Identification of a T lineage-committed progenitor in adult blood

Abstract

With help of a hCD25 reporter controlled by pre-T cell receptor alpha (Ptcra) regulatory elements, T cell precursors were identified in peripheral blood. Sca-1(+)IL-7Ralpha(+)Flt3(-) precursors that were c-kit(lo)Thy-1(hi) generated T lineage cells when cultured on OP9-DL1 stromal cells and upon transfer into Rag2(-/-)Il2rg(-/-) mice. No B cells were generated in vivo and only few in vitro. These cells, which we call circulating T cell progenitors (CTP), were found at the same frequency in Foxn1(nu/nu) thymus-deficient mice and wild-type mice, indicating that they were pre- rather than postthymic. Inhibition of Notch-dependent transcription in vivo reduced the frequency of intrathymic early T cell progenitors (ETP), but not CTP, indicating that the latter are less Notch dependent. Thus, CTP represent T lineage-committed T cell precursors linking extrathymic with intrathymic lymphopoiesis in adult mice.

Figure 1

Circulating lin⁻hCD25⁺ precursors. (A) Lineage depleted BM and blood cells from hCD25 transgenic and non-transgenic mice were stained for lineage markers, hCD25, c-kit and B220. The lower panels show the expression of c-kit and B220 of electronically gated lin⁻hCD25⁺ cells. Numbers in FACS plots indicate percentages of cells within gates or quadrants. (B) Expression of pre-TCRα in circulating lin⁻hCD25⁺ cells. RT-PCR was performed on 250 CTP cells. The same amount of cDNA from hCD25⁺ DN3 cells and DN3 cells from Ptcra^−/− mice was used as positive and negative controls, respectively. One representative out of 2 independent experiments is shown. (C) Expression of surface markers on different lin⁻hCD25⁺ populations. BM CLP-1 (lin⁻hCD25⁺ckit⁺B220⁻) and CLP-2 (lin⁻hCD25⁺c-kit^−/loB220⁺) cells, blood lin⁻hCD25⁺B220⁺ cells (“B220⁺”) and CTP (lin⁻hCD25⁺B220⁻) and thymic hCD25⁺ DN1 (lin⁻CD25⁻CD44^hihCD25⁺) cells were stained for c-kit, IL-7Rα, Flt3, Sca-1, CD44 and Thy-1.1. Histograms show expression levels of the respective surface markers (blue histograms) or unstained controls (red histograms) of electronically gated populations as indicated above. One representative out of 2 independent experiments is shown.

Figure 2

Developmental potential of circulating lin⁻hCD25⁺ cells. (A) Sorted lin⁻hCD25⁺B220⁻ CTP and lin⁻hCD25⁺B220⁺ cells were co-cultured on OP9-DL1 or OP9-GFP cells for 18 days. Cells were stained for CD4, CD8, CD19, NK1.1 and Thy-1.1 and individual wells were analyzed by FACS. Blue histograms represent specific staining, red histograms represent unstained controls. 800 CD19⁺ (center) and 550 NK1.1⁺ (left) cells were recovered from starting cultures of 200 CTP. One representative out of three independent experiments is shown. (B) Limiting dilution analysis of T, B and NK potential of lin⁻hCD25⁺B220⁻ cells (CTP) from peripheral blood. 1, 5 or 40 cells were directly sorted onto OP9-DL1 or OP9-GFP cells and analyzed by FACS after 18 days. Wells containing >50 (B and NK potential) or >100 (T potential) lineage positive cells were scored positive. (C) Analysis of myeloid potential of CTP cells. 500 CTP, BM derived LSK, CLP-1 and CLP-2 cells were cultured in methylcellulose containing SCF, IL-3, IL-6 and Erythropoietin and colonies were counted microscopically. (D) 1000 sorted CTP or 50 lin⁻hCD25⁺B220⁺ cells (CD45.1) were injected intravenously into irradiated Rag2^−/−γc^−/− recipients and spleens were analyzed 5 weeks after transfer by flow cytometry for expression of TCRβ, NK1.1 and CD19. One representative out of 2 independent experiments with 2 mice per group is shown.

