The stream of precursors that colonizes the thymus proceeds selectively through the early T lineage precursor stage of T cell development

Abstract

T cell development in the thymus depends on continuous colonization by hematopoietic precursors. Several distinct T cell precursors have been identified, but whether one or several independent precursor cell types maintain thymopoiesis is unclear. We have used thymus transplantation and an inducible lineage-tracing system to identify the intrathymic precursor cells among previously described thymus-homing progenitors that give rise to the T cell lineage in the thymus. Extrathymic precursors were not investigated in these studies. Both approaches show that the stream of T cell lineage precursor cells, when entering the thymus, selectively passes through the early T lineage precursor (ETP) stage. Immigrating precursor cells do not exhibit characteristics of double-negative (DN) 1c, DN1d, or DN1e stages, or of populations containing the common lymphoid precursor 2 (CLP-2) or the thymic equivalent of circulating T cell progenitors (CTPs). It remains possible that an unknown hematopoietic precursor cell or previously described extrathymic precursors with a CLP, CLP-2, or CTP phenotype feed into T cell development by circumventing known intrathymic T cell lineage progenitor cells. However, it is clear that of the known intrathymic precursors, only the ETP population contributes significant numbers of T lineage precursors to T cell development.

Figure 1.

The emigration of T cell lineage precursors from bone marrow to blood is selective. (A) Lin⁻ blood cells of heterozygous CCR9-EGFP knock-in mice were fractionated according to their CD117 and CCR9^EGFP expression. The plot is gated on Lin⁻ cells. Percentages are indicated. (B) Lin⁻ fractions with the indicated phenotypes were isolated from blood (as shown in A) and from bone marrow, and were cultured as groups of 100 cells on OP9-DL1 stromal layers for 12 and 19 d. The fraction of wells that contained T lineage–committed DN3/4 thymocytes is plotted for each time point. At least 1,200 cells cultured as 100 cells per well were assayed for each population and time point. For the analysis of T lineage precursor activity of Lin⁻ CD117⁺ CCR9^EGFP− blood cells at day 12, >2,000 cells cultured as 100 cells per well were analyzed.

Figure 2.

A thymus transplantation model for the identification of thymus-settling precursors. (A) CD45.1⁺ newborn donor thymic lobes were transplanted under the kidney capsule of CCR9^EGFP/+//CD45.1/CD45.2 F1 recipient mice. The fraction, as well as the absolute number, of recipient thymocytes found in the ETP, DN2, DN3, and DN4 populations in the transplanted lobes are plotted against the time after transplantation. (right) The stacked area plots display the absolute number and the relative contribution of recipient (gray area) and donor (white area) cells per transplanted lobe for each thymocyte subset. Three lobes were analyzed per time point. Data are means ± SD. (B) Flow cytometric analyses show the Lin⁻ CD44⁺ CD25⁻ DN1 subset present in a newborn CD45.1⁺ thymus, and 5, 7, 9, and 11 d after transplantation of such a thymus under the kidney capsule of a CCR9^EGFP/+//CD45.1/CD45.2 F1 recipient mouse (FACS plots on the left of each column). FACS plots on the right of each column are gated on recipient, CD45.2⁺ ETPs, as shown in FACS plots on the left of each column. Percentages are indicated.

Figure 3.

An inducible lineage-tracing approach shows that the stream of precursors proceeds from the bone marrow selectively through the thymic ETP population. (A) The level of recombination for the indicated stages of T cell development is shown for one representative Flt3∷CreERT2//R26R-EYFP mouse at 1 wk (squares), one representative mouse at 3 wk (diamonds), and one representative mouse at 5 wk (circles) after tamoxifen injection. Each line represents the data from one mouse. The 3-mg 4-hydroxy-tamoxifen dose was titrated such that a single injection did not lead to detectable changes in the hematopoietic system. (B) The absolute number of recombined, EYFP⁺ thymic precursors in Flt3∷CreERT2//R26R-EYFP mice at 1 and 2 wk after a single 3-mg tamoxifen injection are shown. Each bar represents one mouse.

