CD44 and CD27 Expression Delineates Stages of B Precursor Development Reflecting Recombination Status and Propensity to Leukemic Transformation.

The expression of CD27, in the absence of CD44 is found in TEL/AML1+ acute lymphoblastic leukemia (ALL) but not in other B precursor subtypes of ALL (Vaskova et al., Leukemia 19(5), 2005). Since CD27 had not been shown in human B precursors, we searched for such cells among B precursors in nonmalignant bone marrow. In all 14 specimens of children without any evidence of malignant disease, we found CD27+CD44−CD10+CD19+ B precursors (6.3±3.9%). The subpopulation of CD27−CD44+ cells that corresponds to most other ALL subtypes was more frequent (26±14.4%). Two other subpopulations which rarely develop into B precursor leukemia were also present: double-positive (DP) (5.5±1.8%) and double-negative (DN) (61.8±14.9%). We asked, whether the 4 subpopulations represent consequent differentiation stages, therefore the expression of CD27 and CD44 combined with CD10 and CD19 was studied by polychromatic flow cytometry together with the differentiation markers CD34, terminal deoxyribonucleotidyl transferase (TdT), CD20, cytoplasmic IgM and cytoplasmic VpreB (CD179a). The percentage of CD34+ cells is the highest in CD27+CD44− and decreases gradually in DP, CD27−CD44+ and DN subpopulations (73.1±20.7%; 43±16.6%; 13.1±8.2%; 4.4±2.9%). A similar trend is found in the percentage of CD10^bright cells, which become virtually missing in CD27− B precursor stages (41.3±15.4%; 7.9±8.1%; 1.4±1.1%; 2.7±1.8%). This sequence of developmental stages was further supported by a gradual loss of intracellular TdT and VpreB and by the increase of cytoplasmic IgM+ cells. We sorted these subpopulations to compare their recombination potential by measuring TdT and RAG-1 mRNA expression by RQ-RT-PCR. Similarly with the protein level, TdT mRNA expression decreases in concordance with the suggested developmental stages. Interestingly, the DP cells cease to transcript RAG-1, suggesting that these cells are in the stage of suppressed RAG-1 expression after completed immunoglobulin (Ig) heavy chain rearrangement. RAG-1 is re-expressed during Ig light chain rearrangement, seen as the reappearance in the CD27−CD44+ subpopulation. Since the cells with a downregulated RAG-1 are known to be frequent among the large proliferating cells we analyzed the percentage of large cells. The DP subpopulation contains the highest percentage of these cells (63.5±9.9%). In all four subpopulations, heavy chain gene (both segments VH1-3-JH and VH4-7-JH) rearrangements were detected, suggesting that heavy chain genes start to rearrange before or at the CD27+CD44− stage. We investigated the light chain rearrangements using the system detecting the intron RSS-Kde rearrangements, which appear in the late phase of Ig light chain rearrangement. Rearranged light chain genes appear at the CD27−CD44+ stage, whereas they are virtually missing at earlier stages. These results show that CD27 molecule, which is so far regarded a marker of memory B cells is expressed also in the early stage of B cell development during Ig heavy chain rearrangement completion. The expression of CD27 and CD44 define differentiation stages that very tightly correlate with Ig recombination maturity as well as with specific subtypes of B precursor leukemia.