Direct role of FLT3 in regulation of early lymphoid progenitors

Abstract

Given that FLT3 expression is highly restricted on lymphoid progenitors, it is possible that the established role of FLT3 in the regulation of B and T lymphopoiesis reflects its high expression and role in regulation of lymphoid-primed multipotent progenitors (LMPPs) or common lymphoid progenitors (CLPs). We generated a Flt3 conditional knock-out (Flt3^fl/fl) mouse model to address the direct role of FLT3 in regulation of lymphoid-restricted progenitors, subsequent to turning on Rag1 expression, as well as potentially ontogeny-specific roles in B and T lymphopoiesis. Our studies establish a prominent and direct role of FLT3, independently of the established role of FLT3 in regulation of LMPPs and CLPs, in regulation of fetal as well as adult early B cell progenitors, and the early thymic progenitors (ETPs) in adult mice but not in the fetus. Our findings highlight the potential benefit of targeting poor prognosis acute B-cell progenitor leukaemia and ETP leukaemia with recurrent FLT3 mutations using clinical FLT3 inhibitors.

Keywords: FLT3; conditional knock‐out mouse model; haematopoiesis; lymphoid development; lymphoid progenitors.

Figure 1

Role of FLT3 in steady‐state adult haematopoiesis. (A) Representative fluorescence‐activated cell sorting (FACS) profiles showing FLT3 surface expression on Lin⁻SCA‐1⁺KIT⁺ cells and Lin⁻SCA‐1^lowKIT^low cells in Mx1 ^+/+ Flt3 ^fl/fl compared to Mx1 ^cre/+ Flt3 ^fl/fl bone marrow (BM) (numbers represent mean percentages of 6–8 mice per genotype) 4 weeks after polyinositolic polycytidylic acid (pIpC) injection. In addition to isotype control and Fluorescence Minus One (FMO) controls, gates for FLT3 expression were set using long‐term haematopoietic stem cells (LT‐HSCs) as a negative internal reference population (IRP), to improve the reliable detection of FLT3 positive and negative cells, as HSCs have been established to lack cell surface FLT3 expression (Adolfsson et al, 2001). (B–C) Mean percentages (±SD of total BM cells) of (B) LT‐HSCs (Lin⁻SCA⁻1⁺KIT⁺CD48‐CD150⁺), CD48⁻ CD150⁻ short‐term (ST)‐HSCs (Lin⁻SCA‐1⁺KIT⁺CD48⁻CD150⁻), CD48⁺ CD150⁺ multipotent progenitors (MPPs) (Lin⁻SCA‐1⁺KIT⁺CD48⁺CD150⁺), CD48⁺ CD150⁻ MPPs (Lin⁻SCA‐1⁺KIT⁺CD48⁺CD150⁻) and common lymphoid progenitors (CLPs) (Lin⁻SCA‐1^lowKIT^lowIL‐7R⁺), (C) ProB cells (Lin⁻B220⁺ CD43⁺ CD19⁺ CD24⁺ CD93⁺), PreB cells (Lin⁻B220⁺ CD43⁻ CD19⁺IgM⁻) and IgM⁺ B cells (Lin⁻B220⁺ CD43⁻ CD19⁺IgM⁺) in 12‐week‐old Mx1 ^+/+ Flt3 ^fl/fl and Mx1 ^cre/+ Flt3 ^fl/fl mice (n = 6–8 mice per genotype in 3 experiments) 4 weeks after pIpC injection. (D) Mean percentages (±SD of total thymus cells) of early thymic progenitors (ETPs) (Lin⁻ CD4⁻ CD8a⁻ KIT ⁺ CD25⁻), Double Negative 2 (DN2) (Lin⁻ CD4⁻ CD8a⁻ KIT ⁺ CD25⁺) and Double Negative 3 (DN3) (Lin⁻ CD4⁻ CD8a⁻ KIT ⁻ CD25⁺) cells in 12‐week‐old Mx1 ^+/+ Flt3 ^fl/fl and Mx1 ^cre/+ Flt3 ^fl/fl mice (n = 6–8 mice per genotype in 3 experiments) 4 weeks after pIpC injection. (E) Polymerase chain reaction analysis of recombination at the Flt3 locus in ProB cells in Mx1 ^+/+ Flt3 ^fl/fl and Mx1 ^cre/+ Flt3 ^fl/fl mice 4 weeks after pIpC injection. Also shown are Vav1 ^+/+ Flt3 ^fl/fl, Vav1 ^cre/+ Flt3 ^fl/fl and wild type (WT) controls. The upper band represents the deleted Flt3 allele (461 bp), the middle band the floxed Flt3 allele (282 bp) and the lower band the WT allele (205 bp). (F–H) Mean percentages (±SD) contribution of CD45.2 cells to (F) LT‐HSCs, CD48⁺ CD150⁻ MPPs and CLPs, (G) ProB cells, PreB cells and IgM⁺ B cells in BM and (H) ETP, DN2 and DN3 cells in thymus of mice transplanted with 2 × 10⁶ cells unfractionated BM cells from Mx1 ^+/+ Flt3 ^fl/fl (CD45.2) or Mx1 ^cre/+ Flt3 ^fl/fl (CD45.2) mice (n = 6 per genotype) together with 2 × 10⁶ cells unfractionated BM competitor cells from WT CD45.1 mice, analysed 8 weeks post‐transplantation and 4 weeks after pIpC injection. *P < 0·05; **P < 0·01; ***P < 0·001.

