Abstract
IntroductionCommon 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.MethodsIn 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).ResultsThe 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%).ConclusionOur 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.
Highlights
Common Variable Immunodeficiency (CVID) is characterized by defective antibody production and hypogammaglobulinemia
We investigated in detail the pre-GC B-cell maturation compartment in blood of 100 CVID patients, taking advantage of the novel and standardized flow cytometric approaches developed by EuroFlow for this purpose [17,18,19], e.g., the recently proposed EuroFlow primary immunodeficiency (PID) 8-color antibody panels [20] that can be implemented in most diagnostic laboratories worldwide, together with the EuroFlow standard operating procedures (SOPs) for sample preparation, data acquisition and analysis, including innovative data analysis tools recently developed by EuroFlow to assess normal vs altered pre-GC B-cell maturation profiles in blood [17, 20,21,22,23]
Based on the data provided by the PID-orientation tube (PIDOT) [22], and the Pre-GC B-cell tube, detailed characterization of B cells in peripheral blood was achieved for a total of 11 distinct B cell subsets: i) immature/transitional B-cells; ii) mature naïve Bcells; iii) unswitched IgMD+, IgM+-only, and IgD+ -only memory B-cells (MBC)
Summary
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) classswitched memory B-cells (MBC) and severely decreased plasmablast/plasma cell (Pb) counts. Common Variable Immunodeficiency (CVID) is the most prevalent symptomatic primary immunodeficiency (PID) It is characterized by defective antibody production that leads to hypogammaglobulinemia [1,2,3] with an increased susceptibility to infections, associated in some CVID patients with enteropathy, autoimmunity, lymphoproliferation, and/or risk of lymphoid malignancy due to more profound immunological dysregulation [4, 5]. Overall, impaired differentiation of mature post-GC B-cells, consisting of severely reduced circulating class-switched memory B-cells (MBC) and strongly decreased (i.e., undetectable) plasmablast/plasma cell production, are the most consistent defects in CVID. Depending on the specific defects encountered in the post-GC MBC and Pb compartments in blood, and the severity of such defects, distinct CVID patient subgroups, associated with distinct clinical profiles, have been identified [12]
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