We studied the differentiation profiles of B cell precursors Materials and methods(BCP) in normal and post-chemotherapy pediatric bone marrow (BM) using multiparameter flow cytometry. The goal of our Sample description study was to draw a comprehensive phenotypic map of the three major maturational BCP stages in BM. By correlating lineage-associated markers, CD45RA, and several adhesion molPediatric bone marrow (BM) samples (n = 44) were obtained ecules, the stage-specific patterns were found to differ in cerfrom children undergoing diagnostic BM aspiration for the foltain details from previously published concepts. Among the lowing diagnoses in the absence of BM involvement at the earliest BCP, a subset of CD34 + CD10 lo precursors was repeattime of immunologic investigation: suspicion of storage disedly observed in addition to the well characterized ease (n = 1), bacterial lymphadenitis (n = 1), neuroblastoma CD34 + CD10 hi CD19 + majority of cells. Only two-thirds of these CD34 + CD10 lo cells expressed CD19. However, uniformity of (n = 1), severe aplastic anemia in remission (n = 2), single sys- observation time from BM aspiration to final evaluation was 1 2 12 years (range: 1 month to 2 6 12 years). The two patients with the shortest follow-up (1 and 2 months) have been in conIntroduction firmed remission for 3 3 12 years and 4 months, respectively. All leukemia/lymphoma patients described in this paper were The mainstays in defining the phenotypic patterns of B cell treated according to BFM (Berlin-Frankfurt-Mü nster) treatdifferentiation have already been documented during the last ment protocols. In brief, for ALL a multi-agent induction thertwo decades. [1][2][3][4] In brief, the CD19 antigen is expressed apy administered in three cycles over the first six months was throughout all stages of B cell maturation, 1,5 while CD10 is followed by a low-dose maintenance therapy until completion highly expressed in the most immature (CD34 + ) B cell precurof the second therapy year. All data concerning these patients sors (BCP), 3 and is lost at a later stage concomitantly with gain were obtained from the Austrian study center of the of surface expression of CD20, IgM and CD22. 2 More international BFM study group. recently, this classical model of B cell differentiation was challenged, as new antigenic patterns, 6-8 new insights into the sequence of antigen acquisition in B cell ontogeny, 9 and Antibodies tissue-related differences in the coordinate patterns of B lymphoid antigen expression were documented. 10,11 In the present study, our aim was comprehensively to redeFluorescein isothiocyanate (FITC)-, and phycoerythrin (PE)-fine the phenotypic patterns of the early stages of normal B labeled, or unconjugated pure monoclonal antibodies cell differentiation in postnatal bone marrow by multi-(MoAbs) were used: CD7 (DK24-FITC), CD10 (SS2/36-PE), parameter flow cytometry. By comparing normal and postCD11a (MHM24 pure), CD19 (HD37-FITC, pure), CD20 (Bchemotherapy bone marrow, we focused on sample-inherent Ly1-F...

The cell-surface expression of the MIC2 antigen defined by the monoclonal antibody 12E7 was investigated on human leukocytes in bone marrow (BM), thymus, and peripheral blood (PB) using multiparameter flow cytometry and cell sorting. In contrast to preceding reports, we found that the MIC2 antigen is not restricted to T cells and monocytes. We show that it is also expressed in the B cell and in the granulocytic lineage, the levels of expression being related to distinct maturational stages. CD34+ cells of BM were found to express the antigen at high levels. Along the granulocytic maturation pathway from CD34+CD33+ blasts to mature granulocytes, MIC2 densities appeared progressively reduced with a considerable decline at the myelocyte stage. In B lymphopoiesis, the earliest CD34+ CD10+ B-cell precursor (BCP) cells, further subdivided by expression of CD19, displayed the highest MIC2 density of BM leukocytes. All later BCP stages showed lower MIC2 expression levels, with a remarkable reduction concomitant with loss of the CD34 antigen at the CD10+CD20- surface mu-chain- stage, and a subsequent slight upregulation along with maturation to CD10-CD20high surface mu-chain+ BCPs. The brightest MIC2 expression of all cells tested was displayed by the most immature thymic T-lineage cells characterized by the antigenic profile CD34weakor- CD7++ surface CD3-CD1a(weak) CD4weak CD8-or weak. Common thymocytes stained slightly less intense with 12E7, whereas all subsequent stages of T-lineage cells in thymus, PB, or BM showed markedly reduced MIC2 levels. Mature peripheral CD4+ as well as CD8+ T cells displayed a bimodal distribution of MIC2. In the CD4+ population, the distinct MIC2 levels were related to the well-studied functional subdivision by differential expression of CD45 isoforms, the helper-inducer/memory subset showing higher MIC2 expression than helper-suppressor/naive CD4+ T cells. Similarly high MIC2 densities were found on CD16+ natural killer cells and on CD14+ monocytes, whereas mature peripheral B cells exhibited low or intermediate expression, and granulocytes exhibited no or only dim expression. These results document that the MIC2 antigen (1) is expressed on all leukocyte lineages; (2) is differentially expressed during T- and B-lymphoid, as well as granulocytic maturation; (3) shows highest expression in the most immature lymphocytic and granulocytic developmental stages; and (4) is also differentially expressed on functional T-cell subsets. We speculate that these observations imply a functional significance of MIC2 in the network of hematopoietic adhesion pathways.

Summary.We have recently shown that CD99 (MIC2) is differentially expressed during normal early B-cell development in the bone marrow (BM). Since immature B-cell precursors (BCP) are assumed to correspond to some extent to acute lymphoblastic leukaemia (ALL) and non-Hodgkin's lymphoma (NHL) cells with respect to patterns of phenotypic differentiation, we wondered whether the particular maturation-associated expression patterns of CD99 in the normal BCP stages were conserved also in malignant cells. Therefore we compared malignant and physiological B cells from paediatric ALL/NHL and normal BM samples with respect to CD99 expression using selective gating and semi-quantitative flow cytometry. Common-ALLs (n ¼ 45) were similar to their corresponding, very immature BCPs (stage 1) in expressing very high levels of CD99. Most pre-B ALLs (n ¼ 16) were also CD99 hi and thus differed from the patterns found in normal cytoplasmic m-chain). In particular, we found that those pre-B-ALL cases which were CD34 þ also showed higher CD99 expression than the CD34 ¹ cases. This prompted us to investigate the levels of CD99 in those rare normal BCPs which also coexpress CD34 and cm; these cells, which are transitory from stage 1 to stage 2, were found also CD99 hi , thus precisely reflecting the patterns of CD34 þ pre-B ALLs. The blasts of Burkitt-type B-cell ALL/NHL samples (n ¼ 13) expressed considerably less CD99, similarly to the more differentiated BCP stages 2 (cm þ ) and 3 (surface m-chain þ ). In summary, we found that paediatric B-lineage malignancies display remarkable synchrony regarding the levels of CD99 expression compared to their putative normal counterparts.

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