scholarly journals Establishing CD19 B-cell reference control materials for comparable and quantitative cytometric expression analysis

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0248118
Author(s):  
Lili Wang ◽  
Rukmini Bhardwaj ◽  
Howard Mostowski ◽  
Paul N. Patrone ◽  
Anthony J. Kearsley ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Reference Materials ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Preliminary Assessment ◽  
Mean Values ◽  
Calibration Methods ◽  
Independent Calibration ◽  
Cd19 Expression

In the field of cell-based therapeutics, there is a great need for high-quality, robust, and validated measurements for cell characterization. Flow cytometry has emerged as a critically important platform due to its high-throughput capability and its ability to simultaneously measure multiple parameters in the same sample. However, to assure the confidence in measurement, well characterized biological reference materials are needed for standardizing clinical assays and harmonizing flow cytometric results between laboratories. To date, the lack of adequate reference materials, and the complexity of the cytometer instrumentation have resulted in few standards. This study was designed to evaluate CD19 expression in three potential biological cell reference materials and provide a preliminary assessment of their suitability to support future development of CD19 reference standards. Three commercially available human peripheral blood mononuclear cells (PBMCs) obtained from three different manufacturers were tested. Variables that could potentially contribute to the differences in the CD19 expression, such as PBMCs manufacturing process, number of healthy donors used in manufacturing each PBMC lot, antibody reagent, operators, and experimental days were included in our evaluation. CD19 antibodies bound per cell (ABC) values were measured using two flow cytometry-based quantification schemes with two independent calibration methods, a single point calibration using a CD4 reference cell and QuantiBrite PE bead calibration. Three lots of PBMC from three different manufacturers were obtained. Each lot of PBMC was tested on three different experimental days by three operators using three different lots of unimolar anti-CD19PE conjugates. CD19 ABC values were obtained in parallel on a selected lot of the PBMC samples using mass spectrometry (CyTOF) with two independent calibration methods, EQ4 and bead-based calibration were evaluated with CyTOF-technology. Including all studied variabilities such as PBMC lot, antibody reagent lot, and operator, the averaged mean values of CD19 ABC for the three PBMC manufacturers (A,B, and C) obtained by flow cytometry were found to be: 7953 with a %CV of 9.0 for PBMC-A, 10535 with a %CV of 7.8 for PBMC-B, and 12384 with a %CV of 16 for PBMC-C. These CD19 ABC values agree closely with the findings using CyTOF. The averaged mean values of CD19 ABC for the tested PBMCs is 9295 using flow cytometry-based method and 9699 using CyTOF. The relative contributions from various sources of uncertainty in CD19 ABC values were quantified for the flow cytometry-based measurement scheme. This uncertainty analysis suggests that the number of antigens or ligand binding sites per cell in each PBMC preparation is the largest source of variability. On the other hand, the calibration method does not add significant uncertainty to the expression estimates. Our preliminary assessment showed the suitability of the tested materials to serve as PBMC-based CD19+ reference control materials for use in quantifying relevant B cell markers in B cell lymphoproliferative disorders and immunotherapy. However, users should consider the variabilities resulting from different lots of PBMC and antibody reagent when utilizing cell-based reference materials for quantification purposes and perform bridging studies to ensure harmonization between the results before switching to a new lot.

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

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

scholarly journals Optimizing recovery of frozen human peripheral blood mononuclear cells for flow cytometry

PLoS ONE ◽  
2017 ◽  
Vol 12 (11) ◽  
pp. e0187440 ◽  
Author(s):  
Bo Langhoff Hønge ◽  
Mikkel Steen Petersen ◽  
Rikke Olesen ◽  
Bjarne Kuno Møller ◽  
Christian Erikstrup
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

CS1: A Potential New Therapeutic Target for the Treatment of Multiple Myeloma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3457-3457 ◽  
Author(s):  
Eric D. Hsi ◽  
Roxanne Steinle ◽  
Balaji Balasa ◽  
Aparna Draksharapu ◽  
Benny Shum ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cell ◽  
Mononuclear Cells ◽  
Plasma Cells ◽  
Target Cells ◽  
Dependent Manner ◽  
Myeloma Cells ◽  
Effector Cells

