Identification of an early T cell progenitor for a pathway of T cell maturation in the bone marrow

Abstract

We have identified a rare ( approximately 0.05-0.1%) population of cells (Thy-1(hi)CD16(+)CD44(hi)CD2(-)TCRalphabeta(-)B220(-)M ac-1(-)NK1. 1(-)) in the adult mouse bone marrow that generates CD4(+) and CD8(+) TCRalphabeta(+) T cells after tissue culture for 48 hr in the presence of Ly5 congenic marrow cells. The essential stages in the maturation of the progenitors were determined; the stages included an early transition from CD2(-)CD16(+)CD44(hi)TCRalphabeta(-) to CD2(+)CD16(int/-)CD44(int/-)TCRalphabeta(-) cells, and a later transition to CD4(+)CD8(+)TCRalphabeta(+) double-positive T cells that rapidly generate the CD4(+) and CD8(+) single-positive T cells. The maturation of the progenitors is almost completely arrested at the CD2(+)TCRalphabeta(-) stage by the presence of mature T cells at the initiation of cultures. This alternate pathway is supported by the marrow microenvironment; it recapitulates critical intermediary steps in intrathymic T cell maturation.

Figure 1

Identification of Thy-1^hiCD2⁻Lin⁻ T cell progenitors in the adult bone marrow. C57BL/6 Ly5.2 bone marrow cells were enriched for Thy-1⁺ cells with immunomagnetic beads, and cells were subsequently stained for Thy-1 with one fluorochrome; TCRαβ, B220, Mac-1, and NK1.1 with a second fluorochrome; and CD2 with a third fluorochrome. Analysis of Thy-1 vs. B220, Mac-1, TCRαβ, and NK1.1 (A); the box encloses Thy-1^hiTCRαβ⁻B220⁻Mac-1⁻NK1.1⁻ (Thy-1^hiLin⁻) cells. The latter cells were gated and reanalyzed for Thy-1 vs. CD2 (B); boxes 1 and 2 enclose Thy-1^hiCD2⁻Lin⁻ and Thy-1^hiCD2⁺Lin⁻ cells, respectively. Progenitor activity of sorted Thy-1^hiCD2⁻Lin⁻ Ly5.2 marrow cells was tested by incubating 5 × 10⁴ cells with 4 × 10⁶ Ly5.1 T cell-depleted marrow cells for 48 hr. The cultured cells were analyzed for CD4 and CD8 markers (C) or TCRαβ vs. Ly5.2 markers (D). Boxes enclose Ly5.2⁺ T cells. In a repeat experiment, the sorted Thy-1^hiCD2⁻Lin⁻ cells were cocultured with T cell-depleted Ly5.1 marrow cells, and cells were harvested at either 24 hr (E) or 48 hr (F). Gated Ly5.2⁺ cells harvested from the cultures were stained for TCRαβ vs. CD2, and boxes enclose CD2⁺TCRαβ⁻ (E) or CD2⁺TCRαβ⁺ (F) cells. The sorted Thy-1^hiCD2⁻Lin⁻ cells were also cocultured with whole instead of T cell-depleted Ly5.1 marrow cells, and cultures were stained for Ly5.2 vs. CD2 after 24 and 48 hr in G and H, respectively. Gated Ly5.2 cells from the latter cultures were stained for TCRαβ vs. CD2 at 24 and 48 hr (I and J, respectively), and boxes enclose CD2⁺TCRαβ⁺ cells. Expression of CD44 on sorted Thy-1^hiCD2⁻Lin⁻ cells before culture (K) is compared with that after coculture with T cell-depleted Ly5.1 marrow cells for 24 (L) or 48 hr (M). Analyses of CD44 vs. CD2 on gated Ly5.2 cells are shown, and boxes enclose CD44^hiCD2⁺ cells. Each pair of profiles is from a separate representative experiment of at least four replicate experiments.

Figure 2

Progenitor activity of Thy-1^hiCD16⁺CD2⁻Lin⁻ sorted bone marrow cells. Enriched marrow cells from Ly5.2 mice were stained for Thy-1 vs. TCRαβ, B220, Mac-1, NK1.1, and CD2 vs. CD16 with three fluorochromes. The latter cells were analyzed for Thy-1 vs. TCRαβ, B220, Mac-1, NK1.1, and CD2 (A), and the box encloses Thy-1^hiCD2⁻Lin⁻ cells. Cells within the box in A were analyzed for Thy-1 vs. CD16 (B), and the box in B encloses Thy-1^hiCD16⁺ cells. Single-color analysis of the intensity of staining of CD16 is also shown in the open areas of E and F also. The Thy-1^hiCD16⁺ sorted cells (5 × 10⁴) were cocultured with 4 × 10⁶ T cell-depleted Ly5.1 marrow cells for 48 hr, and the newly generated CD4⁺ or CD8⁺ TCRαβ⁺Ly5.2 cells are enclosed in boxes in C and D. Cells were harvested also at 24 and 48 hr, gated on the Ly5.2⁺ subset, and analyzed for CD16 staining. The intensity of staining of the sorted cells before culture was compared with that after 24 hr (E) or after 48 hr (F). Open area shows staining before culture, and shaded area shows staining after culture.

Figure 3

CD4⁺CD8⁺ progeny of cultured T cell progenitors. Sorted Thy-1^hiLin⁻ Ly5.2 marrow cells (2 × 10⁴) were cultured with T cell-depleted Ly5.1 marrow cells (4 × 10⁶), and cells were harvested at 44 and 48 hr (A, C, and B, D, respectively). At each time, Ly5.2⁺ cells were gated and analyzed for CD4/CD8 vs. TCRαβ (A and B) or CD4 vs. CD8 markers (C and D). In A and B, boxes enclose CD4⁺ and/or CD8⁺ TCRαβ⁺ T cells. In C and D, boxes enclose double-positive (CD4⁺CD8⁺) T cells.

Figure 4

Diagram of proposed alternative pathway of T cell maturation in the adult bone marrow. Thy-1^hiTCRαβ⁻CD2⁻CD16^hi/intCD44^hi progenitors rapidly mature into Thy-1^hiTCRαβ⁻CD2⁺CD16^int/−CD44^int/− cells in association with TCR β chain gene rearrangement. Subsequently, TCR α chain gene rearrangement results in the development of CD4⁺CD8⁺ (double-positive) intermediary cells. Single-positive CD4⁺CD8⁻ and CD4⁻CD8⁺ T cells are derived from the double-positive cells during processes of positive and negative selection. The transition from Thy-1^hiTCRαβ⁻CD2⁺ to CD4⁺CD8⁺ intermediary cells is arrested in the presence of mature T cells (feedback inhibition).