An alternate pathway for T cell development supported by the bone marrow microenvironment: recapitulation of thymic maturation

Abstract

In the principal pathway of alpha/beta T cell maturation, T cell precursors from the bone marrow migrate to the thymus and proceed through several well-characterized developmental stages into mature CD4+ and CD8+ T cells. This study demonstrates an alternative pathway in which the bone marrow microenvironment also supports the differentiation of T cell precursors into CD4+ and CD8+ T cells. The marrow pathway recapitulates developmental stages of thymic maturation including a CD4+CD8+ intermediary cell and positive and negative selection, and is strongly inhibited by the presence of mature T cells. The contribution of the marrow pathway in vivo requires further study in mice with normal and deficient thymic or immune function.

Figure 1

CD4, CD8, and TCR-α/β profiles of bone marrow cells before and after culture. Freshly isolated bone marrow cells from C57BL/Ka (A) or BALB/c (F) mice were depleted of cells expressing CD4, CD8, and TCR-α/β markers using the gating boxes in the lower left and reanalyzed in B and G. The depleted marrow cells were cultured for 48 h and the bright TCR-α/β⁺ cells that developed are enclosed in the boxes in C and H. The depleted cultured cells were analyzed for CD4 versus CD8 markers in D and I. Analyses of non–T cell–depleted marrow cells after culture are shown in E and J.

Figure 2

Kinetics of development of CD4⁺CD8⁺, CD4⁺, and CD8⁺ T cells during marrow cultures. C57BL/Ka marrow cells depleted of α/β T cells (A) were cultured, and the CD4/CD8 versus TCR-α/β profiles are shown at 36 (B), 40 (C), 44 (D), and 48 (E) h. Boxes enclose the newly generated T cells. Analyses of CD4 versus CD8 markers are shown in the same cultures for 40 (F), 44 (G), and 48 (H) h, and boxes enclose CD4⁺CD8⁺ T cells. A repeat experiment is shown at 40 (I), 44 (J), and 48 (K) h.

Figure 3

Changes in the expression of CD69 on T cell subsets during marrow cultures. C57BL/Ka marrow cells were depleted of α/β T cells and cultured for 40 (A), 44 (B), and 48 (C) h as was shown in Fig. 2, F–H. Gated CD4⁺CD8^med or CD4⁺CD8⁻ cells (boxes 1, 3, and 5) or CD4⁺ CD8⁺ cells (boxes 2 and 4) or CD4⁻CD8⁺ cells (box 6) were analyzed for the expression of CD69. The profiles and percentages of CD69⁺ cells in each box are shown in corresponding panels 1–6. Shaded areas show the CD69⁺ cells.

Figure 4

Surface markers on CD4⁺ and CD8⁺ T cells harvested at 48 h from C57BL/Ka marrow cultures. After gating of α/β⁺ cells, single color profiles for CD3, Thy1.2, CD5, CD2, CD62L, and CD44 receptors are shown in A–F (solid lines). Normal spleen T cell profiles are shown for comparison in C, E, and F (broken lines). Two-color profiles for CD44 versus CD62L receptors on gated normal spleen cells and on gated marrow-cultured cells are compared in G and H, respectively. In addition, the profile for CD44 versus CD16 on gated marrow cultured cells is shown in I. A two-color profile for CD8α versus CD8β receptors on α/β⁺-gated marrow cultured cells is shown in J. One-color profiles for CD25, NK1.1, HSA, cKit, B220, Gr-1, Mac-1, and Ter119 markers on α/β⁺-gated marrow-cultured cells are shown in K–R.

Figure 5

Add back of marrow T cells to T cell–depleted marrow cultures. Freshly isolated marrow cells (A) were sorted using the gating boxes 1 and 2, and reanalysis of T-depleted cells in box 1 are shown in B. After 48 h of culture of cells in B, newly formed T cells are enclosed in the box in C. Culture of combined cells from boxes 1 and 2 are shown in D. Marrow cells were also sorted for CD4⁺ or CD8⁺ TCR-α/β⁺ (E, box 2), or CD4⁻CD8⁻ TCR-α/β⁺ (E, box 3) T cells. F and G show T cell-depleted marrow cells before and after culture. H and I show T cell-depleted marrow cells cultured in combination with cells from E, box 2 or 3, respectively. Marrow cells were also gated for NK1.1⁻ TCR-α/β⁻ (  J, box 1), NK1.1⁺ TCR-α/β⁺ (  J, box 2), and NK1.1⁻TCR-α/β⁺ (  J, box 3) subsets, respectively. Sorted NK1.1⁻TCR-α/β⁺ (K) cells were cultured alone (L), or with cells from box 2 (M) or box 3 (N).

Figure 6

Addition of lymph node T cells or Thy1.2⁺α/β⁻ marrow cells to marrow cultures. Freshly isolated marrow cells from Ly5.1 congenic mice were depleted of T cells by sorting (A and B), and profiles of CD4/CD8 versus Ly5.2 markers are shown after culture (C). The Ly5.1 versus 5.2 profile of lymph node cells from Ly5.2 congenic mice after gating on TCR-α/β⁺ cells is shown in D. Gated TCR-α/β⁺ cells from D were added to T-depleted marrow cells (B) and cultured for 48 h (E). CD4⁺ and CD8⁺ T cells that are Ly5.2⁻ or Ly5.2⁺ are enclosed in left and right boxes, respectively. The profile of TCR-α/β versus Thy1.2 markers on C57BL/Ka marrow is shown in F, and sorted Thy1.2⁻ TCR-α/β⁻ cells were reanalyzed (G). Cells from G were cultured alone and analyzed (H) or cultured in combination with Thy1.2⁺ TCR-α/β⁻ cells (I).