Abstract Background: To identify genes upregulated in human memory B and plasma cells, naïve B cell cDNA was subtracted from plasma cell and memory B cell cDNA. One gene that was highly expressed in plasma cells encodes CS1 (CD2 subset 1, CRACC, SLAMF7), a cell surface glycoprotein of the CD2 family. CS1 was originally identified as a natural killer (NK) cell marker. Monoclonal antibodies (mAbs) specific for CS1 were used to validate CS1 as a potential target for the treatment of multiple myeloma (MM). Methods: Anti-CS1 mAbs were generated by immunizing mice with a protein comprising of the extracellular domain of CS1. Two clones, MuLuc63 and MuLuc90, were selected to characterize CS1 protein expression in normal and diseased tissues and blood. Fresh frozen tissue analysis was performed by immunohistochemistry (IHC). Blood and bone marrow analysis was performed using flow cytometry with directly conjugated antibodies. HuLuc63, a novel humanized anti-CS1 mAb (derived from MuLuc63) was used for functional characterization in non-isotopic LDH-based antibody-dependent cellular cytotoxicity (ADCC) assays. Results: IHC analysis showed that anti-CS1 staining occurred only on mononuclear cells within tissues. The majority of the mononuclear cells were identified as tissue plasma cells by co-staining with anti-CD138 antibodies. No anti-CS1 staining was detected on the epithelia, smooth muscle cells or vessels of any normal tissues tested. Strong anti-CS1 staining was also observed on myeloma cells in 9 of 9 plasmacytomas tested. Flow cytometry analysis of whole blood from both normal healthy donors and MM patients showed specific anti-CS1 staining in a subset of leukocytes, consisting primarily of CD3−CD(16+56)+ NK cells, CD3+CD(16+56)+ NKT cells, and CD3+CD8+ T cells. Flow cytometry of MM bone marrow showed a similar leukocyte subset staining pattern, except that strong staining was also observed on the majority of CD138+CD45−/dim to + myeloma cells. No anti-CS1 binding was detected to hematopoietic CD34+CD45+ stem cells. To test if antibodies towards CS1 may have anti-tumor cell activity in vitro, ADCC studies using effector cells (peripheral blood mononuclear cells) from 23 MM patients and L363 MM target cells were performed. The results showed that HuLuc63, a humanized form of MuLuc63, induced significant ADCC in a dose dependent manner. Conclusions: Our study identifies CS1 as an antigen that is uniformly expressed on normal and neoplastic plasma cells at high levels. The novel humanized anti-CS1 mAb, HuLuc63, exhibits significant ADCC using MM patient effector cells. These results demonstrate that HuLuc63 could be a potential new treatment for multiple myeloma. HuLuc63 will be entering a phase I clinical study for multiple myeloma.

5-Aza-2-Deoxycytidine Regulates Wnt Beta-Catenin Pathway in B Cell Lymphoma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4810-4810
Author(s):  
Xinyu Li ◽  
Lingyu Geng ◽  
Xiangxiang Zhou ◽  
Kang Lu ◽  
Peipei Li ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Beta Catenin ◽  
Cell Functions ◽  
Protein Expressions

Abstract Introduction: The Wnt/beta-catenin pathway is aberrantly activated in B cell lymphomas, unphosphorylated beta-catenin accumulates and translocates into the nucleus, regulates the expression of c-myc, cyclinD1 and many other target genes which govern fundamental cell functions, such as proliferation, cell cycle regulation and apoptosis. Methylation is a highlight of epigenetic regulation research which also occurred in lymphoma, but the concrete mechanism of how the demethylation drug 5-aza-2-deoxycytidine affect Wnt/beta-catenin pathway is still unknown. This study was designed to illuminate the implications on Wnt/beta-catenin pathway via demethylation 5-aza in B cell lymphoma. Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from samples of 30 primary CLL patients. The PBMCs contained more than 90% of CD19+ B lymphocytes, which were detected by flow cytometry and were referred to as primary CLL cells. The activation of Wnt/beta-catenin pathway and DNMT-1 of B cell lymphoma cells lines (MEC-1, LY8, Jeko-1, Grant519, mino and sp53) and the 30 patients were detected by qPCR and western blot. The expressions of beta-catenin in 20 cases of B cell lymphoma tissues were measured by IHC. The B cell lines and PBMCs from 10 primary CLL patients were given 5-aza-2-deoxycytidine in different concentrations, the effects in the pathway and apoptosis were observed by WB and flow cytometry. Results: The expressions of beta-catenin, c-myc, cyclinD1 and DNMT-1 were aberrantly higher in all cell lines we used ( MEC-1,LY8, Jeko-1, Grant519, mino and sp53 Fig.1-A,B), most primary CLL patients (Fig.1-C), and B cell lymphoma tissues (Fig.1-D). The protein expressions of beta-catenin in MEC-1 were higer than primary CLL patients. 0, 0.5, 1.0, 2.0¦ÌM 5-aza-2-deoxycytidine were given to the B cells lines and PBMCs from primary CLL patients for 48h, beta-catenin were found accumulated, but c-myc and cyclinD1 in the downstream were reduced (Fig.2-A,B,C,D). For further understanding of aberrant accumulation ofbeta-catenin, we extracted the nuclear protein of MEC-1, nuclear beta-catenin protein expressions were found decreased and cytoplasmic were increased (Fig.2-E). After 5-aza treatment, the apoptosis rate increased and caspase pathway were activated (Fig.2-A,F). Conclusions: The enhanced expressions of beta-catenin, c-myc, cyclinD1 in the B cell lines and the B cell lymphoma samples indicated the Wnt/beta-catenin was aberrantly activated. After 5-aza treatment with the cell lines (MEC-1, Jeko-1, LY8) and primary CLL cells, the abnormal accumulation of beta-catenin protein was observed which was discrepancy with previous reports, but the decrease of c-myc and cyclinD1 suggested the pathway was inhibited, apoptosis also occurred. The increase of totalbeta-catenin protein was supposed to be an stress reaction of the 5-aza treatment, however, the redundant beta-catenin protein in B cell lymphoma was speculated to be combined with demethylated genes and resulted in dormancy of this pathway. Our results indicated that 5-aza played a demethylation role through Wnt/beta-catenin pathway in B cell lymphoma. The data are of interest in the context of epigenetic-based therapy in B cell lymphoma. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

