Identifying Candidate Normal and Leukemic B Cell Progenitor Populations with Hierarchical Clustering of 6-Color Flow Cytometry Data - A Better View.

Abstract 6-color flow cytometry allows multiparameter analysis of high numbers of single cells. It is an excellent tool for the characterization of a wide range of hematopoietic populations and for monitoring minimal residual disease. However, analysis of complex flow data is challenging. Gating populations on 28 two-parameter plots is extremely tedious and does not reflect the multidimensionality of the data. Here, we describe a novel approach, employing hierarchical clustering (HCA) and support vector machine (SVM) learning in analyzing flow data. This approach provides a new perspective for looking at flow data and promises better identification of rare and novel subpopulations that escape classic analysis. Our aim was to identify normal and leukemic B cell progenitor/stem cell populations in normal (n=6) and ALL (n=10) bone marrow. Samples were labelled with fluorochrome-conjugated antibodies to 6 CD markers (CD 10, 19, 22, 34, 38, 117) and 104 to 106 events were acquired (FACSCanto, BD Biosciences). To analyze flow data with HCA we developed a new algorithm, better suited for the ellipsoid nature of cell populations than other current HCA metrics. Data exported from DiVa software were externally compensated and Hyperlog transformed to achieve a logarithmic-like scale that displayed zero and negative values. Normalized data were then subjected to HCA employing a scale-invariant Mahalanobis distance measurement for merging clusters. This reflects the extended ellipsoid shape of the populations (here: 8 dimensional ellipsoids). We developed a new adaptive linkage algorithm that smoothly shifts from the Euclidean distance (when clusters are too small to compute Mahalanobis distance) to Mahalanobis distance measurement. This allowed us to build the hierarchy from single events, yet to retain the advantage of Mahalanobis measurement for larger clusters. To build classifiers we used SVM employing polynomial kernel. All work was carried out in MATLAB (MathWorks, Inc.). The resulting hierarchical tree combined with the heatmap of the CD marker expression allows visualization of hierarchically clustered data with all 8 parameters displayed in a single plot (!) as compared to 28 traditional two-parameter plots. HCA has big advantage of providing populations homogenous in their expression pattern of all parameters (without the need for complex sub or back gating). We were able to identify populations corresponding to the different stages of B-cell development. In a normal control bone marrow we could detect the following candidate B-lineage progenitor populations: CD34+117+38+10−22−19− (0.94% of total) progenitor/stem cells, CD34+117−38+10+22+19med (0.26% of total) pro-B cells, CD34−117−38+10+22+19+ (2.77% of total) small pre-B cells (lower FCS values), CD34−117−38+10+22+19+ (1.09% of total) large pre-B cells (higher FCS values) and CD34−117−38lo10−22+19+ (5.94% of total) (immature) B cells. In 10 diagnostic or relapse samples HCA clearly identified the main leukemic population. HCA is able to visualize otherwise “hidden” populations. This was exemplified by a distinct CD38+B-lin− population that overlapped with other populations in all 28 two-parameter plots (most likely T cells). We have built a classifier able to find established populations across samples and in large datasets (106 events) for which HCA would be computationally too demanding. In summary, we show the advantages of using hierarchical clustering analysis for large complex multiparameter flow cytometry datasets.

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Circulating B Cell Clones in Familial Waldenström Macroglobulinemia.

Blood ◽

10.1182/blood.v120.21.2703.2703 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2703-2703

Author(s):

Mark C. Lanasa ◽

Sallie D. Allgood ◽

Lynn R. Goldin ◽

Danielle M. Brander ◽

Mary L. McMaster

Keyword(s):

