p<0.0001), noted in both MGUS and aMM/MM but a progressive reduction with increasing disease activity (MGUS>aMM>MM). Furthermore, a correlation with total IgG levels & memory B-cell numbers is evident (r2=-0.053) & progressive reduction in memory B-cell numbers is seen with advancing cycles of therapy. The ratio of switch:non-switch memory B-cells is unaltered (control 1.05, MGUS 0.53, aMM 1.41 & MM 1.49; 1-way ANOVA p=ns). Conversely, there is a compensatory increase in the percentage of transitional B-cells when increasing disease stage is compared to controls (control 7.38% (95%ci 4.9,9.9) vs MGUS 14.0% (95%ci 7.4, 20.7) vs aMM 14.95% (95%ci 8, 21.9); 1-way ANOVA p<0.001) but a reduction is noted in MM (5.82%, 95%ci 4.5,7.2; p<0.0001), primarily being driven by sequential lines of therapy. As a consequence, the ratio of Memory:transitional B-cells is significantly reduced in aMM/MM compared to MGUS & controls (control 10.35, MGUS 20.46, aMM 7.74 & MM 4.57; 1-way ANOVA p=0.006), associated with increasing incidence of bacterial infections. A non-significant correlation is seen between transitional B-cells and total uninvolved immunoglobulin levels and with recall responses to vaccinations. There is a progressive decrease in the CD19+CD27+CD24hi B-cell subset between control and plasma cell dyscrasias (control 20.4% (95%ci 15.5,25.2), MGUS 14.0% (95%ci 7.4, 20.7), aMM 14.95% (95%ci 8, 21.9) & MM 5.82%, 95%ci 4.5,7.2; p<0.0001), primarily being driven by sequential lines of therapy and associated with increased incidence of infection. This study illustrates that patients with myeloma demonstrate reduced total circulating B-cells primarily as a consequence of reduced memory B-cells, associated with reduced immunoglobulin and recall antibody responses. This is associated with increased incidence of bacterial infections and is worsened by sequential exposure to lymphodepleting therapies. Of particular importance is the identified aberration in B-cell subsets seen in MGUS compared with age-matched control, indicative of humoral immune dysregulation highlighting that MGUS may not be an immunologically inert disorder. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Expression of TLR-9, CD86, and CD95 Phenotypes in Circulating B Cells of Patients with Chronic Viral Hepatitis B or C before and after Antiviral Therapy

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Ping-wei Zhao ◽  
Liang Ma ◽  
Hui-fan Ji ◽  
Lei Yu ◽  
Jun-yan Feng ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Viral Hepatitis ◽  
Differential Expression ◽  
Significant Negative Correlation ◽  
Chronic Viral Hepatitis ◽  
Healthy Controls ◽  
Before And After ◽  
Chronic Hbv ◽  
B Cell Subsets

Aims. This study aimed to assess the differential expression of specific B cell subtypes in patients with chronic viral hepatitis.Methods. The frequencies of differential expression of specific B cell subtypes in patients with chronic viral hepatitis and healthy controls were assessed by flow cytometry using monoclonal antibodies specific for CD38, CD27, CD86, CD95, TLR-9, and IgD. The effect of adefovir treatment on B cell subsets in HBV patients was determined. The values of clinical parameters in the patients were also measured.Results. The frequency of CD86+ B cells was not significantly different in chronic HBV patients but was higher in HCV patients compared with that in healthy controls. CD95 and IgD levels were lower in HBV and HCV patients than in healthy controls. A significant negative correlation occurred between the proportion of CD95+ B cells and HBV DNA viral load. The frequency of TLR-9 on the B cells in HBV and HCV patients was higher compared with that of healthy controls. After treatment with adefovir, the frequency of CD95 and IgD expressed on B cells was increased in HBV patients.Conclusions. Activated B cells and exhausted B cells homeostasis were commonly disturbed in HBV and HCV patients.

scholarly journals Comprehensive Profiling of Disease-Relevant Copy Number Aberrations Improves Risk Assessment and Unveils the Clonal Origin of Relapse in Pediatric Acute Lymphoblastic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1474-1474
Author(s):  
Donát Alpár ◽  
Richárd Kiss ◽  
Ambrus Gángó ◽  
Anne Benard-Slagter ◽  
Bálint Egyed ◽  
...  
Keyword(s):  
Risk Assessment ◽  
B Cell ◽  
Copy Number ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Risk Groups ◽  
Gene Rearrangements ◽  
Disease Course ◽  
Cell Precursor ◽  
Time Points

