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.

• MeSH
• běžná variabilní imunodeficience diagnóza   imunologie   metabolismus   MeSH
• CD antigeny analýza   MeSH
• dospělí MeSH
• fenotyp MeSH
• imunofenotypizace * MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• prekurzorové B-lymfoidní buňky imunologie   metabolismus   MeSH
• průtoková cytometrie * MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• studie případů a kontrol MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Evropa MeSH

B-cell precursors (BCP) arise from hematopoietic stem cells in bone marrow (BM). Identification and characterization of the different BCP subsets has contributed to the understanding of normal B-cell development. BCP first rearrange their immunoglobulin (Ig) heavy chain (IGH) genes to form the pre-B-cell receptor (pre-BCR) complex together with surrogate light chains. Appropriate signaling via this pre-BCR complex is followed by rearrangement of the Ig light chain genes, resulting in the formation, and selection of functional BCR molecules. Consecutive production, expression, and functional selection of the pre-BCR and BCR complexes guide the BCP differentiation process that coincides with corresponding immunophenotypic changes. We studied BCP differentiation in human BM samples from healthy controls and patients with a known genetic defect in V(D)J recombination or pre-BCR signaling to unravel normal immunophenotypic changes and to determine the effect of differentiation blocks caused by the specific genetic defects. Accordingly, we designed a 10-color antibody panel to study human BCP development in BM by flow cytometry, which allows identification of classical preB-I, preB-II, and mature B-cells as defined via BCR-related markers with further characterization by additional markers. We observed heterogeneous phenotypes associated with more than one B-cell maturation pathway, particularly for the preB-I and preB-II stages in which V(D)J recombination takes place, with asynchronous marker expression patterns. Next Generation Sequencing of complete IGH gene rearrangements in sorted BCP subsets unraveled their rearrangement status, indicating that BCP differentiation does not follow a single linear pathway. In conclusion, B-cell development in human BM is not a linear process, but a rather complex network of parallel pathways dictated by V(D)J-recombination-driven checkpoints and pre-BCR/BCR mediated-signaling occurring during B-cell production and selection. It can also be described as asynchronous, because precursor B-cells do not differentiate as full population between the different stages, but rather transit as a continuum, which seems influenced (in part) by V-D-J recombination-driven checkpoints.

• MeSH
• buněčná diferenciace genetika   imunologie   MeSH
• dítě MeSH
• lidé MeSH
• prekurzorové B-lymfoidní buňky imunologie   MeSH
• průtoková cytometrie metody   MeSH
• receptory antigenů B-buněk genetika   imunologie   MeSH
• syndromy imunologické nedostatečnosti genetika   imunologie   MeSH
• těžké řetězce imunoglobulinů genetika   MeSH
• V(D)J rekombinace genetika   imunologie   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Flow cytometric detection of minimal residual disease (MRD) in children with B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) requires immunophenotypic discrimination between residual leukaemic cells and B-cell precursors (BCPs) which regenerate during therapy intervals. In this study, EuroFlow-based 8-colour flow cytometry and innovative analysis tools were used to first characterize the immunophenotypic maturation of normal BCPs in bone marrow (BM) from healthy children, resulting in a continuous multiparametric pathway including transition stages. This pathway was subsequently used as a reference to characterize the immunophenotypic maturation of regenerating BCPs in BM from children treated for BCP-ALL. We identified pre-B-I cells that expressed low or dim CD34 levels, in contrast to the classical CD34(high) pre-B-I cell immunophenotype. These CD34(-dim) pre-B-I cells were relatively abundant in regenerating BM (11-85% within pre-B-I subset), while hardly present in healthy control BM (9-13% within pre-B-I subset; P = 0·0037). Furthermore, we showed that some of the BCP-ALL diagnosis immunophenotypes (23%) overlapped with CD34(-dim) pre-B-I cells. Our results indicate that newly identified CD34(-dim) pre-B-I cells can be mistaken for residual BCP-ALL cells, potentially resulting in false-positive MRD outcomes. Therefore, regenerating BM, in which CD34(-dim) pre-B-I cells are relatively abundant, should be used as reference frame in flow cytometric MRD measurements.

• MeSH
• antigeny CD34 metabolismus   MeSH
• buněčná diferenciace fyziologie   MeSH
• genová přestavba B-lymfocytů imunologie   MeSH
• imunofenotypizace metody   MeSH
• kostní dřeň fyziologie   MeSH
• lidé MeSH
• mladiství MeSH
• pre-B-buněčná leukemie diagnóza   imunologie   patofyziologie   MeSH
• předškolní dítě MeSH
• prekurzorové B-lymfoidní buňky imunologie   patologie   fyziologie   MeSH
• průtoková cytometrie MeSH
• regenerace MeSH
• reziduální nádor MeSH
• těžké řetězce imunoglobulinů imunologie   MeSH
• Check Tag
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Mutations in PIK3CD and PIK3R1 cause activated PI3K-δ syndrome (APDS) by dysregulation of the PI3K-AKT pathway. METHODS: We studied precursor and peripheral B-cell differentiation and apoptosis via flowcytometry. Furthermore, we performed AKT-phosphorylation assays and somatic hypermutations (SHM) and class switch recombination (CSR) analysis. RESULTS: We identified 13 patients of whom 3 had new mutations in PIK3CD or PIK3R1. Patients had low total B-cell numbers with increased frequencies of transitional B cells and plasmablasts, while the precursor B-cell compartment in bone marrow was relatively normal. Basal AKT phosphorylation was increased in lymphocytes from APDS patients and natural effector B cells where most affected. PI3K mutations resulted in altered SHM and CSR and increased apoptosis. CONCLUSIONS: The B-cell compartment in APDS patients is affected by the mutations in PI3K. There is reduced differentiation beyond the transitional stage, increased AKT phosphorylation and increased apoptosis. This B-cell phenotype contributes to the clinical phenotype.