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Family Members ◽

Similar Proportion ◽

Cell Populations ◽

Color Flow ◽

Flow Cytometric ◽

Cell Clones ◽

Genomic Studies

Abstract Abstract 2703 Background and Significance: Lymphoplasmacytic lymphoma (LPL) is an indolent but incurable B cell lymphoproliferative characterized by the clonal expansion of plasmacytoid lymphocytes in the lymph nodes and bone marrow. The majority of cases are associated with an IgM isotype paraprotein, and when present, is termed Waldenström macroglobulinemia (WM). WM shows familial aggregation suggesting an inherited risk for disease, and also co-aggregates with CLL in families. In our previous work, we have shown that clonal populations of peripherally circulating B cells can be identified in 18% of unaffected family members from CLL kindreds. Most of these clonal populations have a typical CLL immunophenotype and have been termed CLL-like monoclonal B cell lymphocytosis (MBL). Because CLL and WM have related gene expression profiles and appear to have shared genetic risk, we hypothesized that unaffected family members of WM kindreds would have detectable circulating clonal B cell populations. Further, we undertook systematic flow cytometric screening of familial WM and IgM MGUS cases to determine the prevalence, immunphenotype, and biologic characteristics of circulating malignant B cells. Methods: A diagnosis of LPL / WM or IgM MGUS was determined using standard WHO criteria. All patients and unaffected family members were ascertained at the National Cancer Institute and provided informed consent. Peripheral blood mononuclear cells were isolated using density centrifugation and viably frozen in DMSO containing media. We developed a two tube, nine color flow cytometric assay: the first tube allowed for detection of CLL-like clones based upon co-expression of CD5, CD20, and CD23; the second tube targeted WM populations based upon expression on CD19, CD20, CD25, CD38, and surface IgM. Cell populations were considered clonally restricted if the κ: λ was > 3.0 or < 0.3. Clonal populations were then isolated using fluorescence activated cell sorting (FACS). RNA and genomic DNA were extracted for genetic and genomic studies using phenol: chloroform purification. Results: A total of 155 individuals were analyzed: 54 WM / LPL, 17 IgM MGUS, 1 IgG MGUS, 1 IgA MGUS, 1 NHL, and 81 unaffected family members. Twenty of 54 WM patients had detectable peripherally circulating populations. Thirteen WM patients had no detectable B cells, of these, 11 patients had prior treatment. As such, among the 41 WM patients with a B cell compartment that was analyzable by flow cytometry, 49% (20 of 41) had peripherally circulating B cell clones detected. Four of these 20 cases showed two immunophenotypically distinct clonal B cell populations. The immunophenotype was somewhat heterogenenous: 18 cases expressed surface IgM, CD38 was variable but expressed in most cases, CD25 was not detected in any case, and 4 cases showed a CLL like (CD5+CD20dimCD23+) immunophenotype. Interestingly, we detected peripherally circulating B cell clones in 9 of 17 cases (53%) of IgM MGUS, a proportion nearly identical to that identified in WM / LPL. Three of 9 were “CLL-like” with co-expression of CD5 and CD23, while the majority of clones were CD5negIgM+CD38+. Among unaffected family members, we identified B cell clones in only 4 of 81 (5%). All 4 cases expressed CD5, and 3 showed a CLL-like phenotype, consistent with these individuals having MBL. Among all study subjects, 20 clonal B cell populations of > 104 B cells were FACS purified from 18 different cases: 12 WM, 4 IgM MGUS, 1 IgA MGUS, and 1 unaffected family member. Conclusions: Peripherally circulating B cell clones with an immunophenotype similar to that of LPL can be identified in approximately half of patients with WM / LPL. We observed for the first time that a similar proportion of patients with IgM MGUS have detectable clonal populations with an immunophenotype similar to that observed in WM patients. Genetic and genomic studies to determine the lineage of these populations are currently underway. The frequency of MBL among unaffected family members with WM is lower than that observed in CLL kindreds. CLL-like MBL can be detected at very low numbers because the cell population is immunophenotypically abnormal. The peripherally circulating clones identified in WM / LPL patients have an otherwise normal B cell immunophenotype and can only be detected by light chain restriction. This likely significantly limits the ability of flow cytometry to detect pre-clinical CD5neg IgM expressing clonal populations. Disclosures: No relevant conflicts of interest to declare.

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ZAP-70 Enhances Migration of Malignant B Cells towards Lymphoid Organs in a Burkitt Lymphoma Xenograft Model

Blood ◽

10.1182/blood.v118.21.2844.2844 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2844-2844

Author(s):