Figure 2

Role of FLT3 in adult lymphoid‐committed progenitors. (A) Representative fluorescence‐activated cell sorting (FACS) profiles showing FLT3 surface expression on Lin⁻SCA‐1⁺KIT⁺ cells and Lin⁻SCA‐1^lowKIT^low cells in Rag1 ^+/+ Flt3 ^fl/fl compared to Rag1 ^cre/+ Flt3 ^fl/fl bone marrow (BM) (numbers represent mean percentages of 7 mice per genotype). Gates were set using long‐term haematopoietic stem cells (LT‐HSCs) as a negative internal reference population (IRP). (B) Mean percentages (±SD of total BM cells) of CD48⁻ CD150⁺ LT‐HSCs, CD48⁻ CD150⁻ short‐term (ST)‐HSCs, CD48⁺ CD150⁺ multipotent progenitors (MPPs), CD48⁺ CD150⁻ MPPs and common lymphoid progenitors (CLPs) in 12‐week‐old Rag1 ^+/+ Flt3 ^fl/fl and Rag1 ^cre/+ Flt3 ^fl/fl mice (n = 7 mice per genotype in 2 experiments). (C–D) Mean percentages (±SD of total BM cells) FLT3⁻ and FLT3⁺ subsets of (C) CD48⁻ CD150⁻ ST‐HSCs, CD48⁺ CD150⁺ MPPs and (D) CD48⁺ CD150⁻ MPPs and CLPs in 12‐week‐old Rag1 ^+/+ Flt3 ^fl/fl and Rag1 ^cre/+ Flt3 ^fl/fl mice (n = 7 mice per genotype in 2 experiments). (E) Mean percentages (±SD of total BM cells) of ProB cells, PreB cells and IgM⁺ B cells in 12‐week‐old Rag1 ^+/+ Flt3 ^fl/fl and Rag1 ^cre/+ Flt3 ^fl/fl mice (n = 7 mice per genotype in 2 experiments). (F) Mean percentages (±SD of total thymus cells) of early thymic progenitor (ETP), Double Negative 2 (DN2) and Double Negative 3 (DN3) cells in 12‐weekold adult thymus from Rag1 ^+/+ Flt3 ^fl/fl and Rag1 ^cre/+ Flt3 ^fl/fl mice (n = 7 mice per genotype in 2 experiments). *P < 0·05; **P < 0·01; ***P < 0·001; ns, not significant.