Figure 3

Developmental progression and expansion of CTP. (A) 100 blood derived LSK cells and 100 CTP were sorted and co-cultured on OP9-DL1 cells. Cell numbers were assessed by FACS. Data are shown ± SEM (n=4). (B) 100 blood derived LSK cells and 100 CTP were co-cultured on OP9-DL1 cells. After 4, 7, and 11 days cells were analyzed for the expression of CD4 and CD8 (right panels) and electronically gated CD4⁻CD8⁻ DN cells for the expression of CD44 and CD25 (left panels). One representative out of 2 independent experiments is shown.

Figure 4

CTP express thymus homing markers and home to the thymus. (A) BM derived CLP-1 and CLP-2 cells, CTP (identified as described in Figure 1) and thymic ETP (hCD25⁺CD44⁺c-kit^hiCD25⁻) were stained with P-selectin-Ig fusion proteins in the presence (blue histograms) and absence (red histograms) of free Ca²⁺. (B) BM derived CLP-1 and CLP-2 cells, CTP and thymic ETP were stained with anti-CCR9 antibodies (blue histograms). Red histograms represent staining with an isototype control. One representative out of 3 independent experiments is shown. (C) 1.5 × 10³ CTP or LSK cells from blood (CD45.1⁺CD45.2⁻) were injected intravenously into sub-lethally irradiated (FVB × C57BL/6)F1 mice (CD45.1⁺CD45.2⁺). Thymi were analyzed 2 weeks after transfer by flow cytometry for expression of CD45.1, CD45.2, CD4 and CD8.

Figure 5

CTP are present in peripheral blood of Foxn1^nu/nu mice. (A) CTP from hCD25 transgenic Foxn1^nu/nu mice and hCD25 transgenic Foxn1^wt littermates were analyzed by FACS. Cells were stained with antibodies against lineage markers, hCD25, c-kit, B220 and Thy-1.1 to reveal the frequency of lin⁻hCD25⁺ CTP. Numbers in FACS plots indicate percentages of cells within gates or quadrants. One representative out of 4 independent experiments is shown. (B) 200 CTP from hCD25 transgenic Foxn1^nu/nu mice and hCD25 transgenic Foxn1^wt littermates were sorted and co-cultured on OP9-DL1 cells. After 14 days cells were analyzed for the expression of CD4 and CD8. Numbers in FACS plots indicate percentages of cells within quadrants. One representative out of 3 independent experiments is shown (C) 100 CTP from hCD25 transgenic Foxn1^nu/nu mice were sorted and co-cultured on OP9-DL1 cells. After 4, 7, and 11 days cells were analyzed for the expression of CD4 and CD8 (lower panels) and electronically gated CD4⁻CD8⁻ DN cells for the expression of CD44 and CD25 (upper panels). One representative out of 2 independent experiments is shown.

Figure 6

CTP do not require Notch-induced transcription. Sorted lineage negative cells from hCD25 transgenic BM were infected with DN-MAML or MigR1 control retrovirus and transferred into irradiated hosts. BM, peripheral blood and thymus were analyzed by FACS 6 - 10 weeks after transfer. Numbers in histograms indicate the frequency of eGFP⁺ cells. (A) eGFP expression in different populations of one representative experiment of 6 mice per group. (B) Analysis of Thy-1.1 expression in DN-MAML or MigR1 transduced blood derived lin⁻B220⁻c-kit^lo cells. Numbers in FACS plots indicate percentages of cells within gates or quadrants. Blue gates and histograms indicate eGFP⁺ cells; red histograms indicate eGFP⁻ cells. (C) Statistical analysis of 4 independent experiments. The percentage of eGFP positive cells was normalized to the percentage of eGFP positive cells of total BM for each independent experiment. Data are shown ± SEM.