scholarly journals Quantitative Assessment of the Evolution of Therapeutic Target Antigen Expression Level in Diffuse Large B-Cell Lymphoma in Response to Treatment

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4367-4367
Author(s):  
Agrima Mian ◽  
Narendra Bhattarai ◽  
Wei Wei ◽  
Manishkumar S. Patel ◽  
Paolo F. Caimi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Research Funding ◽  
Geometric Mean ◽  
B Cell Lymphoma ◽  
Antigen Expression ◽  
Target Antigen ◽  
Clinical Variables ◽  
Large B Cell Lymphoma ◽  
Cd19 Expression

Abstract Introduction: Approximately one-third of patients (pts.) with diffuse large B-cell lymphoma (DLBCL) develop relapsed or refractory (R/R) disease after frontline rituximab-(R) based chemo immunotherapy. Variability in expression of the B-cell surface antigen CD20, and possibly CD19, is thought to be an important mechanism of treatment failure, but there is vast heterogeneity in the reported incidence, with most reports utilizing Immunohistochemical (IHC) staining which is non-quantitative in nature. New therapeutics including CAR T-cells, antibodies (Ab) (tafasitimab) and Ab-drug conjugates (loncastuximab teserine) target CD19, while several bispecific engager Abs. in development target CD20. Understanding the evolution of target antigen expression in R/R DLBCL can provide information regarding selection and sequencing of therapies. We sought to quantitate the change in surface expression of CD19 and CD20 in R/R DLBCL after R-based chemo immunotherapy relative to pretreatment levels, to provide insight into the relative stability of these targets. Methods: Pts. diagnosed with DLBCL after January 2014, who were R/R to frontline R-based chemo immunotherapy (R-CHOP or R-EPOCH) were retrospectively identified. Pt. and disease related clinical variables at diagnosis (Dx) and first R/R were recorded. Data from IHC were obtained, as were archival flow cytometry assays performed on tumor biopsies at Dx and in the R/R setting. Using FCS Express® software, neoplastic cells were gated, and fluorescence intensity (FI) of CD19 and CD20 expression were reported (using fluorochromes APC-A for CD19 and PECy7 for CD20, respectively). Multivariate linear mixed model was used to compare median and geometric mean FI of CD19 and CD20 between Dx and R/R, while adjusting for other clinical variables. Results: A total of 51 flow cytometry assays (26 Dx and 25 R/R) were analyzed for 33 pts. Median age at Dx was 64 (range, 41-76) yrs., 24 pts. (73%) were male and 29 (88%) had IPI ≥ 2. 11 (33%) pts. had a prior indolent lymphoma. Cell of origin at Dx was GCB in 16 (49%) and non-GCB in 12 (36%) pts., while two had a double-hit rearrangement at diagnosis. Treatment was R-CHOP in 27 cases (82%) and R-EPOCH in 6 (18%). Median time to R/R was 10.4 months. There was a significant reduction in median FI of CD20 from Dx [median: 40,610 (range: 167 - 259,962)] to R/R [median: 11,596 (range: 63 - 79,592)], representing a mean reduction of 63% at R/R relative to Dx (P= 0.01; 95% CI: 20-73%). Similar change was observed in geometric mean FI of CD20, which was reduced 65% at R/R relative to Dx (P< 0.01; 95%CI: 31-82%). In comparison, on IHC, CD20 was reported to be negative at R/R relative to Dx in only one of 16 pts. for whom IHC results were available. Median and geometric mean FI of CD19 at R/R were 38% and 20% lower compared to Dx, respectively, but these differences were not statistically significant (P= 0.08 and 0.39, respectively) (Table). When examining the relative change in FI at R/R in individual cases, compared to the mean FI of all Dx cases, we observed that 21 out 25 R/R cases (84%) had reduction of CD20 whereas only 14/25 (56%) had reduction of CD19. Interestingly, 7/25 (28%) R/R cases had an increase in CD19 expression by >80% (Figure).When adjusting for clinical variables such as age, sex, presence of B symptoms, bone marrow (BM) involvement, PS and prior indolent lymphoma, the change in CD20 median FI from Dx to R/R maintained statistical significance (p=0.01). Reduction in CD20 geometric mean FI from Dx to R/R was significantly associated with age >60 years (p=0.04), BM involvement (p <0.01) and >1 site of extra nodal involvement (p= 0.03) at Dx. Conclusions: Quantitative assessment by flow cytometry revealed a significant decline in expression of CD20 at R/R compared to Dx in the majority of patients with DLBCL treated with R-based chemo immunotherapy. CD19 expression was unchanged in most R/R cases but was found to be dramatically upregulated in a subset of R/R cases. Given the role of CD19 mediated pathways in B-cell NHL and its association with PI3K pro-survival signaling, these data merit further exploration as a potential mechanism of treatment resistance. These findings also highlight the importance for repeat tissue biopsy at the time of suspected R/R DLBCL, with focus on therapeutic target expression, as it may influence treatment decisions or enrollment in clinical trials exploring efficacy of newer agents. Figure 1 Figure 1. Disclosures Caimi: TG Therapeutics: Honoraria; Seattle Genetics: Consultancy; Amgen Therapeutics.: Consultancy; XaTek: Patents & Royalties: Royalties from patents (wife); Verastem: Consultancy; Genentech: Research Funding; Kite Pharmaceuticals: Consultancy; ADC Theraputics: Consultancy, Research Funding. Hill: Celgene (BMS): Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Kite, a Gilead Company: Consultancy, Honoraria, Other: Travel Support, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding; AstraZenica: Consultancy, Honoraria; Epizyme: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Gentenech: Consultancy, Honoraria, Research Funding; Incyte/Morphysis: Consultancy, Honoraria, Research Funding; Beigene: Consultancy, Honoraria, Research Funding.