Noelia Purroy ◽

Eva Calpe ◽

Pau Abrisqueta ◽

Cecilia Carpio ◽

Carles Palacio ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

B Cells ◽

Tumor Growth ◽

B Cell ◽

Cell Lines ◽

Ectopic Expression ◽

Xenograft Model ◽

Lymphoid Organs ◽

Transfected Cells

Abstract Abstract 2844 Introduction. ZAP-70 (ξ-associated protein) is a protein tyrosine kinase of the Syk/ZAP family that plays a crucial role in cellular activation in T and NK cells. High expression of ZAP-70 protein in malignant cells from Chronic Lymphocytic Leukemia (CLL) correlates with adverse clinical prognostic features, such as unmutated IgHV genes, short time to progression, and short survival. Moreover, ZAP-70 protein has been related to aggressive features of the CLL cells, such as enhanced B-cell receptor (BCR) signaling and higher migration capacity. To further investigate into the mechanisms by which ZAP-70 protein influences the clinical outcome of patients with CLL, we analyzed the functional consequences of ZAP-70 ectopic expression in malignant B-cells. For this, Ramos and Raji (Burkitt) B-cell lines were stably transfected with a ZAP-70 expressing vector (pEGFP-N2ZAP-70). Raji transfectant showed constitutively phosphorylated ZAP-70 protein, whilst Ramos cells required stimulation with 5 μg/ml F(ab') 2 anti-IgM to get ZAP-70 activated. ZAP-70 expression induced the upregulation of the chemokine receptor CCR7, thus giving the cells the ability to better respond and migrate towards CCL21 (own data, Blood 2011 pre-published). CCR7 ligands (chemokines CCL21 and CCL19) are mainly expressed in high endothelial venules and the T zones from secondary lymphoid organs. The aims of this study were firstly to evaluate in vivo the migratory/invasive capability of pEGFP-N2ZAP-70 transfected Raji and Ramos cell lines compared to pEGFP Raji and Ramos cell lines; and later, to compare the overall survival (OS) of mice injected with pEGFP-N2ZAP-70 transfected cells to those injected with only pEGFP transfected cells. Methods. For this, a total of 27 7- to 8-week old SCID (CB17Crl) mice were used. Mice were inoculated intravenously with 5×106 cells of each cell line (6 mice with Raji-GFP, 5 mice with Raji-GFP-ZAP-70, 5 mice with Ramos-GFP and 10 mice with Ramos-GFP-ZAP-70). Mice were observed for the onset of hind legs paralysis, dyspnea, or evidence of tumor growth, once symptoms appeared, mice were euthanized and lymphoid and non-lymphoid organs were obtained for further analysis of the presence of GFP-positive cells by flow cytometry and immunohistochemistry. Results. Twenty-six out of twenty-seven injected mice were included in the analysis. The excluded mouse was found dead before it could be euthanized to obtain the organs. In the Raji xenograft model, 11/11 (100%) of mice had hind legs paralysis as the first symptom to appear. The median survival was 19 days for GFP-ZAP-70 and 16 days for GFP injected mice. There were no statistically significant differences between survival of GFP-ZAP-70 and GFP injected mice (OS was 66.7% [95% CI 38.4–100] vs 33.3% [95% CI 0–71.1], p=0.784, at 19 and 16 days, respectively). In the Ramos xenograft model, 6/15 (40%) of mice showed hind legs paralysis as the first symptom to appear, as well as evidence of abdominal tumor growth in 6/15 (40%), whereas in 3/15 (20%) the established event was dyspnea. The median survival in Ramos xenograft model was 40 days for GFP-ZAP-70 and 38 days for GFP injected mice. Again there were no statistically significant differences between survival of GFP-ZAP-70 and GFP Ramos injected mice (OS was 50% [95% CI 18.4–81.6] vs 40% [95% CI 0–83.8], p=0.180, at 40 and 38 days, respectively). By flow cytometry analysis of GFP cells we found that in the Raji xenograft model there were statistically significant differences between the migration of GFP-ZAP-70 and GFP injected cells towards bone marrow (21.5% vs 5.17, p=0.011), spleen (0.08% vs 0.01%, p=0.006) and thymus (0.00% vs 0.02%, p=0.037). The highest percentages of GFP positive cells were found in bone marrow samples (mean, 9.85%), whereas in spleen and thymus the percentages of GFP positive cells were all below 0, 1%. There was no statistically significant difference between the cellular migration in the Ramos xenograft model in any of the organs analyzed. Conclusion. In conclusion, malignant B-lymphocytes with ectopic expression of activated ZAP-70 protein show enhanced ability to migrate towards and infiltrate lymphoid organs in a xenograft model, specially the bone marrow, although it does not translate into a worse survival of the animals. Further specific immunohistochemical assays to determine infiltrated areas by ZAP-70 expressing lymphocytes are in process. Disclosures: No relevant conflicts of interest to declare.

