Multi-Parameter Flow Cytometric Analysis of Ikaros Family Expression in Human T Cell Development and T-ALL

The Ikaros family transcription factors are critical regulators of T cell development and leukemogenesis. Loss of function of all five family members leads to an early block in murine lymphocyte development, whereas reduced function results in the development of T cell leukemias. Loss of a single family member has only minimal affects on T cell development, suggesting compensating functions of the family members, and emphasizing a need to study expression and function of the whole Ikaros family. We analyzed the expression of all five family members in human and murine thymocytes as they progressed from the CD4–CD8– double negative (DN) to the CD4+CD8+ double positive (DP) developmental stages, and found differences in the expression of Helios and Eos mRNA between the two species. Further, whereas Ikaros and Aiolos mRNA levels increased in both species, protein levels only increased in murine thymocytes. These data suggest that regulation of Ikaros family expression during T cell development differed between the two species. To further examine expression of Ikaros family members during human T cell development, we used multi-parameter flow cytometry to identify subpopulations of human thymocytes. We defined seven populations of CD3– CD4+CD8– immature single positive (ISP) and CD3– DP cells to identify when TCRβ is expressed. We were able to delineate the pre-β-selection ISP1 and DP1 populations and the TCRβ expressing ISP2 and DP2 populations using expression of CD1a, CD28, and CD44. The ISP2 and DP2 populations had a higher percentage of proliferating cells, consistent with these being post-β-selection stages. Protein levels of Ikaros, Helios, and Aiolos all increased with β-selection, however this increase was transient for Ikaros and Helios levels. We further identified 22 populations of thymocytes that express CD3 and showed that CD4 expression is down-regulated after positive selection to create a CD8+CD4–/lo transitional single positive (TSP) developmental stage. Commitment to the CD8 T cell lineage occurs in TSP thymocytes and is marked by expression of CD27. Among CD4+ cells, expression of CD27 can first be seen in MSP2 CD4+ thymocytes, suggesting that commitment to the CD4 lineage occurs at this stage. Ikaros levels increased during the DP9 stage, and Helios and Aiolos expression increased in TSP thymocytes. The increase in Helios was transient, and Helios levels decreased as cells developed through the MSP CD4+ and MSP CD8+ stages. Aiolos levels increased in cells proceeding from the TSP to the MSP CD8+ stage. Within MSP CD4+ thymocytes, Aiolos levels increased transiently beginning in the MSP2 CD4+ thymocytes suggesting that increases in Aiolos protein are associated with CD4/CD8 lineage commitment. Pediatric T cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous disease that develops as a result of clonal expansion of thymocyte populations that bypass regulatory selection steps. We analyzed expression of the Ikaros family members in the leukemic cells from pediatric T-ALL patients and compared them to normal thymic populations, finding that mRNA levels of at least one Ikaros family member were elevated in the cells from every T-ALL patient analyzed. By comparing the ratio of the Ikaros family mRNA levels, we were able to classify the T-ALL patients into groups that showed diverse expression of surface markers. The lack of correlation between Ikaros and Aiolos mRNA and protein levels within the T-ALL suggest that further studies are needed to determine the significance of both mRNA ratios and protein levels in T-ALL patients. Further, studies are required to identify the roles of the Ikaros family members during the key selection steps of normal thymic development in order to understand how altered expression may contribute to leukemogenesis