Dissection of Complementary Vs Unique Cntributions of Kit, Flt3 and IL-7R Signaling to Lymphohematopoietic Development in Vivo

Lymphohematopoietic development requires partially complementary cytokine signals mediated by the receptor tyrosine kinases Kit and Flt3, while lymphoid development also depends on the Type I cytokine receptor IL-7R. Common lymphoid progenitors (CLP) and the earliest thymic progenitors (ETP) uniquely express all three receptors. Complicating our understanding of the roles of these three cytokine pathways are synergistic cross-receptor interactions by which the RTK can activate non-cognate Type I cytokine receptors, e.g., IL-7R. To unravel the complex interplay between these three pathways, particularly in lymphopoiesis, we analyzed marrow, thymus and spleen populations in mice with loss of function of each of these pathways, alone or in combination (Kit^W41/W41 mutant, Flt3L^-/-, and IL-7^-/- knockouts (KO) so that the respective receptors Kit, Flt3 and IL-7R could be used for phenotypic analyses of progenitor populations). As expected, mice with mutations of Kit, alone or in combination with Flt3L and/or IL-7 KO, showed ~85% loss of both long-term (LT) and short-term (ST) HSC, and numbers of subsequent marrow and thymic progenitor populations (multipotent progenitor (MPP) 2, MPP3, CD62L+ lymphoid primed MPP (LMPP), common myeloid progenitor (CMP), granulocyte-macrophage progenitor (GMP), megakaryocyte-erythroid progenitor (MEP), CLP, ETP) decreased proportionately. The earliest effects of Flt3L loss occurred at the MPP3 stage (~80% loss); surprisingly, the number of myeloid progenitors was increased and CLP decreased. IL-7 KO showed declines in lymphoid committed cells at the LMPP through the CLP and ETP stages. Again unexpectedly, IL-7 loss resulted in increased CMP. Mice deficient for both Kit and Flt3L were viable, had further additive reduction in MPP2 numbers, but showed profound, synergistic reductions in MPP3, LMPP, GMP, CLP, and ETP populations. The combination of IL-7 KO with either Kit^W41/W41 or Flt3L KO had no effect on the MPP2, but caused additive declines in LMPP and CLP. Mice triply deficient for Kit, Flt3L and IL-7 (“TKO” mice) were also viable, but showed profound (>99%) loss of all lymphoid progenitors (LMPP, CLP and ETP). To account for the downstream effects of the reduced HSC compartment in Kit^W41/W41 mice, the committed progenitor numbers were normalized to the input HSC numbers in the respective mouse strains. In the absence of Kit signaling, Flt3L and IL-7 loss had ~2-3 log greater effects on lymphopoiesis than myelopoiesis. Consistent with the analyses of numbers of lymphoid vs myeloid progenitors, gene expression analyses of sorted progenitors also showed that in the absence of either Kit and Flt3L, Kit and IL-7, or Kit, Flt3L, and IL-7, MPP3 showed a global shift away from expression of lymphoid developmental pathways and towards myeloid pathways. In addition to expression by CLP and ETP, a subset of MPP3 and LMPP also expressed IL-7R at low levels. These results demonstrate that while Kit has global effects on HSC and their progeny, and Flt3 and IL-7R signals are required only by committed progenitors, 1) Flt3 and IL-7R synergize with and complement Kit in directing lymphoid commitment and/or differentiation of MPP; and 2) that Flt3 and IL-7R signal at the MPP3 or LMPP stages to promote lymphoid development, i.e., earlier than phenotypic CLP. These results can be explained by the co-expression of the RTKs and signaling competent IL-7R in LMPP prior to the phenotypic CLP stage, and the previously described RTK-mediated activation of the IL-7R by non-cognate cross-receptor interactions. Analyses of these complex cytokine interactions may be useful in the elucidation of lymphoid committed progenitor subpopulations and lineage fate decisions among MPP, and the development of therapeutic strategies for expansion of specific progenitor cell populations.