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IL-21 and IL-6 Mediate Interactions Between T Cells and Malignant B Cells in the Bone Marrow Microenvironment in Waldenstrom's Macroglobulinemia

Blood ◽

10.1182/blood.v120.21.1554.1554 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1554-1554

Author(s):

Lucy S. Hodge ◽

Steve Ziesmer ◽

Frank J Secreto ◽

Zhi-Zhang Yang ◽

Anne Novak ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

T Cell ◽

B Cells ◽

Tumor Microenvironment ◽

B Cell ◽

Bone Marrow Microenvironment ◽

Waldenström’S Macroglobulinemia ◽

Waldenström's Macroglobulinemia

Abstract Abstract 1554 T cells in the tumor microenvironment influence the biology of malignant cells in many hematologic malignancies, often through cytokine-mediated interactions. Recent studies involving healthy B cells and CD4+T cells identified an interplay between IL-6 and IL-21, whereby IL-6 increased IL-21 production by T cells, driving the differentiation and IL-6 secretion of nearby B cells. In addition to their known effects on healthy B cell function, IL-6 and IL-21 have also been implicated in the pathology of various lymphomas. In Waldenstrom's macroglobulinemia (WM), IL-6 is elevated in the bone marrow and is associated with increased IgM production. However, the function of IL-21 in the WM tumor microenvironment and its relationship to IL-6 is poorly understood. Our objective in this study was to characterize IL-21 production and function in WM and to examine the role of IL-6 and IL-21 in regulating interactions between malignant B cells and T cells in the tumor microenvironment. Immunohistochemistry revealed significant IL-21 staining in bone marrows of patients with WM (n=5), but the areas of infiltration by WM in the bone marrow sections appeared negative for IL-21 staining. To better understand the origin of IL-21 in in the tumor microenvironment, IL-21 expression was assessed by PCR in the CD19−CD138− fraction of cells remaining in patient bone marrow aspirates after positive selection for malignant B cells (n=5). IL-21 transcript was detected in 4/5 samples. CD19−CD138− cells activated with anti-CD3 and anti-CD28 antibodies expressed higher levels of IL-21 transcript and secreted significantly higher levels of IL-21 protein compared to unstimulated cells, suggesting that IL-21 in the WM bone marrow is derived from activated T cells. Intracellular expression of IL-21 protein was confirmed in CD4+ and CD8+ cells within the CD19−CD138− population using flow cytometry. Furthermore, dual staining of WM bone marrow sections with antibodies against IL-21 and CD3 or CD20 revealed co-staining of IL-21 with CD3+ T cells but not with CD20+ B cells. The response of WM B cells to T-cell derived IL-21 was then assessed in positively selected CD19+CD138+ WM B cells (n=5) and in the MWCL-1 cell line. Using flow cytometry, both the IL-21 receptor and the required common gamma chain subunit were detected on all patient samples as well as on MWCL-1 cells. Treatment of MWCL-1 cells with IL-21 (100 ng/mL) for 72 h increased proliferation by 35% (p<0.05) and IgM secretion by 80% (p<0.005). Similarly, in primary CD19+CD138+ WM cells (n=5), proliferation increased on average by 38% and IgM secretion by 71%. No apoptotic effects were associated with IL-21 in WM. Characterization of STAT activation in response to IL-21 revealed significant phosphorylation of STAT3 in both CD19+CD138+ WM cells and MWCL-1 cells and was associated with increases in BLIMP-1 and XBP-1 protein and decreases in PAX5. As STAT3 activation is known to regulate IL-6, we assessed the effect of IL-21 on B cell-mediated IL-6 secretion using ELISA. IL-21 significantly increased IL-6 secretion by both primary CD19+CD138+ WM cells (n=4) and MWCL-1 cells (87.9 +/− 10.9 ng/mL vs. 297.8 +/− 129.2 ng/mL, p<0.05). Treatment with IL-6 and IL-21 together had no additional effect over IL-21 alone on proliferation or IgM secretion in MWCL-1 cells, but culturing anti-CD3/anti-CD28-activated CD19−CD138−cells from WM bone marrows with IL-6 significantly increased IL-21 secretion (n=3). Overall, these data indicate that T-cell derived IL-21 significantly promotes growth and immunoglobulin production by malignant WM B cells and that subsequent IL-6 secretion by malignant B cells may enhance the secretion of IL-21 by T cells within the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

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Impaired Precursor B-Cell Maturation/Production In The Bone Marrow: Association With Molecular and Immunophenotypic Markers Of Myeloid Dysplasia In Suspected Low-Grade MDS

Blood ◽

10.1182/blood.v122.21.4943.4943 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4943-4943

Author(s):

Charles Repetti ◽

Hsueh-Hua Chen ◽

Yongbao Wang ◽

Vanessa A Jones ◽

Albert K Ho ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Somatic Mutation ◽

