Tyrosine Kinase Inhibitor Therapy Induced Changes in Humoral Immunity in Patients with Chronic Myeloid Leukemia

Abstract Abstract 1699 Background: By affecting not only BCR-ABL1 but also a wide variety of other tyrosine kinases, tyrosine kinase inhibitors (TKIs) have potential impact on the immune defense system. As patients could use TKIs even for decades, immunological off-target effects may be of significance in clinical practice. The aim of this project was to study the effects of TKI therapy on B-cell-mediated immunity in vivo in CML patients. PATIENTS AND METHODS: Peripheral blood (PB) and bone marrow (BM) samples were collected from newly diagnosed chronic phase CML patients and follow-up samples were obtained during TKI therapy at 1, 3, 6, and 12 months after therapy start (imatinib n=21, dasatinib n=15 and nilotinib n=7). Samples from healthy volunteers served as controls (n=10). Plasma immunoglobulin (Ig) levels were measured with an immunoturbidometric method and BM and PB lymphocyte subclasses were analyzed with multicolor flow cytometry. Ig heavy chain rearrangement PCR according to BIOMED-2 protocol was used to identify clonal B-cell populations among sorted CD19pos B lymphocytes. RESULTS: At diagnosis, before TKI therapy, plasma Ig levels were similar to healthy controls. During imatinib treatment IgA and IgG levels decreased continuously and were significantly lower at 12 months compared either to diagnostic samples or to healthy controls: median IgA level at 12 months 1.22 vs. 1.51 g/l at diagnosis (p=0.040) or vs. 1.61 g/L in healthy controls (p=0.023); median IgG at 12 months 7.00 vs. 8.80 g/l at diagnosis (p=0.009) or vs. 8.55 g/l in healthy controls (p=0.009). Similarly plasma IgM levels decreased: 0.59 g/ at 12 months vs. 1.06 g/l at diagnosis (p=0.0002), but did not differ significantly from healthy controls (0.75 g/l, p=0.331). Positive correlation was found between plasma IgG and IgM levels at 3 months (r=0.678, p=0.045). One imatinib patient developed severe constant hypogammaglobulinemia, which was first noticed at 3 months, and imatinib was discontinued at 15 months due to recurrent respiratory infections. In dasatinib-treated patients, only plasma IgM levels decreased notably, and at 12 months, they were significantly lower (0.39 g/l) compared to diagnosis (0.86 g/l, p=0.011) or healthy controls (0.75 g/l, p=0.037). No statistically significant changes were observed in nilotinib-treated patients, possibly due to small number of patients. The CD45 expression analyzed by flow cytometry was used to detect different B-cell subclasses (CD45high as mature B-cells, CD45intermediate as maturating B-cells and CD45low as immature B-cells). In imatinib treated patients the proportion of mature B-cells in BM from all CD19+ cells was significantly lower at 3 months than in dasatinib group (55% vs. 84%, p<0.05), whereas the proportion of maturating B-cells was higher (34% vs. 12%, p<0.05). This could be related to the observed low plasma Ig levels in imatinib patients, as only mature B-cells are capable of secreting immunoglobulins. BM B-cell profile of nilotinib patients resembled that of imatinib patients. Although clonal T-cells are found in the majority of CML patients (Kreutzman et al. Blood 2010), Ig heavy chain rearrangement analysis did not reveal any clonal B-cell populations in CML patients at diagnosis (n=2) or during imatinib (n=2) or dasatinib (n=9) treatment. Similarly, no marked abnormalities were observed in serum electrophoresis or immunofixation analysis. CONCLUSIONS: Imatinib-treated patients have reduced plasma Ig levels and the proportion of mature immunoglobulin-secreting B-cells was lower in BM. Unexpectedly, dasatinib-induced changes were much less prominent, although dasatinib inhibits a broader spectrum of kinases, some of which are clearly involved in B lymphocyte biology, such as the Lyn-kinase. Further studies are needed to understand which kinase inhibition plays the major role in imatinib-induced reduction of Ig levels. However, the measurement of plasma Ig levels is warranted in patients with recurrent respiratory infections and possibility of hypogammaglobulinemia needs to be taken into account. Disclosures: Koskela: Novartis: Honoraria. Richter:Bristol-Myers Squibb: Honoraria; Novartis: Honoraria. Hjorth-Hansen:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria. Porkka:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria. Mustjoki:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria.