• MeSH
• agamaglobulinemie genetika   imunologie   MeSH
• aktivace lymfocytů genetika   imunologie   MeSH
• B-lymfocyty imunologie   MeSH
• buněčná diferenciace genetika   imunologie   MeSH
• dítě MeSH
• dospělí MeSH
• fosfatidylinositol-3-kinasy genetika   MeSH
• fosforylace genetika   MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mutace genetika   imunologie   MeSH
• plazmatické buňky imunologie   MeSH
• předškolní dítě MeSH
• prekurzorové B-lymfoidní buňky imunologie   MeSH
• přesmyk imunoglobulinových tříd genetika   imunologie   MeSH
• protoonkogenní proteiny c-akt genetika   MeSH
• recidiva MeSH
• signální transdukce genetika   MeSH
• somatická hypermutace imunoglobulinových genů genetika   imunologie   MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• Asijci MeSH
• běloši MeSH
• buněčná diferenciace MeSH
• buňky kostní dřeně imunologie   patologie   MeSH
• chronická lymfatická leukemie etnologie   imunologie   patologie   MeSH
• imunofenotypizace MeSH
• lidé MeSH
• počet lymfocytů MeSH
• prekurzorové B-lymfoidní buňky imunologie   patologie   MeSH
• studie případů a kontrol MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem MeSH

B cells play an important role in the immune responses which affect the outcomes of kidney allografts. Dynamic changes of B-cell compartments in clinical kidney transplantation are still poorly understood. B-cell subsets were prospectively monitored using flow cytometry for 1 year in 98 kidney transplant recipients. Data were correlated with immunosuppression and clinical outcomes. An increase in the total population of B lymphocytes was observed during the first week after transplantation. The level of IgM(high) CD38(high) CD24(high) transitional B cells reduced significantly up until the third month, with partial repopulation in the first year. Lower numbers of transitional B cells in the third month were associated with higher risk of graft rejection. IgM(+) IgD(+) CD27(-) naive B cells did not change within follow-up. IgM(+) CD27(+) nonswitched memory B cells and IgM(-) CD27(+) switched memory B cells increased on post-operative day 7. IgM(-) CD38(high) CD27(high) plasmablasts showed similar kinetics during the first post-transplant year, similar to transitional B cells. In conclusion, sensitized kidney transplant recipients as well as those with either acute or chronic rejection within the first post-transplant year exhibited lower levels of transitional B cells. Therefore, these data further support the hypothesis that transitional B cells have a protective role in kidney transplantation.

• MeSH
• alografty MeSH
• antigen CD24 metabolismus   MeSH
• antigeny CD27 metabolismus   MeSH
• antigeny CD38 metabolismus   MeSH
• časové faktory MeSH
• dítě MeSH
• dospělí MeSH
• imunologická paměť imunologie   MeSH
• imunosupresivní léčba MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• plazmatické buňky imunologie   MeSH
• podskupiny B-lymfocytů imunologie   MeSH
• předškolní dítě MeSH
• prekurzorové B-lymfoidní buňky imunologie   MeSH
• příjemce transplantátu MeSH
• prospektivní studie MeSH
• rejekce štěpu imunologie   MeSH
• senioři MeSH
• senzitivita a specificita MeSH
• transplantace ledvin * MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

The expression of CD27 and CD44 correlate with the genotype of B-precursor acute lymphoblastic leukemia (ALL). Based on the expression of these antigens, we identified counterparts of TEL/AML1(pos) and TEL/AML1(neg) leukemic cells in nonmalignant bone marrow. Although CD27 is known as a marker of mature memory B cells, we recently showed that CD27 is also expressed by malignant and nonmalignant B precursors. Here, we show that CD27 and CD44 delineate stages of B-precursor development. Well-established differentiation markers showed that the developmental sequence starts from undetermined progenitors, expressing CD44. Upon B-lineage commitment, cells gain CD27 and lose CD44. The CD27(pos)CD44(neg) (CD27 single positive, 27SP) cells are the earliest stage within CD10(pos)CD19(pos) B precursors and express RAG-1 and TDT. These cells correspond to TEL/AML1(pos) ALL (1/4 pediatric B-precursor ALL). The development follows to CD27/CD44 double-positive (27/44DP) stage, 44SP stage and CD27/CD44 double-negative (27/44DN) stage. Before exit to periphery, CD44 is reexpressed. The 27/44DP cells are mostly large and profoundly suppress RAG-1. Despite their presumably high proliferation potential, 27/44DP cells rarely dominate in leukemia. At 44SP stage, which corresponds to TEL/AML1(neg) leukemias, RAG-1 is reexpressed and Ig light chain gene starts to be rearranged.

• MeSH
• antigeny CD27 biosyntéza   fyziologie   genetika   MeSH
• antigeny CD44 biosyntéza   fyziologie   genetika   MeSH
• dítě MeSH
• financování organizované MeSH
• genová přestavba B-lymfocytů imunologie   MeSH
• imunofenotypizace MeSH
• leukemie B-buněčná diagnóza   genetika   imunologie   MeSH
• lidé MeSH
• lymfopoéza genetika   imunologie   MeSH
• prekurzorové B-lymfoidní buňky cytologie   imunologie   patologie   MeSH
• vývojová regulace genové exprese MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• srovnávací studie MeSH