Propensity of adult lymphoid progenitors to progress to DN2/3 stage thymocytes with Notch receptor ligation

Abstract

Notch family receptors control critical events in the production and replenishment of specialized cells in the immune system. However, it is unclear whether Notch signaling regulates abrupt binary lineage choices in homogeneous progenitors or has more gradual influence over multiple aspects of the process. A recently developed coculture system with Delta 1-transduced stromal cells is being extensively used to address such fundamental questions. Different from fetal progenitors, multiple types of adult marrow cells expanded indefinitely in murine Delta-like 1-transduced OP9 cell cocultures, progressed to a DN2/DN3 thymocyte stage, and slowly produced TCR(+) and NK cells. Long-term cultured cells of this kind retained some potential for T lymphopoiesis in vivo. Adult marrow progressed through double-positive and single-positive stages only when IL-7 concentrations were low and passages were infrequent. Lin(-)c-Kit(low)GFP(+)IL-7Ralpha(+/-) prolymphocytes were the most efficient of adult bone marrow cells in short-term cultures, but the assay does not necessarily reflect cells normally responsible for replenishing the adult thymus. Although marrow-derived progenitors with Ig D(H)-J(H) rearrangements acquired T lineage characteristics in this model, that was not the case for more B committed cells with V(H)-D(H)J(H) rearrangement products.

Figure 1. Fetal/adult disparity in T lymphopoietic potential in OP9-DL1 co-cultures

A. E14 Fetal liver progenitors and adult bone marrow Lin⁻RAG1/GFP⁺c-Kit^loIL-7Rα⁺ cells were cultured with 5 ng/ml IL-7 and 5 ng/Flt3-L on OP9-DL1 for fourteen days. Representative plots are shown for cultured cells stained for TCRβ and TCRγδ. Similar results were obtained in experiments with 8 other fractions of adult bone marrow. B. Percentages of TCRβ⁺ and TCRγδ⁺ cells present at the end of cultures are shown along with yields per input progenitor. The values are means of duplicate cultures ± S.D. in this typical experiment.

Figure 2. Delta1 mediated Notch signals inhibit B and myeloid lineage differentiation from adult progenitors in short term (9 days) cultures

A. Six subsets of adult progenitors were sorted according to expression of RAG1/GFP, c-Kit and Sca-1 after lineage depletion before culture on OP9-Vector (top row) and OP9-DL1 (bottom row) for 9 days (5 ng/ml IL-7 and 5 ng/ml Flt-L). Recovered cells were then stained for CD11c/Mac-1 and CD19 and results from four of the major populations are illustrated here. B. Yields of B lineage cells (left panel) and myeloid cells (right panel) per input progenitor were calculated for all six groups of cultures. Open bars represent cells recovered from OP9-Vector cultures, while shaded bars correspond to OP9-DL1 cultures. The values are means of duplicate cultures ± S.D. in this representative of three similar experiments.

Figure 3. A range of adult bone marrow progenitors representing various stages of differentiation expand under the influence of Delta 1

A. Six fractions of bone marrow progenitors were sorted on the basis of RAG1/GFP expression and cultured for 5 weeks on stromal cells plus 5 ng/ml IL-7 and 5 ng/ml of FL. The yields per input were calculated for each weekly expansion. B. Light scatter properties of cultured cells are compared to freshly isolated lymphocytes (left panel). May-Grunwald-Giemsa staining of a cytocentrifuge preparation is also shown (right panel). C. Representative flow cytometry results are shown for Lin⁻ c-Kit^lo IL-7Rα⁺ marrow cells expanded on OP9-DL1 for four weeks and then stained for progenitor and lineage specific markers. Identical results were obtained when eight other fractions of bone marrow were cultured in this way. D. Expanded cells were first stained for surface lineage markers (VCAM-1 for stromal cells, DX-5 for NK cells, CD3ε for T cells, Mac-1 for myeloid cells), along with CD25 and CD44. They were then permeabilized and stained for cytoplasmic T lineage stage-specific proteins. Quadrants were set based on isotype matched controls (ISO) and positive controls using bone marrow and thymocytes (data not shown). Cultured cells were gated for VCAM-1⁻ DX-5⁻ CD3ε⁻ Mac-1⁻ cells, Isotype control stainings for cytoplasmic (cyt) TdT (Rabbit IgG and FITCGoat anti-rabbit IgG), cyt CD3ε (FITC IgG1), cyt TCRβ (PE IgG2) were also prepared with cultured cells and are shown in the upper panel. Cytoplasmic TdT, CD3ε and TCRβ are shown in the lower panel

