Identification of a human B-cell/myeloid common progenitor by the absence of CXCR4

Abstract

CXCR4 is a chemokine receptor required for hematopoietic stem cell engraftment and B-cell development. This study found that a small fraction of primitive CD34(+)/CD19(+) B-cell progenitors do not express CXCR4. These CD34(+)/CD19(+)/CXCR4(-) cells were also remarkable for the relative lack of primitive myeloid or lymphoid surface markers. When placed in B-lymphocyte culture conditions these cells matured to express CXCR4 and other surface antigens characteristic of B cells. Surprisingly, when placed in a myeloid culture environment, the CXCR4(-) B-cell progenitors could differentiate into granulocyte, macrophage, and erythroid cells at a high frequency. These data define a novel B-cell/myeloid common progenitor (termed the BMP) and imply a less restrictive pathway of myeloid versus lymphoid development than previously postulated.

Figure 1.

Flow cytometric analysis of CXCR4 expression in B-cell progenitor maturation. (A) Representative flow histograms of CD34⁺/CD19⁺ B-cell progenitor (gate R2) and CD34^-/CXCR4⁺ later B cells (gate R3) sorted and analyzed for CCXCR4 and CCR7 expression. There was a surprisingly large fraction of CD34⁺/CD19⁺ B-cell progenitors that did not express the chemokine receptor CXCR4. More mature CD34^-/CD19⁺ BM B cells express CXCR4 at a high level. The percentage of each subpopulation relative to the total number of cells in gates R2 or R3 is shown. (B) Representative flow histograms of CD34⁺/CD19⁺/CXCR4^- cells cultured for 3 days in B-lymphoid conditions showing that the majority of cells lose expression of CD34 and a gain expression of CXCR4. The percentage of each subpopulation relative to the total number of cells is shown. (C) The majority of CD34⁺/CD19⁺/CXCR4^- cells cultured in B-lymphoid conditions gain expression of κ/λ light chains. Text data are the average of 3 distinct experiments; this figure shows a representative histogram.

Figure 2.

Flow cytometric characterization of the expression of other primitive hematopoietic surface markers on CD34⁺/CD19⁺/CXCR4^- cells. Low-density BM cells were stained for 6-color immunofluorescence as described in “Materials and methods.” Representative histograms of the flow cytometric phenotypic characterization of the CD34⁺/CD19⁺/CXCR4^- cells are shown in dot plots A-E. Refer to Table 1 for the averaged fraction of the CD34⁺/CD19⁺/CXCR4^- cells expressing combinations of primitive hematopoietic surface antigen. Dot plot A depicts CD34⁺CD19⁺ cells within R2. Cells contained in R2 were then analyzed for the expression of CXCR4 (CD184 in the figure) and CD133 (VEGFr2) in dot plot B. Essentially no CD34⁺/CD19⁺/CXCR4^- cells were CD133⁺. CD34⁺/CD19⁺/CXCR4^- cells were also analyzed for the expression of CD16 and CD90 (dot plot C). Whereas a small fraction of these cells expressed CD16, very few expressed CD90. Dot plot D depicts the expression of CD33 fraction of CD34⁺/CD19⁺/CXCR4^- cells. Dot plot E shows the expression of CD127 and CD117 in the CD34⁺/CD19⁺/CXCR4^- population. CD117 analysis found a significant increase in the mean fluorescence intensity, but the gated fraction of cells expressing CD177 is still small, as shown in Table 1.

Figure 3.

B-cell progenitors in myeloid colony formation assays. (A) Reanalyzing the sorted CD34⁺/CD19⁺ cells in each experiment for the presence of any CD34⁺/CD19^- cells that might form myeloid colonies showed that 100% of sorted cells expressed CD19. The percentage of each subpopulation relative to the total number of cells is shown. (B) Myeloid colony formation per 1000 cells. A significant fraction of CD34⁺/CD19⁺/CXCR4^- BM cells was able to form myeloid colonies containing granulocytes, macrophages, or erythrocytes (top) as compared to CD34⁺/CD19⁺/CXCR4⁺ cells (middle) and total CD34⁺ cells (bottom). □ indicates granulocyte-macrophage colony-forming units (CFU-GMs); , BFU-E; and ▪, CFU-GEMM. Note the different scales showing the colony numbers between the graphs; n = 3 each performed in triplicate. Error bars indicate standard error (SE).

Figure 4.

Two models of hematopoietic stem cell development consistent with the data presented here. In stem cell differentiation model A, the hematopoietic stem cell could form 3 common progenitors, the common myeloid progenitor, the common lymphoid progenitor, and the B-cell/myeloid common progenitor described here. In stem cell differentiation model B, the stem cell differentiates to only the common lymphoid progenitor; however, along with differentiating into Band T cells, the CLP can give rise to a BMP cell, which can differentiate into B cells and myeloid cells. HSC indicates hematopoietic stem cell; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; BMP, B-cell/myeloid progenitor.