A developing picture of lymphopoiesis in bone marrow

Abstract

The earliest progenitors of lymphocytes are extremely rare and typically present among very complex populations of hematopoietic cells. Additionally, it is difficult to know how cells with any given set of characteristics are developmentally related to stem cells and maturing lymphoid precursors. However, it is now possible to divide bone marrow into progressively smaller fractions and exploit well-defined culture systems to determine which ones contain cells that can turn into lymphocytes. Analysis of steroid hormone sensitive cells and use of two-step cultures is providing additional information about the most likely differentiation pathways for B and natural killer cell lineage lymphocytes. A newly identified category of early lymphoid progenitors can now be sorted to high purity from RAG1/GFP knock in mice. Furthermore, the same experimental model makes it possible to image lymphoid progenitors in fetal and adult hematopoietic tissues.

Fig. 1. Localization of B and NK lineage progenitor activity in bone marrow

The small subset that lacks markers associated with differentiated cells (Lin⁻) can be subdivided into many fractions. Here we depict the relative sizes of the populations (width) and yields (height) of B or NK lineage cells that were obtained when cells of each type were placed in defined short term cultures.

Fig. 2. The main routes to B and NK cell lineages

Lin⁻ c-kit^Lo pro-lymphocytes were cultured 3 days with SCF, FL and IL-7 under defined conditions and harvested. The CD19⁻ Mac-1⁻ fraction was then sorted according to the expression of CD122 and CD45R as shown. Each of the resulting four subsets was then cultured for an additional 4 days with SCF, FL, IL-7 and IL-15. The figures given in black represent numbers of cells obtained in each subset divided by the starting cell numbers (yield). Yields of B and NK cells after the 2^nd step cultures (shown in green and orange, respectively) were also calculated in the same way but multiplied by the yields in the representative 1st step culture to show how many B or NK cells were generated from one pro-lymphocyte through each pathway. When β-estradiol was included in the 2nd step cultures, differentiation of B cells (red arrows) was more significantly reduced than formation of NK cells.

Fig. 3. Early stages of NK lineage differentiation

The acquisition and loss of markers by differentiating NK cell progenitors is shown in this summary of our culture studies.

Fig. 4. What type of cell normally seeds the thymus?

Three types of bone marrow cells are known to be able to colonize the thymus under experimental circumstances. Early lymphoid progenitors in marrow most closely resemble the least differentiated, triple negative (TN1) thymocytes and both are steroid hormone sensitive. The Pax-5 transcription factor supports cells committed to the B lineage, while signals from Notch receptors are required for T lymphopoiesis and suppress the B lineage option.

Fig. 5. Remarkable distribution of lymphoid progenitors in tissues

GFP⁺ cells (green) are shown along with CD11_b/Mac-1+ cells (red) in 15 day fetal liver (A) or 16 day bone marrow (B) from heterozygous RAG-1/GFP knock in mice. These images of stained viable preparations were recorded as single optical sections with a Zeiss confocal microscope.

Fig. 6. Expression of RAG-1 by lymphoid progenitors in the fetal thymus

Bright field (A, C) and fluorescent (B, D) images were made of a 13 day heterozygous RAG-1/GFP knock in embryo. White arrowheads indicate the fetal thymus, while the heart (H) and left arm (LA) are also marked (panels A and B). The thymus and surrounding connective tissue (CT) was then dissected to illustrate the rich population of GFP⁺ cells (C, D). Corresponding FACS analysis of similar lobes revealed that approximately 80% of cells expressed GFP at that stage (data not shown).

Fig. 7. Lymphoid progenitors are dispersed in adult bone marrow

Frozen sections of femoral bones were prepared from heterozygous (A) or homozygous (B) RAG-1/GFP knock in mice. Before sacrifice, TRITC-labeled BS-1 lectin was injected to provide red staining of the vasculature, and asterisks indicate the presence of the central sinus in each section. The photographs represent 12–22 adjacent confocal optical sections, which were assembled to form the final montage. Adult marrow plugs were then prepared, stained and examined as viable preparations. Dispersed RAG-1 expressing cells (green) are contrasted with those stained red for CD19 (C) or CD11_b/Mac-1 (D). These images were also recorded with a Zeiss confocal microscope.

Fig. 7. Lymphoid progenitors are dispersed in adult bone marrow

Frozen sections of femoral bones were prepared from heterozygous (A) or homozygous (B) RAG-1/GFP knock in mice. Before sacrifice, TRITC-labeled BS-1 lectin was injected to provide red staining of the vasculature, and asterisks indicate the presence of the central sinus in each section. The photographs represent 12–22 adjacent confocal optical sections, which were assembled to form the final montage. Adult marrow plugs were then prepared, stained and examined as viable preparations. Dispersed RAG-1 expressing cells (green) are contrasted with those stained red for CD19 (C) or CD11_b/Mac-1 (D). These images were also recorded with a Zeiss confocal microscope.