Conflicts Of Interest ◽

Statistical Significance ◽

Sequence Variant ◽

Low Grade ◽

Precursor B Cells

Abstract Rationale Myelodysplastic syndromes (MDS) are clonal stem cell disorders that disrupt orderly maturation of multiple hematopoietic lineages. Several studies have suggested that maturation of precursor B cells (hematogones) is also abnormal in MDS. As a result, the presence of normal numbers or increased precursor B cells in bone marrow (BM) is frequently used as a diagnostic feature arguing against a diagnosis of MDS. We compared the presence of myeloid-associated gene mutations and myeloid maturation abnormalities with qualitative and quantitative precursor B cell findings in BM samples submitted for workup of cytopenias or MDS. Methods Seventeen BM aspirate samples with <5% blasts submitted for cytopenia or MDS evaluation were compared with 10 samples having 5% or more blasts and changes diagnostic of MDS or AML. Mutation analysis was performed on genomic DNA using a targeted exome sequencing assay. This assay employs a TruSeq custom amplicon design on the MiSeq platform (Illumina, San Diego, CA). The assay covers the commonly mutated areas of 19 myeloid-associated genes. Somatic mutation status was assigned based on mutation levels, previous association with myeloid neoplasia, and no prior identification in public or internal databases as a normal sequence variant. Flow cytometry using 6-color (CD19/CD34) and 8-color (CD19/10) formats was used to assess lymphoblasts; CD34/13 was used to assess myeloblasts; and CD11b, CD13, CD16, and CD38 were used to assess abnormalities in myelopoiesis. Results Among the 17 BM samples submitted for cytopenia or MDS evaluation that had <5% blasts, 7 (41%) had immunophenotypic myeloid maturation abnormalities. Ten (59%) of the 17 cases had at least one myeloid-associated somatic mutation, with TET2 and ASXL1being the most commonly mutated genes. The ratio of myeloblasts to B-lymphoblasts, calculated using either CD10 or CD19, was >10:1 in 10/17 (59%) cases. Nine of the 17 (53%) cases had virtually no precursor B cells detected. Discrete abnormalities in more mature myeloid forms were seen in 7/10 (70%) cases with low numbers of B-lymphoblasts but in none of the 7 cases with significant numbers of B-lymphoblasts. MDS-associated mutations were more common in cases with rare B-lymphoblasts (7/9) than in those with higher percentages of precursor B cells (3/8), but the difference did not reach statistical significance (P = 0.15). Genes mutated in the group with B-lymphoblasts present included ASXL1 (3 cases), DNMT3A (2), TET2 (1) and TP53 (2). Two of these mutated cases presented with isolated thrombocytopenia. By comparison, myeloblast/lymphoblast ratios were >50:1 in all 10 unequivocal MDS/AML samples (>5% blasts); 8 (80%) of these cases had MDS-associated mutations, and 4 (50%) had mutations in multiple genes. Conclusions Decreases in BM precursor B cells in cases of possible low-grade MDS were usually, but not always, associated with the presence of MDS-associated mutations. However, cases with normal or increased precursor B cell numbers also showed MDS-associated mutations although immunophenotypic evidence of myeloid maturation abnormalities was not seen in this group. The identification of a subgroup of cytopenic patients with likely pathogenic mutations in bone marrow precursors but minimal phenotypic evidence of myeloid dysplasia may indicate clonal abnormalities primarily located outside the granulocyte or common stem precursor populations, e.g. restricted to the megakaryocytic lineage. Therefore, the presence of intact precursor lymphoblast and myeloid maturation by higher-dimensional flow cytometry as a primary criterion to argue against a diagnosis of low-grade MDS needs further evaluation, especially when granulocytopenia is absent. Disclosures: No relevant conflicts of interest to declare.

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Clonal B-cell populations in patients with idiopathic thrombocytopenic purpura

Blood ◽

10.1182/blood.v76.11.2321.bloodjournal76112321 ◽

1990 ◽

Vol 76 (11) ◽

pp. 2321-2326 ◽

Cited By ~ 39

Author(s):

D van der Harst ◽

D de Jong ◽

J Limpens ◽

PM Kluin ◽

Y Rozier ◽

...