Figure 4.

The ETP population in mice after Notch1 ablation contains Notch1-deficient T cell lineage precursors. (A) In the absence of Notch1, only very few ETPs can be isolated for the detection of T cell lineage precursors. The FACS plots are gated on Lin⁻ CD44⁺ CD25⁻ DN1 thymocytes derived from Notch1^lox/lox//CCR9^EGFP/+ and Notch1^lox/lox//MxCre//CCR9^EGFP/+ mice 2 wk after pIpC injection. Percentages are indicated. (B) Single TMPs were sorted from Notch1^lox/lox//CCR9^EGFP/+ and Notch1^lox/lox//MxCre//CCR9^EGFP/+ mice 4 wk after pIpC injection on OP9-DL1 stromal layers and cultured for 12–14 d. Cells falling into the right half of the ETP gate shown in A were considered TMPs. DN3/4 thymocytes developed on OP9-DL1 stroma even in the absence of Notch1, as previously described (reference 20). Percentages are indicated. (C) Genomic PCR was performed on DNA purified from the OP9-DL1 culture of DN3/4 thymocytes generated from a single TMP of a Notch1^lox/lox//CCR9^EGFP/+ and a Notch1^lox/lox//MxCre//CCR9^EGFP/+ mouse 4 wk after pIpC injection. Two sets of primers were used, with one detecting the wild-type (N1 wild-type) and the floxed (N1^lox allele) Notch1 allele (left lane) and the other detecting the deleted allele of Notch1 (Δ N1; right lane). Because the added hematopoietic cells did not carry a wild-type Notch1 allele, this band is thought to be derived from OP9-DL1 stroma. PCR fragment sizes in base pairs are given in parenthesis.

Figure 5.

A rare Flt3^low CCR9^EGFP+ LSK population shows the highest level of recombination among LSK subsets in the Ftl3-driven lineage-tracing model. The LSK population was analyzed by FACS in CCR9^EGFP/+//Flt3∷Cre//R26R-EYFP mice. The top left plot is gated on LSK bone marrow cells, and the histogram plots are gated on the indicated regions of interest. The fraction of EYFP⁺, recombined cells is shown for each subset. Percentages are indicated.

Figure 6.

Flt3^low CCR9^EGFP+ LSKs are phenotypically identical and functionally closely related to TMPs. (A) LSKs were fractionated according to their Flt3 and CCR9-EGFP expression. The FACS plot on the left is gated on Lin⁻ cells, and the plot on the right is gated on LSK cells (as shown in the plot on the left). LSK subpopulations were sorted from the indicated regions. Percentages are indicated. (B) 100 cells of the indicated precursors were sorted onto OP9-DL1 stroma, cultured for 9 d, and analyzed by FACS. The levels of CCR9-EGFP and CD44 expression are indicative of the maturity of each precursor, as T lineage–biased precursors are expected to progress faster, whereas precursors without a T lineage bias or those biased toward a non-T fate will take longer to develop to DN3 thymocytes. The histogram plots are gated on DN2/3 thymocytes in which CCR9-EGFP expression increases with the progression from DN2 to DN3 thymocytes (reference 8). The results of three independent wells are shown in each histogram plot. Contour plots are gated on Lin⁻ CD90⁺ cells, and the numbers in each quadrant indicate the mean frequency of each population from three independent experiments. (C) The results of a representative clonogenic myeloid progenitor assay are shown for a 96-well plate seeded with 5, 10, 20, or 40 cells of the indicated precursors. The number of detectable myeloid colonies for each well is given. (D) Semiquantitative RT-PCR was performed on fivefold dilutions of cDNA prepared from the indicated precursors with primers specific for the indicated transcripts. Primers specific for hypoxanthin-phosphoribosyl-transferase (HPRT) transcript were used as a loading control. PCR fragment sizes in base pairs are given in parenthesis.