Introduction: Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy characterized by a heterogeneous genomic landscape. Copy number aberrations (CNA) emerge during the development, progression and treatment resistance of ALL, and can serve as genomic markers for prognostic classification of patients or for scrutinizing clonal evolution associated with relapse. While identification of distinct CNAs with well-characterized prognostic significance has its own value, uncovering the co-segregation of driver aberrations in individual patient samples could allow for a more personalized risk assessment and treatment response prediction. Methods: Disease-relevant CNAs were profiled in children with B- or T-cell precursor ALL using a next-generation sequencing based digital multiplex ligation-dependent probe amplification (digitalMLPA) assay containing 598 probes specific for 54 genes with key relevance in ALL. Besides the diagnostic samples of 91 patients treated according to the BFM protocols, 14 matching samples drawn at the time of first or second relapse were comparatively analyzed. Clonal relationship between B-cell precursor cell populations prevailing at different time points during the disease course was also investigated by screening immunoglobulin heavy-chain gene rearrangements in matching diagnostic and relapse samples using Illumina deep-sequencing with &gt;20,000x coverage. Results: Whole chromosome gains and losses, subchromosomal CNAs as well as alterations conferring intrachromosomal gene fusions were simultaneously identified by digitalMLPA with results available within 36 hours. Aberrations were observed in 96% of diagnostic patient samples and increased numbers of CNAs were detected in individual samples at the time of relapse as compared to diagnosis. DigitalMLPA results were successfully validated by conventional MLPA, FISH and PCR data. Comparative scrutiny of 24 matching diagnostic and relapse samples from 11 patients harboring CNAs revealed three different patterns of clonal relationships with (i) one patient displaying identical CNA profiles at diagnosis and relapse, (ii) six patients showing clonal evolution with all lesions detected at diagnosis being present at relapse and (iii) four patients displaying conserved as well as lost or gained CNAs at the time of relapse, suggestive of the presence of a common ancestral cell compartment giving rise to clinically manifest leukemia at different time points during the disease course. Time between diagnosis and first relapse of T-ALL patients displaying altered CNA profiles suggested a prolonged time requirement of clonal evolution, and of the development of manifest leukemia from an ancestral clone compared to the quick return of an identical clone at the time of relapse. Comparison of the IGH gene rearrangements identified at diagnoses and relapse revealed identical compositions of the most abundant clonotypes in all but one B-ALL patients analyzed; hence, IGH repertoire did not reveal an additional depth of clonal history in our cohort, e.g. by demonstrating the presence of an ancestral clone as the major source of clonal expansion at disease progression in a patient with altered CNA profiles suggesting direct clonal evolution between diagnosis and relapse. Copy number profiles acquired by digitalMLPA were used for determining CNA-based risk groups (Table 1) which were combined with karyotyping and molecular cytogenetic data in order to establish an extended prognostic classifier for patients with B-cell precursor ALL. This novel classifier distinguished four combined genetic risk groups showing significantly different 5-year survival rates (GR: 97%, IR: 84%, IHR: 63% and PR: 13%). Conclusions: DigitalMLPA allows for a rapid, scalable and highly optimized copy number profiling of genomic regions recurrently altered by driver aberrations in pediatric ALL. Based on the comparison of CNA profiles at diagnosis and relapse, clonal evolution and emergence of relapse from an ancestral clone are the predominant driving mechanisms of disease progression. Comprehensive copy number profiling by digitalMLPA identifies distinct prognostic groups for risk assessment in B-cell precursor ALL. Supporting grants : LP95021, K_16 #119950, NVKP_16-1-2016-0004, KH17-126718, BO/00320/18/5, FK_19 #131476, ÚNKP-19-4-SE-77 Disclosures Benard-Slagter: MRC Holland: Employment. de Groot:MRC Holland: Employment. Savola:MRC Holland: Employment.

Deletion Of The Von Hippel-Lindau Gene Interferes With The Development Of Peripheral B-Cell Subsets

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3666-3666
Author(s):  
Kathrin Händschke ◽  
Stefanie Weber ◽  
Mandy Necke ◽  
Anita Hollenbeck ◽  
Bertram Opalka ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cxcr4 Expression ◽  
Hypoxic Response ◽  
Hematopoietic Stem ◽  
Von Hippel Lindau ◽  
Cell Numbers ◽  
Immature B Cells ◽  
B Cell Subsets