scholarly journals Regulation of Human NK Cell Activation by Expression of HLA Class I Molecules in Pig Endothelial Cells

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Benjamin Obando ◽  
Arthur Cross-Najafi ◽  
Kevin Lopez ◽  
Deepthi Thadasina ◽  
Wenjun Zhang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Human Peripheral Blood ◽  
Flow Cytometric Analysis ◽  
Organ Shortage ◽  
Major Barrier ◽  
Xenograft Rejection ◽  
Hla Class I Molecules

Background:   Pig-to-human xenotransplantation (XTx) is a promising solution to the organ shortage. Genetically engineered pigs lacking major xenoantigens have reduced hyperacute rejection and prolonged xenograft survival. Despite these advancements, acute xenograft rejection (AXR) remains a major barrier to clinical XTx. AXR is mediated by multiple immune cells, of which natural killer (NK) cells play a crucial role. Previous studies have shown that human HLA-E suppresses NK cell activation through the inhibitory receptor NKG2A. We seek to improve pig-to-human compatibility by expressing HLA-E in a genetically modified pig endothelial cell (pEC) line. This cell line 5GKO/ HLA-G+ has mutations in five genes encoding for xenoantigens and expresses HLA-G, an inhibitory ligand of the NK cell receptor KIR2DL4. In this study, the 5GKO/HLA-G+/HLA-E+ pEC line was established to examine whether co-expression of inhibitory ligands promotes NK cell tolerance.      Methods:   The HLA-Eα/pCDNA3.1 plasmid containing the HLA-E α-chain (HLA-Eα) cDNA driven by a CMV promoter was linearized and introduced into 106 cells of the 5GKO/HLA-G+ pEC line by electroporation. After 48 hours, HLA-E expression was analyzed by flow cytometry. HLA-E+ pECs were isolated by flow cytometry sort and co-cultured with human peripheral blood mononuclear cells (PBMCs) stimulated by IL-2. NK cell degranulation was compared between the 5GKO/HLA-G+ and 5GKO/HLA-G+/HLA-E+ pEC lines by measuring CD107a expression in the CD3- CD56+ cell population.          Results:   HLA-E molecules were successfully expressed on the pECs surface, indicating the HLA-E a chain can pair with the existing b2-microglobulin (B2M). The transfection efficiency was 38.2%. Three weeks later, the 5GKO/HLA-G+/HLA-E+ pEC was successfully established, confirming via flow cytometric analysis. The analysis of NK cell degranulation (CD107a) is underway.     Conclusion:   We established a 5GKO/HLA-G+/HLA-E+ pEC line, which is a valuable tool to study human-to-pig xenoreactive immune response in vitro, with the goal of improving pig-to-human xenograft immunotolerance. 

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