Interleukin 3 Stimulation Enhances Tyrosine Phosphorylation and Proliferative Activity of Human Fetal Thymocytes and Leukemic T-cell Precursors at Multiple Developmental Stages of T-Cell Ontogeny

Abstract

The purposes of this study were to elucidate the biologic effects of recombinant human interleukin 3 (rhIL-3) on leukemia T-cell precursors and fetal thymocytes corresponding to discrete and sequential developmental stages of human T-cell ontogeny, and to examine the biochemical nature of the IL-3 receptor linked transmembrane signal in human T-cell precursor populations. The specific binding of biosynthetically labeled ³⁵S-rhIL-3 to leukemic T-cell precursors from T-lineage ALL patients was initially investigated. In 5 of 9 cases, the binding of ³⁵S-rhIL-3 was significantly blocked by excess cold rhIL-3, and the percentage of inhibitable binding ranged from 40% to 64% (mean ± SE = 53 ± 4%). In these cases, 5–13 femtomols (mean + SE = 7.0 ± 1.5 fms) of ³⁵S-rhIL-3/10⁷ cells were specifically bound. rhIL-3 stimulated in a dose dependent fashion the in vitro clonal proliferation of leukemic T-cell precursors with composite immunophenotypes corresponding to the developmental stages of prothymocytes/double negative immature thymocytes (3 of 5 cases) and corticothymocytes/double positive CD4⁺CD8⁺ immature thymocytes (4 of 4 cases). By contrast, leukemic T-cell precursors from 2 T-lineage ALL cases with a composite immunophenotype of medullary thymocytes/single positive CD4⁺CD8⁻/CD4⁻CD8⁺ mature thymocytes did not show an enhanced proliferative activity after stimulation with increasing concentrations of rhIL-3. All of the 5 cases with significant ³⁵S-rhIL-3 binding and none of the 4 cases with no ³⁵S-rhIL-3 binding showed a proliferative response to rhIL-3. Thus, there was a high correlation between ³⁵S-rhIL-3 binding and proliferative response of leukemic T-cell precursors in colony assays, indicating that functional IL-3 receptors were detected in ligand binding assays. rhIL-3 also stimulated the proliferation of immature double positive CD4⁺CD8⁺ thymocytes from 9 of 10 fetal thymuses without inducing differentiation and with no selective advantage for the development of CD4⁺CD8⁻ or CD4⁻ or CD4⁻CD8⁺ single positive thymocytes. The observed differences in IL-3 responsiveness among T-cell precursor populations at distinct developmental stages indicates that IL-3 receptors may be expressed only during the early stages of human T-cell ontogeny preceding the negative or positive selection events within the thymic microenvironment. Stimulation of fetal thymocytes with rhIL-3 resulted in enhanced tyrosine phosphorylation of 8 distinct cellular substrates with molecular weights of 44 kDa, 55 kDa, 60 kDa, 69 kDa, 98 kDa, 123 kDa, 150 kDa and 190 kDa, but it did not result in stimulation of phosphoinositide turnover and increased Ins-1,4,5-P₃ production. The induction of tyrosine phosphorylation by rhIL-3 was augmented by further crosslinking surface bound IL-3 molecules with an anti-IL-3 antibody and it was abrogated by the tyrosine kinase inhibitor genistein. The ligation of the CD45 protein tyrosine phosphatase markedly suppressed the tyrosine phosphorylation of specific substrates induced by IL-3 stimulation. Thus, the mitogenic transmembrane signal triggered by the engagement of the IL-3 R on human T-cell precursors is linked to a functional protein tyrosine kinase (PTK)/protein tyrosine phosphatase (PTP) regulatory pathway. Taken together, these results indicate that IL-3 may have an important growth regulatory role in early stages of human T-cell ontogeny.

Key Words:

• Interleukin 3
• Tyrosine phosphorylation
• proliferative activity
• foetal lymphocytes
• leukemic T-precursors
• T-cell ontogeny