Abstract The development of hematopoietic cells occurs in highly specialized microenvironments within bone marrow, thymus and spleen. Hematopoietic stem and progenitor cells are positioned at the lowest end of a bone marrow oxygen gradient, which implies a role for the hypoxic-response pathway in regulating hematopoiesis. In this pathway the von Hippel-Lindau protein (pVhl) is the central negative regulator and continuously mediates the proteasomal destruction of the hypoxia-inducible factor-1α (Hif-1α). Under hypoxic conditions Hif-1α destruction is inhibited and results in the expression of hypoxia-inducible genes. In order to study the role of pVhl in hematopoiesis we crossed Vhl conditional mice with vav-iCre mice to induce a constitutive hematopoiesis-specific Vhl deletion (VhlKOvav). As expected, we observed a dramatic expansion of spleen erythropoiesis, however bone marrow lin-Sca-1+c-kit+CD48-CD150+ hematopoietic stem cell numbers of VhlKOvav mice did not significantly differ from control mice. The most striking observation in VhlKOvav mice was that B-cell numbers in bone marrow and spleen were decreased by 53% and 78%, respectively. In order to exclude that the loss of B-cells in VhlKOvav mice was due to a B-cell extrinsic effect we crossed Vhl conditional mice to the B-cell specific deleter lines CD19-Cre and Mb1-Cre (VhlKOCD19 and VhlKOMb1 mice). Flow cytometric analysis also revealed decreased peripheral B-cell numbers in VhlKOCD19 mice and an even more pronounced B-cell loss in VhlKOMb1 mice (B-cells spleen, mean±SEM; control (n=9), 77±2.4x106; VhlKOMb1 (n=12), 1.3±0.2x106; p<0.001). This demonstrated that pVhl is cell-autonomously required for the normal development of the B-cell compartment. To more accurately define the Vhl-null B-cell developmental defect we analyzed bone marrow B-cell subsets of VhlKOMb1 and control mice. Early B-cell progenitor numbers defined by the surface markers B220, CD43, CD24, BP-1 (Hardy’s classification, fractions A-D) of VhlKOMb1 mice were not altered. In contrast, we observed a significant 41% reduction of the immature B220+CD43-IgM+IgD- and a 46% reduction of the mature B220+CD43-IgM+IgD+ bone marrow VhlKOMb1 B-cell numbers compared to controls. In peripheral blood VhlKOMb1 immature and mature B-cell numbers were even more decreased (by 81% and 86%, respectively). We hypothesized that increased CXCR4 expression, which is negatively regulated by pVhl, could be responsible for the decreased bone marrow egress of immature bone marrow B-cells. Indeed, we observed a more than twofold increase of CXCR4 expression of VhlKOMb1 compared to control bone marrow immature B-cells (MFI, mean±SEM; control (n=3), 72±15; VhlKOMb1 (n=3), 162±23; p<0.05). Strikingly, VhlKOMb1 spleens were almost devoid of follicular B220+CD21/35intmCD23+IgD+IgM+ and marginal zone B220+CD21/35highCD23-IgMhigh B-cells and accordingly follicular structures could not be observed in histological sections. In VhlKOMb1 lymph nodes the follicular B-cell numbers were also dramatically decreased. Next, we flow sorted residual splenic VhlKOMb1 B-cells and were able to confirm deletion of the Vhl-gene by PCR. Target genes of the hypoxic-response pathway such as Pgk1, Vegf and Bnip3 were 10- to more than 100-fold higher expressed in sorted VhlKOMb1 compared to control B-cells. As a possible reason for the low VhlKOMb1 peripheral B-cell numbers we identified a more than twofold reduction in CD62L expression by immature blood B-cells (MFI, mean±SEM; control (n=3), 3127±250; VhlKOMb1 (n=3), 1528±66; p<0.05) which presumably impaired their homing ability to peripheral lymphoid organs. Additionally, we detected an increased B-cell apoptosis rate of VhlKOMb1 B-cells in the spleen. Finally, we were able to show that decreased follicular splenic B-cell numbers of VhlKOCD19 mice could be completely rescued by additionally breeding Hif-1α conditional alleles into the system (Hif-1αVhlKOCD19mice). In summary, we identified pVhl as a key regulator of peripheral B-cell maturation. We show that pVhl-mediated negative regulation of the hypoxic-response pathway is required for normal peripheral B-cell differentiation. Our data suggest that B-cell pVHL loss-of-function leads to decreased bone marrow egress and decreased lymphoid organ homing of immature B-cells mediated by the dysregulation of CXCR4 and CD62L. Disclosures: No relevant conflicts of interest to declare.

