Flk2+ common lymphoid progenitors possess equivalent differentiation potential for the B and T lineages

Abstract

Mature blood cells develop from multipotent hematopoietic stem cells through a series of sequential intermediates in which the developmental potential for particular blood lineages is progressively extinguished. We previously reported the identification of one of these developmental intermediates, the common lymphoid progenitor (CLP), which can give rise to T cells, B cells, dendritic cells (DCs), and natural killer cells (NKs), but lacks myeloid and erythroid potential. Recently, several studies have suggested that the T-cell and DC potential of CLP is limited or absent, and/or that CLP contains significant myeloid potential. Here, we show that the originally identified CLP population can be divided into functionally distinct subsets based on the expression of the tyrosine kinase receptor, Flk2. The Flk2(+) subset contains robust in vivo and in vitro T-cell, B-cell, DC, and NK potential, but lacks myeloid potential and, therefore, represents an oligopotent, lymphoid-restricted progenitor. This population of cells does not appear to be B cell-biased and robustly reconstitutes both B and T lineages in vivo, consistent with its being a physiologic progenitor of both of these subsets. Thus, Flk2 expression defines a homogeneous, readily obtainable subset of bone marrow CLP that is completely lymphoid-committed and can differentiate equivalently well into both B and T lineages.

Figure 1

CLPs can be divided into Flk2⁺ and Flk2⁻ fractions. (A) Strategy originally used to isolate CLPs by first gating on Lin⁻IL-7Rα⁺ cells (left panel) and subsequently on c-Kit^intSca-1^int cells (middle panel) reveals heterogeneous expression of Flk2 (right panel). (B) Changing the gating strategy by first gating on lineage negative cells (not shown) reveals 2 distinct IL-7Rα⁺ populations (left panel). The Flk2⁺ fraction shows homogeneous expression of c-Kit and Sca-1 (middle panel) whereas the Flk2⁻ fraction shows a more heterogeneous expression pattern (right panel). Numbers on plots are percentages of gated cells. (C) Surface expression of CD27, CD62L and CD93/AA4.1 on CLP^F (bold lines) in comparison to MPP (solid thin lines), or isotype controls (dashed lines). (D) FACS plot overlay of CLP^F (black dot plot) and lineage negative (excluding B220, gray contour plot) populations in C57Bl/6 BM. AA4.1^hi cells are predominantly B220⁺.

Figure 2

Only CLP^F have multilymphoid potential in vivo. (A-D) FACS analysis of spleens 3 weeks after intravenous transplantation of 10³ CLP^F or CLP^F− into Ly5 congenic, sublethally (400 rad) irradiated hosts and control mice that received sham transplants. (A) Host cells are Ly5.1⁺ whereas donor-derived cells are Ly5.2⁺. (B,C) FACS plots showing T cell (TCRαβ⁺), B cell (CD19⁺), or NK cell (Nk1.1⁺) cell reconstitution of the spleen after pregating on either Ly5.1⁺ host cells (left panels) or Ly5.2⁺ donor-derived cells (middle and right panels). Both Flk2⁻ and Flk2⁺ CLPs gave rise to B cells but donor-derived T cells and NK cells were only detectable in CLP^F reconstituted mice. (D) FACS plots showing absence of myeloid cells (Mac-1⁺Gr-1⁺) from donor-derived Flk2⁻ fraction cells or CLP^F. (E) Thymi from irradiated (400 rads) host Ly5.2⁺ animals 3 weeks after intravenous (i.v.) transplantation of 10³ CLP^F or Flk2⁻ fraction cells in comparison to animals that received sham transplants (PBS) were analyzed for donor derived Ly5.1⁺ progeny. 3 weeks after transplantation CLP^F derived thymocytes were responsible for roughly two-thirds of the thymus cellularity in mice that received transplants whereas the Flk2⁻ fraction yielded no thymic cells. (F) Thymic reconstitution after intrathymic injection of 10³ Ly5.1⁺ CLP^F or Flk2⁻ fraction cells into irradiated (400 rads) Ly5.2⁺ recipient mice. Numbers on plots are percentages of gated cells.

