Multiple extrathymic precursors contribute to T-cell development with different kinetics

Abstract

T-cell development in the thymus depends on continuous supply of T-cell progenitors from bone marrow (BM). Several extrathymic candidate progenitors have been described that range from multipotent cells to lymphoid cell committed progenitors and even largely T-lineage committed precursors. However, the nature of precursors seeding the thymus under physiologic conditions has remained largely elusive and it is not known whether there is only one physiologic T-cell precursor population or many. Here, we used a competitive in vivo assay based on depletion rather than enrichment of classes of BM-derived precursor populations, thereby only minimally altering physiologic precursor ratios to assess the contribution of various extrathymic precursors to T-lineage differentiation. We found that under these conditions multiple precursors, belonging to both multipotent progenitor (MPP) and common lymphoid progenitor (CLP) subsets have robust T-lineage potential. However, differentiation kinetics of different precursors varied considerably, which might ensure continuous thymic output despite gated importation of extrathymic precursors. In conclusion, our data suggest that the thymus functions to impose T-cell fate on any precursor capable of filling the limited number of progenitor niches.

Figure 1

A depletion approach to analyze thymocytopoiesis under competitive conditions with near-normal precursor ratios. (A) Outline of the experimental approach. For details see “Results.” (B) Postsort analysis of lin⁻ BM cells depleted of populations expressing various surface markers by FACS sorting (right panels). (Left panels) Sorted lin⁻ BM cells. Numbers in gates and quadrants indicate the percentage of cells. (C) T-cell precursors are confined to CD27- and CD135-expressing cells under competitive conditions. Lin⁻ BM cells from B6 CD45.1 mice (test) were depleted of CD27⁺ cells, CD135⁺ cells, or both, mixed with lin⁻ BM cells from B6 CD45.2 mice (competitor), and transferred intravenously into Il7ra-deficient hosts. Ctl indicates mixture of nondepleted B6 CD45.1 lin⁻ BM cells with B6 CD45.2 lin⁻ BM cells. Donor-derived thymocytopoiesis was analyzed flow cytometrically 2 and 4 weeks after transfer. Representative FACS plots of test versus competitor ratios 2 weeks (top panels) and 4 weeks (bottom panels) after transfer. Numbers in gates indicate the percentage of cells. Combined analysis of 2 independent experiments with 2 mice per group.

Figure 2

Depletion of CD117^hi, CD127⁺, or CD90⁺ precursors does not abrogate T-cell differentiation. Lin⁻ BM cells from B6 CD45.1 mice (test) were depleted of CD117^hi cells, CD127⁺ cells, CD90⁺ cells, or all of them combined (triple), mixed with lin⁻ BM cells from B6 CD45.2 mice (competitor), and transferred intravenously into Il7ra-deficient hosts. Ctl indicates mixture of nondepleted B6 CD45.1 lin⁻ BM cells with B6 CD45.2 lin⁻ BM cells. Donor-derived thymocytopoiesis was analyzed by flow cytometry 2 and 4 weeks after transfer. (A) Representative FACS plots of test versus competitor ratios 2 weeks (top panels) and 4 weeks (bottom panels) after transfer. Numbers in quadrants indicate the percentage of cells. (B) Thymic reconstitution 2 weeks after transfer. Combined analysis of 5 independent experiments with 2 mice per group. Proportions of test populations were normalized to ctl. Data are shown as mean ± SEM; ***P < .001 (C) Thymic reconstitution 4 weeks after transfer. Combined analysis of 5 independent experiments with 2 mice per group. Proportions of test populations were normalized to ctl. Data are shown as mean ± SEM; *P < .02; **P < .01.