Figure 4. Expanded cells have properties similar to DN2/DN3 stage thymocytes

Lin⁻Sca-1⁺c-Kit⁺ cells were prepared by sorting cells recovered from 28 day cultures of Lin⁻ c-Kit^lo IL-7Rα⁺ bone marrow progenitors. This eliminated the small numbers of stromal, T and NK cells present at that time Highly enriched lymphocytes from the long term cultures were then compared to freshly sorted CD3ε⁻ CD4⁻ CD8⁻ NK1.1⁻ Mac-1⁻ CD19⁻ adult thymocytes that were further resolved into DN subsets according to expression patterns of CD44 and CD25. RT-PCR analyses were performed to detect transcripts that vary in a stage dependent fashion (22). The plots depict values obtained with freshly isolated thymocytes and the stars show results obtained with cultured cells.

Figure 5. Expanded cells retain the potential to become T cells and natural killer cells

A. Long-term (28 days) cultured lymphocytes derived from the Lin⁻ c-Kit^lo IL-7Rα⁺ subset of marrow were purified by sorting for Lin⁻c-Kit⁺Sca-1⁺ cells. They were then re-cultured on OP9-DL1 for another week before staining for NK1.1, Mac-1, TCRβ, TCRγδ, c-Kit and Sca-1 antigens. B. A total of 10⁵ sorted c-Kit⁺Sca-1⁺ cells were mixed with the same amount of host type, whole bone marrow cells and intravenously injected into lethally irradiated Ly5.1 mice. Twenty-eight days after transplantation, thymocytes from non-transplanted host and transplanted animals were collected and assayed for thymus reconstitution. The left panels show gating for donor Ly5.2⁺ lymphocytes, while the middle and right panels illustrate their properties.

Figure 6. T lineage differentiation from marrow progenitors is influenced by IL-7 concentration and subculture

A. Lin⁻c-Kit^loIL-7Rα⁺ pro-lymphocytes were cultured first on OP9-DL1 with 5 ng/ml IL-7 and 5 ng/ml Flt3-Lfor two weeks. They were then subcultured to fresh stromal cells with the indicated concentrations of IL-7 plus Flt3-L and subcultured again one week later. After a total of four weeks of culture, cells were harvested and stained for flow cytometry analysis. B. HSC, LSP, ELP and CLP fractions were cultured on OP9-DL1 with 1 ng/ml IL-7 and 5 ng/ml Flt3-L for eight days. They were then subcultured under the same conditions for another 16 days before harvest and staining for CD4 and CD8. C. Total yields of CD4⁺ CD8⁺ (DP) cells were calculated per input HSC, LSP, ELP or CLP in cultures that were maintained for the whole time on OP9-DL1 with Flt3-L and either 1 ng/ml IL-7 or 0.2 ng/ml IL-7. After the first eight days, all groups were subcultured with the original conditions for another 16 days before harvest and analysis. The absolute numbers of DP cells per sorted progenitor were calculated by multiplying yields of the first culture interval with that of the second interval and the frequency of DP cells. D. HSC were cultured on OP9-DL1 with Flt3-L and 1 ng/ml IL-7 for 8 days, and then subcultured in two groups . One group (shown by open bars) was harvested and stained on day 27, The parallel group (closed bars) was maintained in the same way, but passaged one additional time on day 24. Numbers of total cells, CD4⁻ CD8⁻ ( DN) and DP cells are shown.

Figure 7. Long-term OP9-DL1 co-cultures selectively expand bone marrow cells that lack immunoglobulin V_H-D_HJ_H rearrangements

B220⁺IgM⁻ B progenitors from adult bone marrow were sorted into CD43⁺CD19⁻, CD43⁺CD19⁺and CD43⁻CD19⁺ fractions before culture for 4 weeks. CD19⁻c-Kit⁺Sca-1⁺ cells were then sorted to exclude differentiated and stromal cells. Genomic DNA was extracted and D_H-J_H (upper panel) and V_H-D_HJ_H immunoglobulin gene rearrangement were tested and compared with those prepared from initiating progenitors. Germline and rearrangement products are labeled, while two artifact bands are designated with asterisks.