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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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Patients With DLBCL Commonly Have B-Cell Lymphopenia and Circulating Clonal B-Cell Populations With a Phenotype Concordant With The Underlying Tumour

Blood ◽

10.1182/blood.v122.21.3013.3013 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3013-3013

Author(s):

Ruth M de Tute ◽

Sharon Barrans ◽

Andy C. Rawstron ◽

Peter W.M. Johnson ◽

Andrew J Davies ◽

...

Keyword(s):

Gene Expression ◽

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Gene Expression Profiling ◽

Peripheral Blood ◽

Expression Profiling ◽

Common Finding ◽

Cell Populations ◽

The Impact

Abstract Clonal B-cell populations with either a CLL or a non-CLL phenotype are a common finding in normal individuals but uncertainty remains about how this relates to the development of clinically significant disease. The aim of this study was to investigate the frequency of peripheral blood clonal B-cell populations and B-cell subset abnormalities in newly presenting DLBCL patients and to determine whether the incidence of these abnormalities differed between the GCB and ABC subtypes, which are regarded as having distinct pathogenesis. The study was carried out using peripheral blood samples collected from patients entered in the UK-REMoDL-B trial. This trial is testing the hypothesis that the ABC subtype of DLBCL responds preferentially to R-CHOP- Bortezomib. Gene expression profiling is performed on the diagnostic tissue biopsy (FFPE) using the Illumina WG-DASL assay prior to randomisation classified as GCB, ABC or unclassified (UN). The availability of GEP data allows meaningful comparison with the phenotype of clonal populations detected by flow cytometry. Peripheral blood taken prior to first treatment was analysed using multi-colour flow cytometry. Following red cell lysis with ammonium chloride, samples were incubated with a panel of antibodies comprising of a CD19 and CD20 backbone, with Kappa, Lambda, CD5, CD45, CD49d, LAIR-1, CXCR5, CD31, CD95, CD38 and CD10, supplemented in some cases by CD81, CD79b, and CD43. A minimum of 500,000 events were acquired on a FacsCanto II flow cytometer (Becton Dickinson). B-cells were enumerated and any monoclonal populations identified were classified as CLL, germinal centre (GC), non-GC or not otherwise specified (NOS) where the phenotype was indeterminate. 358 samples were eligible for inclusion from patients with an average age of 62.2years (range 22.9-86.1). Abnormalities were detected in 52% of cases (B-lymphopenia ((<0.06 x 109/l) 33%, B-lymphocytosis (>1 x 109/l) 2.8%, CLL clone 3.6%, GC clone 9.8%, non-GC clone 9.8%, clonal population NOS 2.2%). Gene expression profiling results were available for 278 individuals; 51% GCB, 32% ABC and 17% unclassified. The relationship between peripheral blood B-cell findings and the GEP determined phenotype of the tumour is shown in the table:TableB-lymphopeniaCLL CloneMonoclonal GC typeMonoclonalNon-GC typeMonoclonal NOSNormalB-cellGCB n=14241/142 (29%)5/142 (3.5%)21/142 (15%)8/142 (5.6%)2/142 (1%)72/142 (51%)ABC n=8927/89 (30%)2/89 (2%)2/89 (2%)12/89 (13.5%)2/89 (2%)49/89 (55%)Unclassified n=4726/47 (55%)0/50 (0%)2/47 (4%)6/47 (12%)6/47 (5%)14/47 (30%) In patients where clonal populations were detected in the peripheral blood there was striking concordance between the phenotype of the clone and the GEP of the underlying tumour. Presence of a GC-population by flow was highly predictive of GCB GEP (84% GC–type populations detected were in GCB cases). The number of discordant cases and the number of CLL clones detected approximate to the numbers that would be expected in a normal population of a similar age. It is, therefore, likely that in most cases circulating tumour cells or a closely related precursor clone are being detected. The similarity between the results of the ABC and unclassified GEP groups suggest that these are biologically related. An unexpected finding in this study was the high incidence of B-lymphopenia at a level that might be expected to be associated with increased risk of infection. This may reflect suppression of normal B-cells by the neoplastic clone or be a marker of underlying immune dysfunction that may predispose to the development of the tumour. Immuosuppression has a role in the pathogenesis of DLBCL in the elderly and this study suggests that this may also be a factor in the wider patient population. These results may have implications for prognostic assessment and may offer opportunities for early diagnosis and possibly response assessment in some patients. The impact on outcome will be assessed in the course of the trial. Disclosures: Jack: Roche /Genentech: Research Funding.

