To clarify the phenotypes of various classes of human hematopoietic progenitor cells, we used a multicolor staining protocol in conjunction with CD34 and a newly developed mouse antihuman c-kit proto-oncogene product (KIT) monoclonal antibody (MoAb). We characterized three cell fractions in CD34+ cells that express KITlow and KIThigh cells in addition to KIT- cells. A clonogenic assay showed that most granulocyte- macrophage colony-forming cells (GM-CFC) were present in CD34+KIThigh populations, whereas erythroid burst-forming cells (BFU-E) were detected mainly in the CD34+KITlow population. CD34(+)-KIT- fraction contained a small number of BFU-E. Morphologic analysis showed that blast-like cells were more enriched in the CD34+KITlow fraction. KITlow cells contained CD34+CD38- cells that were considered to be very primitive progenitor cells, as determined by a replating assay. To clarify the biologic differences between both fractions, we examined the more primitive progenitor cell functions by assessing long-term culture-initiating cells (LTC-IC) on the stromal cells. At week 2, more CFC recovered from the culture in the fraction initiated with a CD34+KIThigh population. However, more LTC-IC were present during weeks 5 to 9 in the CD34+KITlow population. These results indicate that primitive progenitors are more enriched in the KITlow population and that the KIThigh population contains many GM-committed progenitor cells. We also showed that anti-KIT MoAb inhibited the ability of CD34+ cells to generate CFC on the stromal layer in the LTC system. This suppressive effect was more evident in the generation of BFU-E by CD34+KITlow cells. Moreover, we confirmed that CD34+KIThigh cells emerged from CD34+KITlow cells during coculture with allogeneic stromal cells or from liquid culture in the presence of stem cell factor (SCF), interleukin-6, and erythropoietin. These results emphasize the pivotal role of the KIT and SCF interaction in hematopoiesis and indicate that KITlow cells are more primitive than KIThigh cells.

T lymphoblasts of rat origin were (a) surface labeled with 125I and (b) internally labeled with 3H-marked sugars. Cell lysates were purified by immunoabsorption using the putative anti-rat interleukin 2 (IL2) receptor monoclonal antibody ART18 . The purified material was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis. Two specific membrane components were detected: a 50-kDa major and a 36-kDa minor component in reducing and a 45-kDA major and a 72-kDa minor component in nonreducing conditions, respectively. Both components were found to be susceptible to trypsinization and to neuraminidase treatment. 3H-labeled sugars were incorporated into the major component. The results indicate that the rat IL2 receptor is either a 50-kDa glycoprotein or a 36-kDa molecule, or that both components are part of the receptor molecule.

We have cloned cDNA encoding the mouse interleukin-2(IL-2) receptor from a murine T cell line, CTLL using human IL-2 receptor cDNA as probe. COS 7 cells transfected with the cDNA expressed the antigen recognized by the monoclonal antibody against the murine IL-2 receptor. The cDNA identified 4 species of mRNA (4.5, 3.5, 2.2 and 1.5 kb) of the mouse IL-2 receptor in CTLL cells. Difference in the length of mRNA seems to be ascribed to the variable length of the 3' untranslated sequence. Total nucleotide sequence ('1400 bp) of this cDNA was determined and compared with the human receptor. The nucleotide and amino acid sequences of the IL-2 receptor are 70% and 60%, respectively, homologous in average between the two species. The comparison has revealed several conserved regions localized to particular exons such as transmembrane and cytoplasmic portions, suggesting that these regions are important for receptor function and its regulation.

In this report we have summarized our findings on the IL-2 receptor and our attempts to find an IL-2 receptor targeted immunosuppressive therapy. IL-2 receptors are detectable in two different forms: as monomeric, surface expressed, and as dimeric, presumably non-surface expressed molecules. The dimeric form seems to be non-covalently bound to an as yet undefined 110 KD molecule. The functions of the monomeric versus the dimeric form, as well as that of the noncovalently bound molecule, and their relationship to high and low affinity IL-2 receptors are not yet clear and remain to be elucidated. Upon antigenic or mitogenic stimulation, IL-2 receptors became expressed at the surface of T-lymphocytes. Receptor expression is accompanied by the capacity of the cells to proliferate in response to IL-2. Resting T-cells do not proliferate in response to IL-2. IL-2 dependent proliferation of cells without external stimulation is either due to the presence of a small number of IL-2 receptor bearing cells in the respective population or due to a small number of IL-2 receptors present on the surface of cells. IL-2 itself does not induce IL-2 receptor expression on resting cells but has been shown to up-regulate its own receptor once expressed. In contrast to resting lymphocytes, some leukemic cells and early embryonic thymocytes in the species tested constitutively express IL-2 receptors. The role of such constitutively expressed receptors is not yet clear. As demonstrated in mice, the requirement(s) for induction of IL-2 receptor expression for the helper/inducer subset (Lyt-2+) are different from those of the cytotoxic/suppressor subset (L3T4+). In contrast to Lyt-2+ cells, the accessory cell requirement for L3T4+ cells could not be replaced by cytokines. Whether Lyt-2+ cells require an additional, not yet defined receptor inducing factor (RIF) besides IL-2 in order to become IL-2 receptor positive and to consequently proliferate in response to IL-2, is a matter of controversy. There is evidence that interleukin-1 and some functionally related factors produced by leukemic cells enhance expression and/or function of the IL-2 receptors. IL-2 receptors of the high and low affinity type expressed upon antigenic stimulation are cleared from the cell surface. As demonstrated in this report, the vast majority of them, probably those of low affinity type, are released from the cells continuously. The mechanism of their release and the possible immunoregulatory role of circulating receptors found in the serum of animals is not yet clear.(ABSTRACT TRUNCATED AT 400 WORDS)

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