Figure 3

Depletion of various precursors results in thymocytopoiesis progressing with different kinetics. CD4 versus CD8 profiles of experiments performed as described in Figure 2. (A) Analysis 2 weeks after transfer. One representative of 8 individual mice is shown. (Top panels) Competitor population. (Bottom panels) Test population. Numbers in quadrants indicate the percentage of cells. (B) Analysis 4 weeks after transfer. One representative of 8 individual mice is shown. (Top panels) Competitor population. (Bottom panels) Test population. Numbers in quadrants indicate the percentage of cells. (C) Proportions of DP (left panel) and SP (right panel) thymocytes within test (black bars) and competitor (gray bars) populations 4 weeks after transfer. Data are shown as mean ± SEM; *P < .05; ***P < .001; n = 8.

Figure 4

Differential intrathymic differentiation kinetics of MPPs and CLPs do not depend on differential thymus-seeding capacity. (A-B) Sorted lin⁻CD27⁺CD117^hiSca-1⁺CD135⁺ MPPs and lin⁻CD27⁺CD117^loSca-1^loCD127⁺-CD135⁺ CLPs from B6 CD45.2 mice were intrathymically injected into B6 CD45.1 mice. Donor-derived cells were analyzed by FACS for the expression of CD4 and CD8 after 7, 14, 21, and 28 days. (A) Representative FACS plots of donor-derived (CD45.1⁺) thymocytes. Numbers in quadrants indicate frequency of donor-derived cells. (B) Analysis of donor-derived thymic subsets of at least 3 mice per group (DN: CD4⁻CD8⁻; DP: CD4⁺CD8⁺; SP: CD4⁺CD8⁻ and CD4⁻CD8⁺). (C) Lin⁻ BM cells from B6 CD45.1 mice (test) were depleted of CD117^hi cells, CD127⁺ cells, or both (double), mixed with lin⁻ BM cells from B6 CD45.1/CD45.2 mice (competitor), and transferred intravenously into Il7ra-deficient hosts. Ctl indicates mixture of nondepleted B6 CD45.1 lin⁻ BM cells with B6 CD45.1/CD45.2 lin⁻ BM cells. Five days after transfer thymocytes were harvested, cultured on OP9-DL1 cells for an additional 14 days, and analyzed for the expression of CD45.1 and CD45.2. Numbers indicate frequencies of cells in adjacent gates.

Figure 5

Depletion of circulating CD117^hi or CD127⁺ precursors does not abrogate T-cell differentiation. (A) Flow cytometric analysis of lin⁻ cells from BM (top panels) and blood (bottom panels). Cells were stained with antibodies against lineage markers CD90, CD117, CD127, CD27, Sca-1, and CD135. Numbers indicate frequencies of cells within gates. BM cells from 5 mice and blood cells from 10 mice were pooled for analysis. Plots are representative of 3 independent experiments. (B-C) Lin⁻ blood cells from 20 B6 CD45.2 mice (blood) were depleted of CD117^hi cells or CD127⁺ cells, mixed with lin⁻ BM cells from B6 CD45.1 mice (BM), and transferred intravenously into sublethally irradiated Il7ra-deficient hosts. Ctl indicates mixture of nondepleted B6 CD45.2 lin⁻ blood cells with B6 CD45.1 lin⁻ BM cells. Donor-derived thymocytopoiesis was analyzed flow cytometrically by staining for CD45.1 and CD45.2 (B) as well as CD4 and CD8 (C). Numbers indicate frequencies of cells within gates or quadrants. Data are representative of 2 independent experiments with 2 to 3 recipients per group.

Figure 6

Characterization of putative lin⁻CD117^−/loCD127⁻CD90⁻ BM-derived T-cell precursors. Flow cytometric analysis of lin⁻CD117^−/loCD127⁻CD90⁻ cells from BM. Cells were stained with antibodies against lineage markers CD90, CD117, CD127, CD27, and CD135 (A), and Sca-1 (B), CCR9 (C), CCR7 (D), or with P-selectin Ig to reveal expression of PSGL-1 (E). Cells from CCR9-deficient and CCR7-deficient mice were used as controls in panels C and D, respectively. For staining of PSGL-1, P-selectin-Ig was used in the presence (negative control) and absence of 10 mM EDTA (ethylenediaminetetraacetic acid). Numbers indicate frequencies of cells within gates. Data are representative of 2 independent experiments.