Keyword(s):

Flow Cytometry ◽

B Cells ◽

Autoimmune Diseases ◽

B Cell ◽

Malignant Lymphoma ◽

Idiopathic Thrombocytopenic Purpura ◽

Clinical Manifestations ◽

Cell Populations ◽

Dna Rearrangement ◽

Thrombocytopenic Purpura

Idiopathic thrombocytopenic purpura (ITP) may be associated with other autoimmune diseases and the development of lymphoproliferative malignancies. In Sjogren's disease, Graves' disease, and essential mixed cryoglobulinemia, which are also associated with the development of B-cell neoplasia, clonal B-cell expansions have been detected. Eleven patients with ITP were investigated for the presence of a clonal excess (CE) using kappa-lambda flow cytometry and DNA analysis for rearrangement of immunoglobulin heavy and light chain genes in blood and/or spleen lymphocytes. In 10 of 11 patients, clonal B-cell populations were found by one or both tests. In three of these patients, oligoclonal B-cell populations were suggested by the combined findings. In all four patients with a small paraproteinemia, the isotype was confirmed by either flow cytometry or DNA rearrangement analysis. Our data suggest that the oligoclonal expansions are not restricted to CD5+ B cells, as in the majority of patients this subset was below the detection level of flow cytometry or DNA rearrangement analysis. None of the patients developed clinical manifestations of malignant lymphoma during a follow-up period of 10 to 44 months after sampling. We conclude that clonal excess populations of B cells are not a unique feature of malignant lymphoma, but may occur in autoimmune diseases, suggesting a benign (oligo)clonal B-cell proliferation.

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CCR5-binding chemokines modulate CXCL12 (SDF-1)–induced responses of progenitor B cells in human bone marrow through heterologous desensitization of the CXCR4 chemokine receptor

Blood ◽

10.1182/blood-2002-01-0248 ◽

2002 ◽

Vol 100 (7) ◽

pp. 2321-2329 ◽

Cited By ~ 46

Author(s):

Marek Honczarenko ◽

Yi Le ◽

Aleksandra M. Glodek ◽

Marcin Majka ◽

James J. Campbell ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Chemokine Receptor ◽

Calcium Mobilization ◽

Human Bone ◽

Human Bone Marrow ◽

Cell Populations ◽

B Lymphopoiesis ◽

Heterologous Desensitization

Although the SDF-1 (CXCL12)/CXCR4 axis is important for B-cell development, it is not yet clear to what extent CC chemokines might influence B lymphopoiesis. In the current study, we characterized CC chemokine receptor 5 (CCR5) expression and function of primary progenitor B-cell populations in human bone marrow. CCR5 was expressed on all bone marrow B cells at levels between 150 and 200 molecules per cell. Stimulation of bone marrow B cells with the CCR5-binding chemokine macrophage inflammatory protein 1β (MIP-1β; CCL4) did not cause chemotaxis, but CCL4 was able to trigger potent calcium mobilization responses and activation of the mitogen-activated protein kinase (MAPK) pathway in developing B cells. We also determined that CCR5-binding chemokines MIP-1α (CCL3), CCL4, and RANTES (CCL5), specifically by signaling through CCR5, could affect all progenitor B-cell populations through a novel mechanism involving heterologous desensitization of CXCR4. This cross-desensitization of CXCR4 was manifested by the inhibition of CXCL12-induced calcium mobilization, MAPK activation, and chemotaxis. These findings indicate that CCR5 can indeed mediate biologic responses of bone marrow B cells, even though these cell populations express low levels of CCR5 on their cell surface. Thus, by modulation of CXCR4 function, signaling through CCR5 may influence B lymphopoiesis by affecting the migration and maturation of B-cell progenitors in the bone marrow microenvironment.

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Collaboration of histoincompatible T and B lymphocytes using cells from tetraparental bone marrow chimeras.

Journal of Experimental Medicine ◽

10.1084/jem.142.4.989 ◽

1975 ◽

Vol 142 (4) ◽

pp. 989-997 ◽

Cited By ~ 100

Author(s):

H von Boehmer ◽

L Hudson ◽

J Sprent

Keyword(s):

Bone Marrow ◽

T Cells ◽

B Cells ◽

B Cell ◽

B Lymphocytes ◽

Parental Strain ◽

Secondary Response ◽

Cell Populations ◽

Sheep Erythrocytes ◽

Bone Marrow Chimeras

T-B collaboration has been studied in a secondary response to sheep erythrocytes using either syngeneic or allogeneic T- and B-cell combinations. T cells prepared from tetraparental bone marrow chimeras (TBMC), carrying H-2 determinants of one parental strain only, cooperated with syngeneic, as well as with allogeneic B cells carrying the alloantigens to which the T cells had been tolerized in the chimeric environment. When TBMC-derived cells of a single H-2 specificity were transferred with a mixture of TBMC-derived B cells of both H-2 types of the parental strains, no preference for syngeneic cooperation was found. The data therefore suggest that the presence of differing H-2-complex determinants on the allogeneic T- and B-cell populations of the two different strain combinations tested do not interfere with T-B collaboration when the cell populations studied are mutually tolerant.

