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Tie2 is a receptor-type tyrosine kinase expressed on hematopoietic stem cells and endothelial cells. We used cultured embryonic stem (ES) cells to determine the function of Tie2 during early vascular development and hematopoiesis. Upon differentiation, the ES cell-derived Tie2+ Flk1+ fraction was enriched for hematopoietic and endothelial progenitor cells. To investigate lymphatic differentiation, we used a monoclonal antibody against LYVE-1 and found that LYVE-1+ cells derived from Tie2+ Flk1+ cells possessed various characteristics of lymphatic endothelial cells. To determine whether Tie2 played a role in this process, we analyzed differentiation of Tie2-/- ES cells. Although the initial numbers of LYVE-1+ and PECAM-1+ cells derived from Tie2-/- cells did not vary significantly, the number of both decreased dramatically upon extended culturing. Such decreases were rescued by treatment with a caspase inhibitor, suggesting that reductions were due to apoptosis as a consequence of a lack of Tie2 signaling. Interestingly, Tie2-/- ES cells did not show measurable defects in development of the hematopoietic system, suggesting that Tie2 is not essential for hematopoietic cell development.

The interleukin-2 (IL-2) receptor gamma chain is indispensable for IL-2-, IL-4-, IL-7-, IL-9-, and IL-15-mediated signaling. Mutations of the human gamma chain cause the X-linked severe combined immunodeficiency (XSCID), showing that T and natural killer cells absolutely require the gamma chain for their development in humans. To elucidate the roles of the gamma chain in hematopoiesis, we have generated mice, by gene targeting, that express a form of the gamma chain lacking the cytoplasmic region. Male mice carrying the truncated gamma-chain mutant, which mimics mutations in patients with XSCID, showed a decrease in the number of lymphocytes and an increase in monocytes; the number of T cells was profoundly reduced and no natural killer cells were detected, which is similar to the characteristic of human XSCID. Unlike human XSCID, the levels of B cells were also reduced. In spite of the severe decrease in CD45R+/sIgM+ B cells, the level of IgM in serum of the 8-week-old mutant mice was higher than that of control littermates. Interestingly, the stem cell population with surface phenotypes of CD34, c-kit, and Sca-1 was significantly increased. Furthermore, the colony-forming assay showed that the mutant mice had 15-fold higher numbers of hematopoietic progenitor cells in the spleen as compared with that of controls. These results indicate that functional loss of the gamma chain causes significant effects on the immunological system in mice.

We previously demonstrated the existence of a third component, p64, of IL-2 receptor (IL-2R), tentatively named the gamma chain of IL-2R. Our recent studies provided evidence suggesting that the gamma chain endows the beta chain of IL-2R with IL-2 binding ability. The gamma chain was detected in lymphoid transfectants of IL-2R beta cDNA, which showed the intermediate-affinity IL-2R, but not in nonlymphoid transfectants of IL-2R beta cDNA, which showed no IL-2 binding activity. The comparative study between two subclones of lymphoid MOLT4 transfectant of IL-2R beta cDNA demonstrated that the amount of the gamma chain coprecipitated with IL-2R beta was proportional to numbers of the IL-2 binding sites. These results suggest the possibility that the gamma chain associates with IL-2R beta and has an important role in formation of the intermediate-affinity IL-2R complex. On the other hand, we have also demonstrated the association of IL-2R beta with a certain tyrosine kinase, of which activation by IL-2 could be indispensable process at the initial pathway of signal transduction.

We previously established a monoclonal antibody, TU11 mAb, which is specific for human IL-2 receptor (IL-2R) beta chain (p75) and does not inhibit IL-2-binding to IL-2R beta. Using TU11 mAb, we first demonstrated the existence of a third component, p64, of IL-2R, tentatively named the gamma chain of IL-2R. TU11 mAb precipitated not only the beta chain but also the alpha and gamma chains in the lysates of cells bearing the high-affinity IL-2R in the presence of IL-2 without any chemical crosslinker. The gamma chain was also detected in lymphoid MOLT alpha beta and MOLT beta cells, which were stably transfected with both alpha and beta cDNA, and with beta cDNA alone, respectively, but not in fibroblastoid COS alpha beta and COS beta cells, which were stably transfected with both alpha and beta cDNA, and with beta cDNA alone, respectively. Furthermore, IL-2-mediated growth signals were transduced in the lymphoid transfectant cells but not in the fibroblastoid transfectant cells, suggesting the possibility that the gamma chain along with the beta chain has an essential role in the transduction of IL-2-mediated growth signals. Using TU11 mAb, we secondly demonstrated that IL-2 rapidly induces tyrosine phosphorylation of both the beta and gamma chains in an IL-2-dose-dependent manner. The tyrosine phosphorylation of beta and gamma chains were also detected in the lymphoid transfectant cells but not in the fibroblastoid transfectant cells, indicating the correlation between tyrosine kinase activation and IL-2-mediated growth signaling. The beta chain was phosphorylated in in vitro on serine, threonine and tyrosine residues, but the gamma chain was phosphorylated in in vitro predominantly on tyrosine residues, suggesting the possibility that the gamma chain itself is a tyrosine kinase molecule.