Figure 3

Role of FLT3 in fetal lymphoid‐committed progenitors. (A) Representative fluorescence‐activated cell sorting (FACS)profiles showing FLT3 surface expression on Lin⁻SCA‐1⁺KIT⁺ cells and Lin⁻SCA‐1^lowKIT^low cells in Rag1 ^+/+ Flt3 ^fl/fl compared to Rag1 ^cre/+ Flt3 ^fl/fl E14.5 fetal liver (FL) cells (numbers represent mean percentages of 8–11 embryos per genotype). (B) Mean percentages (±SD of total FL cells) of CD48⁻ CD150⁺ long‐term haematopoietic stem cells (LT‐HSCs), CD48⁻ CD150⁻ short‐term (ST)‐HSCs, CD48⁺ CD150⁺ multipotent progenitors (MPPs), CD48⁺ CD150⁻ MPPs and common lymphoid progenitors (CLPs) in E14.5 Rag1 ^+/+ Flt3 ^fl/fl and Rag1 ^cre/+ Flt3 ^fl/fl embryos (n = 8–11 embryos per genotype in 2 experiments). (C–D) Mean percentages (±SD of total FL cells) FLT3⁻ and FLT3⁺ subsets of (C) CD48⁻ CD150⁻ ST‐HSCs, CD48⁺ CD150⁺ MPPs and (D) CD48⁺ CD150⁻ MPPs and CLPs in E14.5 Rag1 ^+/+ Flt3 ^fl/fl and Rag1 ^cre/+ Flt3 ^fl/fl embryos (n = 8–11 embryos per genotype in 2 experiments). (E) Mean percentages (±SD of total FL cells) of ProB cells in E14.5 Rag1 ^+/+ Flt3 ^fl/fl and Rag1 ^cre/+ Flt3 ^fl/fl embryos (n = 8–11 embryos per genotype in 2 experiments). (F) Mean percentages (±SD of total fetal thymus cells) of early thymic progenitor (ETP) and Double Negative 2 (DN2) cells in E14.5 Rag1 ^+/+ Flt3 ^fl/fl and Rag1 ^cre/+ Flt3 ^fl/fl embryos (n = 4–7 embryos per genotype in 1 experiment). (G–I) Mean percentages (±SD) contribution of CD45.2 cells to (G) LT‐HSCs, CD48⁺ CD150⁻ MPPs and CLPs, (H) ProB cells, PreB cells, and IgM⁺ B cells in bone marrow and (I) ETP, DN2 and Double Negative 3 (DN3) cells in thymus of lethally‐irradiated CD45.1 mice transplanted with 2 × 10⁶ cells unfractionated E14.5 FL cells from Rag1 ^+/+ Flt3 ^fl/fl (CD45.2) or Rag1 ^cre/+ Flt3 ^fl/fl (CD45.2) mice (n = 5 per genotype) together with 2 × 10⁶ cells unfractionated E14.5 FL competitor cells from wild type CD45.1 embryos, analysed 8 weeks post‐transplantation. *P < 0·05; **P < 0·01; ***P < 0·001.

Figure 4

Role of FLT3 in generation of distinct subsets of B cells. (A) Representative fluorescence‐activated cell sorting (FACS)profiles of Follicular B2 cells (CD19⁺ CD93⁻ CD5⁻ CD43⁻ CD23⁺ CD1d⁻), Marginal Zone B cells (MZB: CD19⁺ CD93⁻ CD23‐CD1d⁺) and B1a cells (CD19⁺ CD93⁻ CD5⁺ CD43⁺ CD23⁻ CD1d⁻) in spleen in 12‐week‐old Rag1 ^+/+ Flt3 ^fl/fl and Rag1 ^cre/+ Flt3 ^fl/fl mice (numbers in gates represent percentages of total spleen cells). (B) Mean percentages (±SD of total spleen cells) of follicular B2, MZB and B1a cells in 12‐week‐old Rag1 ^+/+ Flt3 ^fl/fl and Rag1 ^cre/+ Flt3 ^fl/fl mice (n = 6 mice per genotype in 2 experiments). (C) Representative FACS profiles of B2 cells (CD19⁺ CD5⁻ CD43⁻ CD23⁺ CD11b⁻) and B1a cells (CD19⁺ CD5⁺ CD43⁺ CD23⁻) in the peritoneal cavity (PC) of 12‐week‐old Rag1 ^+/+ Flt3 ^fl/fl and Rag1 ^cre/+ Flt3 ^fl/fl mice (numbers in gates represent percentages of total PC cells). (D) Mean percentages (±SD of total PC cells) B2 cells and B1a cells of 12‐week‐old Rag1 ^+/+ Flt3 ^fl/fl and Rag1 ^cre/+ Flt3 ^fl/fl mice (n = 6 mice per genotype in 2 experiments). (E–F) Mean percentages (±SD) contribution of CD45.2 cells to (E) follicular B2 cells, MZB cells and B1a cells in the spleen and to (F) B2 cells and B1a cells in the PC of CD45.1 wild type (WT) mice transplanted with 2 × 10⁶ unfractionated E14.5 FL cells from Rag1 ^+/+ Flt3 ^fl/fl (CD45.2) or Rag1 ^cre/+ Flt3 ^fl/fl (CD45.2) mice (n = 2–5 per genotype) together with 2 × 10⁶ cells unfractionated E14.5 FL WT CD45.1 competitor cells, analysed 8 weeks post‐transplantation.