Figure 3

Kinetics of CLP^F-driven T cell reconstitution. (A) Kinetic analyses of B-cell (squares) and T-cell (diamonds) reconstitution of spleen (left panel) and blood (right panel) of sublethally irradiated mice that received transplants of 10³ CLP^F (black) or Flk2⁻ fraction cells (white). Results shown are mean values of 3 to 5 animals analyzed per time point. For blood analysis, the same animals were bled periodically for 10 weeks. (B) FACS plots illustrating the CD4/CD8 profile of host (top row) and CLP^F derived donor cells (bottom row) as well as reconstitution with thymic B cells (B220⁺), γδ T cells, and NK cells showing the lymphoid potential of CLP^Fs. (C) FACS plots illustrating the T-lineage development of intravenously injected CLP^F in the thymus, from 1 to 5 weeks. Detailed analysis of CD4/CD8 development (top row), and within the CD4/CD8 double negative fraction, CD44 and CD25 development (middle row) or CD25 and c-Kit development (bottom row), after previously gating on lineage negative cells. Shown are representative FACS plots from 3 mice that received transplants per time point. (D) Thymic reconstitution 6 days after intrathymic (i.t.) injection transfer of 10³ CLP^F into congenic unirradiated mice. Events shown are gated on lineage negative donor-derived cells. FACS analysis reveals generation of c-Kit^hi DN1 (CD25⁻c-Kit⁺) and c-Kit^hi DN2(CD25⁺c-Kit⁺) directly derived from CLP^F. Numbers on plots are percentages of gated cells.

Figure 4

CLP^F produce Mac-1⁺ B1b cells and Mac-1⁺CD11c⁺ dendritic cells, but not myeloid cells. (A) Congenic sublethally irradiated mice received transplants of 10³ CLP^F cells. Three weeks after the transfer spleens were analyzed for donor-derived cells. FACS plots shown are previously gated on recipient (left) or donor-derived cells (right) and are B220⁻CD19⁻Gr-1⁻. Percentage of Mac-1⁺CD11c⁺ dendritic cells are shown. (B) Analysis of Mac-1⁺ B-1b cell differentiation from CLP^F in the spleen 4 weeks after transplantation. Cells are pregated on CD5⁻, and the percentage of Mac-1⁺IgD⁻ B-1b cells are shown. Numbers on plots are percentages of gated cells. (C) Methylcellulose myeloid differentiation assay. c-Kit⁺Lin⁻Sca-1⁺Flk2⁺ (MPP^F) cells, CLP^F, and Flk2⁻ fraction cells (F⁻ frac.) from bone marrow were double sorted and plated out at 100 cells (MPP^F) and 500 to 1000 cells (CLP^F and F⁻ frac.) in triplicates in methyl cellulose medium containing SCF, Flt3L, GM-CSF, Epo, Tpo, IL-3, IL-6, and IL-11. The average number of colonies yielded and their lineage content as colony forming units (CFU) from 2 combined experiments are shown. G, granulocyte; E, erythrocyte; M, macrophage; B, B cell; and Σ, sum.

Figure 5

CLP^F clonally give rise to B and T cells. (A) B-cell (top panel) and T-cell (bottom panel) progenitor frequency of CLP^F was assessed by culturing them on either S17 (for B cells) and delta-like 1 expressing OP9 (for T cells) stroma cells in the presence of IL-7, SCF, and Flt3L for 10 days in 96-well plates at densities between 0 and 8 cells per well. The left panel shows the percentage of failure of detection of either CD19⁺ or Thy1.1⁺ progeny by FACS. The middle panel shows the number of progeny cells obtained from a single CLP^F in these assays with the black bar marking the average burst size. Right panel shows a representative CD19/Thy1.1 FACS plot of a B- and T-cell culture. Horizontal bars in the middle panels represent means. Numbers on right plots are percentages of gated cells. (B) Limiting dilution analysis of donor cells after transfer of CLP^F into sublethally irradiated hosts. For B cells, CLP^F were intravenously injected and spleens analyzed at 4 weeks. For T cells, CLP^F were intrathymically injected and thymi analyzed at 4 days after injection. Three to 5 mice per cell dose were analyzed and numbers shown within the plots represent the calculated limiting number. (C) Results of a clonal assay to simultaneously detect all 4 lymphoid lineages from a single cell (for experimental design and technical details, see Figure S3). Positive clones are symbolized by black circles and a negative result by white circles. Of the 17 clones analyzed, each derived from a single CLP^F cell, 8 of 17 gave rise to B cells and 12 of 17 to T cells when cultured under appropriate conditions. Of these positive clones a total of 35% showed simultaneous development of T and B cells, demonstrating the bipotential of CLP^F. Furthermore, 7 clones of the 17 underwent 3 or more cell divisions and therefore could also be analyzed for NK, DC, and myeloid potential. Whereas 4 of 7 showed NK cell and 6 of 7 DC potential, none of the clones generated any myeloid cells (0/7). Of the 7 clones analyzed for all 4 lymphoid lineages 2 (28%) simultaneously readout all 4 lymphoid lineages, proving that a single CLP^F can possess the full lymphoid potential.