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THU0040 PROTEINASE 3-REACTIVE B CELL RECONSTITUTION AFTER TREATMENT WITH RITUXIMAB FOR ANCA-ASSOCIATED VASCULITIS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.5169 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 233.1-233

Author(s):

A. Berti ◽

S. Hillion ◽

A. Hummel ◽

E. Carmona ◽

T. Peikert ◽

...

Keyword(s):

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Peripheral Blood ◽

Proteinase 3 ◽

Research Support ◽

Color Flow ◽

Anca Associated Vasculitis ◽

Cell Repopulation

Background:Proteinase 3 (PR3)-reactive B cells are present in PR3-ANCA-associated vasculitis (AAV) at levels higher than healthy controls.Objectives:To evaluate the dynamics of the PR3-reactive B cell repopulation in patients with PR3-AAV after treatment with rituximab, and to analyze possible associations between these immunological changes and long-lasting remissions.Methods:We analyzed all available frozen peripheral blood mononuclear cells (n=148) from 23 randomly-selected PR3-AAV patients who participated in the RAVE trial and achieved complete remission (BVAS=0, prednisone=0) after treatment with rituximab.We measured PR3-reactive B cells and the relative subsets by a multi-color flow cytometry panel including CD19, IgD, CD27, CD38, CD24, and a biotinylated PR3 revealed by fluorescent streptavidin. The clinical data of the trial were correlated with flow-cytometry data.Results:10/23 (43%) patients relapsed during the follow up, 8/10 relapses were severe. At baseline, clinical features, PR3-ANCA levels, % of total PR3-reactive B cells and PR3-reactive B cell subsets were similar between relapsers and non-relapsers. All patients were followed until the end of the trial, for a mean of 44 months (25-75%IQR 31-54), without difference in follow-up time between relapsers and non-relapsers (p=0.98).The majority of patients had B cell repopulation at 12 (range 12-24) months after rituximab. At the time of B cell repopulation, transitional (CD19+CD24+CD38+) and naïve (CD19+CD27+IgD-) B cells were higher compared to baseline, while total plasmablasts (PB) were unchanged, and mature B cells significantly decreased in both relapsers and non relapsers. PR3-reactive B cells reappeared in all the patients, and the % of PR3-reactive of B cells were higher at the B cell repopulation visit compared to baseline (5.82% vs 4.25%, p<0.05), while total B cells were lower (66/μL vs 151/μL, p<0.01), regardless of future relapse.Within PR3-reactive B cells, only the % of PB (CD19+CD27+CD38+PR3+) were higher in relapsers vs. non-relapsers (median [25-75%IQR]; 1.95% [1.315-3.845] vs 0.84% [0.05-1.66], p=0.022) and severe relapsers vs non-severe relapsers (2.165% [1.66-4.315] vs 0.84% [0.1-1.74], p=0.015). Time-to-relapse and time-to severe-relapse were significantly shorter in patients with circulating PR3-PB higher than the median value of the cohort (1.6%) during B cell reconstitution (Figure 1A-B).Conclusion:In PR3-AAV, during B cell reconstitution after rituximab, the total fraction of PR3-B cells increases, due to the expansion of the transitional and naïve B cell compartments. Circulating PR3-PB within PR3-B cells are enriched in the peripheral blood of relapsing and severely relapsing patients compared to non-relapsing patients. Higher levels of PR3-PB after rituximab during B cell reappearance significantly increased the risk of subsequent relapse and severe relapse.References:[1]Cornec D, Berti A, Hummel A, et al. J Autoimmun. 2017Disclosure of Interests:Alvise Berti: None declared, Sophie Hillion: None declared, Amber Hummel: None declared, Eva Carmona: None declared, Tobias Peikert: None declared, Carol Langford: None declared, Peter A. Merkel: None declared, Paul Monach: None declared, Philip Seo: None declared, Robert Spiera Grant/research support from: Roche-Genetech, GSK, Boehringer Ingelheim, Chemocentryx, Corbus, Forbius, Sanofi, Inflarx, Consultant of: Roche-Genetech, GSK, CSL Behring, Sanofi, Janssen, Chemocentryx, Forbius, Mistubishi Tanabe, E. William St. Clair: None declared, Fernando Fervenza: None declared, Kristina Harris: None declared, John H. Stone Grant/research support from: Roche, Consultant of: Roche, Jacques-Olivier Pers: None declared, Ulrich Specks: None declared, Divi Cornec: None declared

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The Complex Role of KDM6A in B-Cell Development and Function