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THU0034 AUTOREACTIVE B CELLS ESCAPE PERIPHERAL CHECKPOINT IN ANCA-ASSOCIATED VASCULITIS AND SJÖGREN’S SYNDROME

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.5748 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 230.3-230

Author(s):

N. Chriti ◽

M. Boudigou ◽

E. Porchet ◽

J. O. Pers ◽

S. Hillion ◽

...

Keyword(s):

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Peripheral Blood ◽

Healthy Subjects ◽

Peripheral Tolerance ◽

Healthy Controls ◽

Autoreactive B Cells ◽

Anca Associated Vasculitis

Background:B cells play a central role in many autoimmune diseases (AIDs) including ANCA-associated vasculitis (AAV) and primary Sjögren’s syndrome (pSS). Most of the research that has been conducted on AID has focused on the production, secretion, and pathogenicity of auto-antibodies, but little is known on the characteristics of autoreactive B cells in humans.Objectives:This study aims at characterizing circulating autoantigen (PR3 ou SSA)-specific B-cells in patients with AAV and pSS compared to healthy subjects to better understand their role in thenatural and pathological autoimmunityand define the mechanisms leading to the breakdown of self-tolerance in patients with AID.Methods:First, we developed a new flow-cytometry method to detect circulating auto-reactive B cell based on the specificity of their B-cell receptor (BCR). To study surface phenotype of specific B cells by flow cytometry, blood samples were collected from patient with PR3-ANCA AAV, pSS and from healthy subjects. Functional analysis of antigen-specific B cells was also elicited by in vitro analysis of their capacity to secrete immunoglobulins against SSA or PR3 antigens by ELISPOTResults:Phenotype analysis showed that antigen-specific B cells in patients have a memory phenotype compared with healthy controls (5 to 9% are IgG-expressing memory B cells). It suggests that in AID, theses auto-reactive cells are able to differentiate into IgG isotype-switched cells and escape peripheral tolerance checkpoint but not in healthy subjects. Interestingly, Naturalauto-reactive Bcells are able to secrete only IgM isotype autoantibodies uponin vitrostimulation but not IgG class switched antibodies. In order to better understand what differentiates auto-reactive B cells and the mechanisms leading to pathological autoimmunity, agenomic analysisof the antibody repertoire as well as atranscriptional profilingof these cells by single-cell RNA seq is ongoing to understand further the differences of these autoreactive B cells between healthy subjects and patients with AIDs.Conclusion:We developed a technology to identify and isolate antigen-specific B cells from the peripheral blood of patients with AID. Our results suggest that autoreactive B cells escape peripheral tolerance checkpoint and are able to differentiate into IgG isotype-switched cells in patients with AIDs but not in healthy subjects.References:[1]D. Cornec, A. Berti, A. Hummel, T. Peikert, J.-O. Pers, et U. Specks, « Identification and phenotyping of circulating autoreactive proteinase 3-specific B cells in patients with PR3-ANCA associated vasculitis and healthy controls »,J. Autoimmun., vol. 84, p. 122–131, 2017[2]P. F. Kerkman et al., « Identification and characterisation of citrullinated antigen-specific B cells in peripheral blood of patients with rheumatoid arthritis », Ann. Rheum. Dis., vol. 75, no 6, p. 1170‑1176, juin 2016, doi: 10.1136/annrheumdis-2014-207182.Acknowledgments:with support of vasculitis foundation, CSL Berhing, SFRDisclosure of Interests:None declared

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Clonal B-cell populations in patients with idiopathic thrombocytopenic purpura

Blood ◽

10.1182/blood.v76.11.2321.2321 ◽

1990 ◽

Vol 76 (11) ◽

pp. 2321-2326 ◽

Cited By ~ 2

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

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

Blood ◽

10.1182/blood.v110.11.1428.1428 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1428-1428 ◽

Cited By ~ 2

Author(s):

Karel Fišer ◽

Tomáš Sieger ◽

Josef H. Vormoor

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Hierarchical Clustering ◽

Mahalanobis Distance ◽

Cell Populations ◽

Flow Data ◽

Color Flow ◽

Two Parameter

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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Over-Expression of IRF4 (MUM1) Protein Predicts Poorer Outcome in Myeloma Patients

Blood ◽

10.1182/blood.v112.11.5127.5127 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5127-5127

Author(s):

Ruth M de Tute ◽

Reuben Tooze ◽

Roger G Owen ◽

Andy C Rawstron

Keyword(s):