B Cell With Regulatory Function Are Enriched Within Transitional and IgM Memory B Cell Subsets In Healthy Donors But Are Reduced and Functionally Impaired In Patients With Chronic Graft-Versus-Host Disease

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4478-4478
Author(s):  
Anushruti Sarvaria ◽  
Ahmad Khoder ◽  
Abdullah Alsuliman ◽  
Claude Chew ◽  
Takuya Sekine ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cd4 T Cells ◽  
Memory B Cells ◽  
Regulatory Function ◽  
Healthy Controls ◽  
Transitional B Cells ◽  
B Cell Subsets

The immunosuppressive function of IL10 producing regulatory B cells (Bregs) has been shown in several murine models of inflammation and autoimmune disease. However, there is a paucity of data regarding the existence of an equivalent regulatory B cell subset in healthy individuals and their potential role in the pathogenesis of chronic graft-versus-host disease (cGVHD) remains unknown. Here, we examined the functional regulatory properties of peripheral blood (PB)-derived human B cell subsets from healthy individuals. In addition, we carried out studies to explore their role in cGVHD, using B cells from patients following allogeneic stem cell transplantation (HSCT). We first determined whether human IL-10 producing B cells are enriched within any othe previously described human B cell subsets: CD19+IgM+CD27+ IgM memory, CD19+IgM-CD27+ switched memory, CD19+IgM+CD27- naive, and and transitional CD19+CD24hiCD38hi. Following in vitro stimulation with CD40 ligand, the majority of IL-10 producing B cells were found within the CD24hiCD38hi transitional and CD19+IgM+CD27+B cell subsets. We next assessed the regulatory properties of the PB-derived B cell subsets, by sort-purifying IgM memory (CD19+IgM+CD27+), switched memory (CD19+IgM-CD27+), naïve (CD19+IgM+CD27-) and transitional (CD19+CD24hiCD38hi) B cells from healthy controls, and cultured them 1:1 with autologous magnetic-bead purified CD4+ T cells. CD3/CD28 stimulated CD4+ T cells cultured with either CD19+IgM+CD27- naïve or CD19+IgM-CD27+ switched memory B cells proliferated to the same extent and produced equivalent amounts of IFN-γ to cultures containing CD4+ T cells alone. In contrast, culture of CD4+ T cells with IgM memory and transitional B cells significantly suppressed CD4+ T cell proliferation [median percent proliferating CD4+ T cells 52.5%; (33%-75%)] and 51% (25%-63%)], respectively when compared with CD3/CD28 stimulated CD4+ T cells (positive control) [89.5% (75%-92%], p=0.0001. The inhibitory effect of IgM memory and transitional B cells on CD4+ T cell proliferation was cell dose dependent with the highest suppression observed at a ratio of 1:1. These data suggest that human PB transitional and IgM memory B cells are endowed with regulatory function. We next examined if the in vitro suppressive effect of transitional and IgM memory B cells is mediated by regulatory T cells (Tregs). For this purpose, CD4+ T cells were depleted of CD127lo CD25hi CD4+ T cells by magnetic cell purification. B cell subsets were cultured with CD3/CD28 stimulated CD4+ CD25- T cells at a ratio of 1:1. IgM memory and transitional B cells were able to significantly suppress the proliferation and Th1 cytokine response by CD4+ CD25- T cells compared to cultures containing CD4+ CD25-T cells alone, indicating that the suppressive activity of Bregs is independent of Tregs. To further understand the underlying mechanims though which Bregs exert T-cell suppression, we used antibody blockade experiments and showed that this suppressive effect was mediated partially via the provision of IL-10, but not TGF-ß. Using transwell experiments, we further determined that the suppressive function of Bregs is also partly dependent on direct T cell/B cell contact. We next assessed whether the activity of Breg cells might be altered in patients with cGVHD. B cells from patients with cGVHD were refractory to CD40 stimulation and produced less IL-10 when compared to patients without cGVHD post-SCT and healthy controls, [1.02% (0.22-2.26) vs.1.72% (0.8-5.52) vs. 2.16 (1.3- 5.6), p=0.001]. Likewise, the absolute number of IL-10 producing B cells was significantly lower in cGvHD patients compared to patients without cGVHD and healthy controls (p=0.007), supporting both a qualitative and quantitative defect in IL-10 producing B cells in cGvHD. Our combined studies provide important new data defining the phenotype of B cell populations enriched in regulatory B cells in healthy humans and provide evidence for a defect in the activity of such cells in patients with cGVHD post-SCT. In association with previous reports showing defects in Treg cell activity in GVHD, our results suggest the existence of a broad range of deficiencies in immune regulatory cell function in cGvHD patients. * Both Anushruti Sarvaria and Ahmad K contributed equally. Disclosures: No relevant conflicts of interest to declare.

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