Blood ◽

10.1182/blood-2018-99-119538 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 513-513

Author(s):

Ling Tian ◽

Monique Chavez ◽

Lukas D Wartman

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Bone Marrow Cells ◽

Cell Development ◽

Young Female ◽

B Cell Development ◽

And Function

Abstract Loss-of-function mutations in KDM6A, an X-linked H3K27 demethylase, occur recurrently in B-cell lymphoid malignancies, including B-cell acute lymphoblastic leukemia and non-Hodgkin lymphoma. Germline inactivating mutations in KDM6A cause a neurodevelopmental disorder called Kabuki syndrome that is associated with recurrent infections and hypogammaglobulinemia.1 The role of KDM6A in normal B-cell development and function, as well as how the somatic loss of KDM6A contributes to B-cell malignancies, has not been completely defined. To address this issue, we generated a conditional knockout mouse of the KDM6A gene (with LoxP sites flanking the 3rd exon) and crossed these mice with Vav1-Cre transgenic mice to selectively inactivate KDM6A in hematopoietic stem/progenitor cells. We characterized normal hematopoiesis from young (6 to 8 week old) and aged (50 to 55 week old) male and female KDM6A conditional KO mice. We found a significant shift from lymphoid to myeloid differentiation in the bone marrow and peripheral blood of these mice. Young, female KDM6A-null mice had mild splenomegaly. Their spleens had an increased number of neutrophils (Gr-1+CD11b+ cells) and erythrocyte progenitors (CD71+Ter119+ cells) and a decreased number of B-cells (B220+ cells). These changes became more pronounced with age and were specific to the female, homozygous KDM6A knockout mice. Furthermore, analysis of B-cell maturation showed that the loss of KDM6A was associated with decreased immature (B220+IgM+ cells) and mature, resting B-cells (B220+IgD+ cells) in the spleen. Similar changes were present in the bone marrow (decreased B220+IgM+ cells and B220+CD19+ cells) and peripheral blood (decreased B220+IgM+, B220+IgD+ and B220+CD19+ cells). Early B-cell development is also altered in KDM6A-null mice. Flow cytometry showed a decrease in multipotent progenitor cells (MPPs) with a decrease in both common lymphoid progenitors (CLPs) and B cell-biased lymphoid progenitors (BLPs) in young, female KDM6A-null mice bone marrow. Next, we performed flow cytometry to catergorize the Hardy fractions of early B-cell development on bone marrow isolated from young, female KDM6A-null mice. B-cell progenitor analysis (Hardy profiles) showed an increase in Fraction A with a concomitant decrease in Fraction B/C and Fraction D, which was likely indicative of an incomplete block in B-cell differentiation after the Fraction A stage. When bulk bone marrow cells isolated from young, female KDM6A-null mice were plated in methylcellulose supplemented with interleukin-7, we observed a significantly decreased colony formation compared with bone marrow cells isolated from wildtype littermates. This pre-B lymphoid progenitor cell plating phenotype was expected given the flow cytometry results of decreased B-cell progenitors outlined above. We examined the effect of the loss of KDM6A expression on germinal center (GC) formation in the spleen following immunization with NP-CGG (4-Hydroxy-3-nitrophenylacetyl-Chicken Gamma Globulin, Ratio 16). Two weeks after NP-CGG immunization, we observed a significant decrease in follicular B-cells (FO) and a significant increase in GC B-cells as compared to wildtype littermates (Figure 1). The result is significant as GC B-cells are thought to be the cell-of-origin of follicular and DLBCL. To determine if inactivation of KDM6A affected antibody production, we measured IgM, IgG, IgE and IgA levels by ELISA from serum isolated from young, female KDM6A-null mice. Results revealed higher levels of IgM and lower levels of IgG in serum from KDM6A-null mice, which is suggestive of a class switch recombination (CSR) defect. Concordant with this result, we observed that the loss of KDM6A impaired CSR to IgG1 in splenic B cells after in vitro stimulation for three days with lipopolysaccharide (LPS), an anti-CD180 antibody and interleukin-4. Moreover, we observed a striking defect in the production of plasma cells from KDM6A-null B-cells after LPS stimulation. Taken together, our data shows that KDM6A plays an important, but complex, role in B-cell development and that loss of KDM6A impedes the B-cell immune response in a specific manner that may contribute to infection and B-cell malignancies.Stagi S, et al. Epigenetic control of the immune system: a lesson from Kabuki syndrome. Immunol Res. 2016; 64(2):345-359. Disclosures No relevant conflicts of interest to declare.

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