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Plasma Cell ◽

Plasma Cells ◽

Cell Populations ◽

Surface Markers ◽

Cell Surface Markers ◽

Over Expression ◽

Cell Purification

Abstract IRF4 (also known as MUM1) is a member of the interferon regulatory factor family of transcription factors whose expression is critical for the transition from pre B-cell to immature B-cell and for differentiation of mature B-cell to plasma cell. It has previously been reported by Heintel el al (Leukemia (2008) 22, 441–445) that over-expression of IRF4 at the mRNA level is associated with a poor outcome in myeloma. Measurement of expression at the protein level using flow cytometry would remove the need for plasma cell purification and also allow evaluation of IRF4 in other cell populations. We have developed a multi-colour flow cytometry assay incorporating an indirect intracellular staining using the goat polyclonal antibody IRF4 (Santa Cruz). This permits simultaneous analysis of IRF4 in combination with five cell surface markers, allowing quantitation of the IRF4 levels present in specific B-cell and plasma cell populations. The aim of this study was to assess the expression profile of IRF4 in neoplastic and normal B-cells and plasma cells and evaluate the relationship between IRF4 protein expression and outcome in multiple myeloma patients. Leucocytes prepared from whole bone marrow using ammonium chloride lysis were incubated with surface markers CD52 PE, CD38 PerCPCy5.5, CD19 Pe-Cy7, CD138 APC and CD20 APC-Cy7. Cells were then fixed and permeabilised before incubation with unconjugated IRF4, washing and incubation with an Alexa488 secondary antibody. IRF4 expression showed the expected profile in samples with normal B-lineage cells. Low levels of expression were found in normal mature B-cells (mean MFI 1033, range 395–2265), higher levels in progenitor B-cells (mean 4.1-fold higher than mature B-cells, mean MFI 4252, range 612–22958) and strong uniform expression in normal BM plasma cells (mean 13.8-fold higher than mature B-cells, mean MFI 14250, range 7391–21914). Plasma cells from untreated myeloma patients showed a mean 1.9-fold higher IRF4 expression level than normal plasma cells (P=<0.001, normal n=16, neoplastic n = 19, mean MFI 27510, range 6807–53662). The myeloma patients were then separated into two groups; those with IRF4 expression in the normal range (n=6, mean MFI 12858, range 4182–21028) and those with high IRF4 expression (n=13, mean MFI 34501, range 24892–53662). After a median follow-up of 19 months, 6 patients had died in the high expression group (46.2%) compared with no deaths in the low expression group; this difference was statistically significant (Cox regression analysis, p=0.036). The assay developed can detect quantitative differences in IRF4 expression between B-progenitors, mature B-cells, normal plasma cells and neoplastic plasma cells. The ability to simultaneously analyse IRF4 expression with other cell surface markers allows rapid enumeration of IRF4 protein levels without plasma cell purification and greatly enhances the potential for IRF4 to be used as a prognostic marker. Neoplastic plasma cells can be categorised as having low or high levels of IRF4 expression with over-expression predicting a significantly shorter overall survival.

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hnRNP F Influences Binding of a 64-Kilodalton Subunit of Cleavage Stimulation Factor to mRNA Precursors in Mouse B Cells

Molecular and Cellular Biology ◽

10.1128/mcb.21.4.1228-1238.2001 ◽

2001 ◽

Vol 21 (4) ◽

pp. 1228-1238 ◽

Cited By ~ 86

Author(s):

Kristen L. Veraldi ◽

George K. Arhin ◽

Kathleen Martincic ◽

Ling-Hsiu Chung-Ganster ◽

Jeffrey Wilusz ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Heavy Chain ◽

