Lucía Del Pino-Molina1, Eduardo López-Granados1, Quentin Lecrevisse2,3, Juan Torres Canizales1, Martín Pérez-Andrés2,3, Elena Blanco2,3, Marjolein Wentink4, Carolien Bonroy5, Jana Nechvatalova6, Tomas Milota7, Anne-Kathrin Kienzler8, Jan Philippé5, Ana E Sousa9, Mirjam van der Burg10, Tomas Kalina11, Jacques J M van Dongen12, Alberto Orfao2,3. 1. Clinical Immunology Department, La Paz University Hospital and Lymphocyte Pathophysiology in Immunodeficiencies Group, La Paz Institute for Health Research (IdiPAZ) and Center for Biomedical Network Research on Rare Diseases (CIBERER U767), Madrid, Spain. 2. Clinical and Translation Research Program, Cancer Research Centre (IBMCC, USAL-CSIC), Department of Medicine, Cytometry Service (NUCLEUS), University of Salamanca (USAL), Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain. 3. Biomedical Research Networking Centre Consortium of Oncology (CIBERONC) Instituto de salud Carlos III, Madrid, Spain. 4. Department of Immunology, Erasmus University Medical Center (Erasmus MC), Rotterdam, Netherlands. 5. Department of Laboratory Medicine, University Hospital Ghent, Ghent, Belgium. 6. Department of Allergology and Clinical Immunology, Faculty of Medicine, Masaryk University and St Anne's University Hospital in Brno, Brno, Czechia. 7. Department of Immunology, Second Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czechia. 8. Nuffield Department of Medicine, Experimental Medicine Division, University of Oxford, Oxford, United Kingdom. 9. Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal. 10. Department of Pediatrics, Laboratory for Immunology, Leiden University Medical Center, Leiden, Netherlands. 11. CLIP - Childhood Leukemia Investigation Prague, Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University and University Hospital Motol, Prague, Czechia. 12. Department of Immunohematology and Blood Transfusion, Leiden University Medical Center (LUMC), Leiden, Netherlands.
Abstract
Introduction: Common Variable Immunodeficiency (CVID) is characterized by defective antibody production and hypogammaglobulinemia. Flow cytometry immunophenotyping of blood lymphocytes has become of great relevance for the diagnosis and classification of CVID, due to an impaired differentiation of mature post-germinal-center (GC) class-switched memory B-cells (MBC) and severely decreased plasmablast/plasma cell (Pb) counts. Here, we investigated in detail the pre-GC B-cell maturation compartment in blood of CVID patients. Methods: In this collaborative multicentric study the EuroFlow PID 8-color Pre-GC B-cell tube, standardized sample preparation procedures (SOPs) and innovative data analysis tools, were used to characterize the maturation profile of pre-GC B-cells in 100 CVID patients, vs 62 age-matched healthy donors (HD). Results: The Pre-GC B-cell tube allowed identification within pre-GC B-cells of three subsets of maturation associated immature B-cells and three subpopulations of mature naïve B-lymphocytes. CVID patients showed overall reduced median absolute counts (vs HD) of the two more advanced stages of maturation of both CD5+ CD38+/++ CD21het CD24++ (2.7 vs 5.6 cells/µl, p=0.0004) and CD5+ CD38het CD21+ CD24+ (6.5 vs 17 cells/µl, p<0.0001) immature B cells (below normal HD levels in 22% and 37% of CVID patients). This was associated with an expansion of CD21-CD24- (6.1 vs 0.74 cells/µl, p<0.0001) and CD21-CD24++ (1.8 vs 0.4 cells/µl, p<0.0001) naïve B-cell counts above normal values in 73% and 94% cases, respectively. Additionally, reduced IgMD+ (21 vs 32 cells/µl, p=0.03) and IgMD- (4 vs 35 cells/µl, p<0.0001) MBC counts were found to be below normal values in 25% and 77% of CVID patients, respectively, always together with severely reduced/undetectable circulating blood pb. Comparison of the maturation pathway profile of pre-GC B cells in blood of CVID patients vs HD using EuroFlow software tools showed systematically altered patterns in CVID. These consisted of: i) a normally-appearing maturation pathway with altered levels of expression of >1 (CD38, CD5, CD19, CD21, CD24, and/or smIgM) phenotypic marker (57/88 patients; 65%) for a total of 3 distinct CVID patient profiles (group 1: 42/88 patients, 48%; group 2: 8/88, 9%; and group 3: 7/88, 8%) and ii) CVID patients with a clearly altered pre-GC B cell maturation pathway in blood (group 4: 31/88 cases, 35%). Conclusion: Our results show that maturation of pre-GC B-cells in blood of CVID is systematically altered with up to four distinctly altered maturation profiles. Further studies, are necessary to better understand the impact of such alterations on the post-GC defects and the clinical heterogeneity of CVID.
Introduction: Common Variable Immunodeficiency (CVID) is characterized by defective antibody production and hypogammaglobulinemia. Flow cytometry immunophenotyping of blood lymphocytes has become of great relevance for the diagnosis and classification of CVID, due to an impaired differentiation of mature post-germinal-center (GC) class-switched memory B-cells (MBC) and severely decreased plasmablast/plasma cell (Pb) counts. Here, we investigated in detail the pre-GC B-cell maturation compartment in blood of CVIDpatients. Methods: In this collaborative multicentric study the EuroFlow PID 8-color Pre-GC B-cell tube, standardized sample preparation procedures (SOPs) and innovative data analysis tools, were used to characterize the maturation profile of pre-GC B-cells in 100 CVIDpatients, vs 62 age-matched healthy donors (HD). Results: The Pre-GC B-cell tube allowed identification within pre-GC B-cells of three subsets of maturation associated immature B-cells and three subpopulations of mature naïve B-lymphocytes. CVIDpatients showed overall reduced median absolute counts (vs HD) of the two more advanced stages of maturation of both CD5+ CD38+/++ CD21het CD24++ (2.7 vs 5.6 cells/µl, p=0.0004) and CD5+ CD38het CD21+ CD24+ (6.5 vs 17 cells/µl, p<0.0001) immature B cells (below normal HD levels in 22% and 37% of CVIDpatients). This was associated with an expansion of CD21-CD24- (6.1 vs 0.74 cells/µl, p<0.0001) and CD21-CD24++ (1.8 vs 0.4 cells/µl, p<0.0001) naïve B-cell counts above normal values in 73% and 94% cases, respectively. Additionally, reduced IgMD+ (21 vs 32 cells/µl, p=0.03) and IgMD- (4 vs 35 cells/µl, p<0.0001) MBC counts were found to be below normal values in 25% and 77% of CVIDpatients, respectively, always together with severely reduced/undetectable circulating blood pb. Comparison of the maturation pathway profile of pre-GC B cells in blood of CVIDpatients vs HD using EuroFlow software tools showed systematically altered patterns in CVID. These consisted of: i) a normally-appearing maturation pathway with altered levels of expression of >1 (CD38, CD5, CD19, CD21, CD24, and/or smIgM) phenotypic marker (57/88 patients; 65%) for a total of 3 distinct CVIDpatient profiles (group 1: 42/88 patients, 48%; group 2: 8/88, 9%; and group 3: 7/88, 8%) and ii) CVIDpatients with a clearly altered pre-GC B cell maturation pathway in blood (group 4: 31/88 cases, 35%). Conclusion: Our results show that maturation of pre-GC B-cells in blood of CVID is systematically altered with up to four distinctly altered maturation profiles. Further studies, are necessary to better understand the impact of such alterations on the post-GC defects and the clinical heterogeneity of CVID.
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