Plasma Cells ◽

Memory B Cells ◽

Negative Regulator ◽

Cleavage Reaction ◽

Hnrnp F ◽

A Site ◽

Ig Heavy Chain

ABSTRACT Previous studies on the regulation of polyadenylation of the immunoglobulin (Ig) heavy-chain pre-mRNA argued fortrans-acting modifiers of the cleavage-polyadenylation reaction operating differentially during B-cell developmental stages. Using four complementary approaches, we demonstrate that a change in the level of hnRNP F is an important determinant in the regulated use of alternative polyadenylation sites between memory and plasma stage B cells. First, by Western analyses of cellular proteins, the ratio of hnRNP F to H or H′ was found to be higher in memory B cells than in plasma cells. In memory B cells the activity of CstF-64 binding to pre-mRNA, but not its amount, was reduced. Second, examination of the complexes formed on input pre-mRNA in nuclear extracts revealed large assemblages containing hnRNP H, H′, and F but deficient in CstF-64 in memory B-cell extracts but not in plasma cells. Formation of these large complexes is dependent on the region downstream of the AAUAAA in pre-mRNA, suggesting that CstF-64 and the hnRNPs compete for a similar region. Third, using a recombinant protein we showed that hnRNP F could bind to the region downstream of a poly(A) site, block CstF-64 association with RNA, and inhibit the cleavage reaction. Fourth, overexpression of recombinant hnRNP F in plasma cells resulted in a decrease in the endogenous Ig heavy-chain mRNA secretory form-to-membrane ratio. These results demonstrate that mammalian hnRNP F can act as a negative regulator in the pre-mRNA cleavage reaction and that increased expression of F in memory B cells contributes to the suppression of the Ig heavy-chain secretory poly(A) site.

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OP0186 CHANGES IN CIRCULATING B CELL LEVELS AND IMMUNOPHENOTYPE ARE ASSOCIATED WITH DEVELOPMENT OF ARTHRITIS FOLLOWING TREATMENT WITH CHECKPOINT INHIBITORS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.908 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 112.2-113

Author(s):

M. Gatto ◽

S. Bjursten ◽

C. Jonell ◽

C. Jonsson ◽

S. Mcgrath ◽

...

Keyword(s):

Flow Cytometry ◽

B Cells ◽

Adverse Events ◽

B Cell ◽

Mononuclear Cells ◽

Checkpoint Inhibitors ◽

Therapy Monitoring ◽

Cell Subset ◽

Peripheral Blood Mononuclear ◽

Transitional B Cells

Background:Inflammatory arthritis (IA) is frequent among rheumatic side effects induced by checkpoint inhibitor (CPI) therapy for metastatic malignancies1. While T cells are likely to sustain the inflammatory process2, fewer data are available concerning the role of B cells3.Objectives:To investigate the phenotype of circulating B cells in patients who develop CPI-induced IA (CPI-IA) and to compare it with features of B cells in patients not developing immune-related adverse events (irAE) upon CPI treatment.Methods:B cell subsets at baseline (before CPI initiation) and during CPI treatment were analyzed in CPI-IA patients and in patients receiving CPI but who did not develop irAE (non-irAE). Peripheral blood mononuclear cells (PBMC) were analyzed by flow cytometry and B cells were identified as CD19+ and divided into naïve (CD27-IgD+), memory (CD27+IgD+/-), double negative (CD27-IgD-) and transitional (CD10+CD24+CD38+/hi) B cells. Levels of CD21, an activation marker on transitional B cells, were also analyzed. Non-parametric tests were used for analysis of differences between groups.Results:Six CPI-IA and 7 non-irAE patients matched for age, gender and CPI treatment were included, who had received CPI treatment due to metastatic melanoma. Flow cytometry revealed a significant increase of circulating B cells (p=0.002) (Figure 1A) and especially of transitional B cells in CPI-IA patients vs. non-irAE (median %, range: 7.8 (4.5-11.4) vs. 3.2 (1.6-4.3),p=0.007) (Figure 1B), while no remarkable changes were seen across other subsets. Transitional B cell levels significantly decreased from active to quiescent CPI-IA in all patients (p=0.008). In two CPI-IA patients for whom baseline sampling was available, the increase of transitional levels occurred early after CPI treatment and before CPI-IA onset. Levels of expression of CD21 on transitional B cells were increased in CPI-IA vs. non-irAE (p=0.01).Conclusion:Transitional B cells are expanded in CPI-IA patients and seem to increase early after start of CPI therapy. Monitoring this B cell subset might lead to closer follow-up and earlier diagnosis of CPI-IA.References:[1]Ramos-Casals M, Brahmer JR, Callahan MK, et al. Immune-related adverse events of checkpoint inhibitors. Nat Rev Dis Primers 2020;6:38[2]Murray-Brown W, Wilsdon TD, Weedon H, et al. Nivolumab-induced synovitis is characterized by florid T cell infiltration and rapid resolution with synovial biopsy-guided therapy. J Immunother Cancer 2020;8:e000281[3]Das R, Bar N, Ferreira M, et al. Early B cell changes predict autoimmunity following combination immune checkpoint blockade. J Clin Invest. 2018;128:715-2Disclosure of Interests:None declared

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