Extra-Thymic Physiological T Lineage Progenitor Activity Is Exclusively Confined to Cells Expressing either CD127, CD90, or High Levels of CD117

T cell development depends on continuous recruitment of progenitors from bone marrow (BM) to the thymus via peripheral blood. However, both phenotype and functional characteristics of physiological T cell precursors remain ill-defined. Here, we characterized a putative CD135+CD27+ T cell progenitor population, which lacked expression of CD127, CD90, and high levels of CD117 and was therefore termed triple negative precursor (TNP). TNPs were present in both BM and blood and displayed robust T lineage potential, but virtually no myeloid or B lineage potential, in vitro. However, TNPs did not efficiently generate T lineage progeny after intravenous or intrathymic transfer, suggesting that a physiological thymic microenvironment does not optimally support T cell differentiation from TNPs. Thus, we propose that physiological T cell precursors are confined to populations expressing either CD127, CD90, or high levels of CD117 in addition to CD135 and CD27 and that TNPs may have other physiological functions

TNPs display limited non-T lineage potential.

<p>BM derived MPPs, CLPs, TNPs and thymic ETPs were sorted from C57BL/6 mice and 500 cells of each population were cultured on OP9 cells in the presence of 5 ng/ml IL-7 and 5 ng/ml Flt3L. A) Cells were analyzed for the expression of CD11c and CD11b at day 7 of culture. B) Cells were analyzed for the expression of CD19 and B220 at day 11 of culture. C) Relative expansion of myeloid cells after 7 days of culture (from 500 input cells). D) Relative expansion of B cells after 11 days of culture (from 500 input cells). C, D) Numbers above columns indicate fold expansion. E) TNP-derived cultures. Analysis was performed as in panel A) after 17 and 24 days. F) TNP-derived cultures. Analysis was performed as in panel B) after 17 and 24 days. A, B, E, F) One representative experiment out of 2 independent experiments is shown. C, D) Combined data of two independent experiments with 6 wells per experiment. Error bars indicate SEM.</p

TNPs do not efficiently differentiate upon intrathymic transfer.

<p>BM derived MPPs, CLPs and TNPs from C57BL/6 mice were sorted and 20,000 cells were intrathymically injected into non-irradiated CD45.1 mice or CD45.1/2 mice. A) Donor derived cells were analyzed in thymus by FACS for the expression of CD45.1 and CD45.2 at 3 weeks after transfer. B) Donor derived cells from MPPs, CLPs and TNPs were also analyzed for CD4 and CD8 surface expression for identification of DN (CD4⁻CD8⁻), DP (CD4⁺CD8⁺) and SP (CD4⁺ or CD8⁺) cells. C) Donor derived electronically gated DN cells from MPPs, CLPs and TNPs were analyzed for CD117 and CD25 surface expression for identification of DN subsets (DN1: CD117⁺CD25⁻; DN2: CD117⁺CD25⁺; DN3: CD117⁻CD25⁺). One representative experiment out of three independent experiments with 3 mice each is shown. D) Combined analysis of 3 experiments with each 3 mice per group. Error bars indicate SEM.</p

Characterization of TNPs from BM and blood.

<p>Lineage depleted BM and blood cells from C57BL/6 mice were stained for lineage markers, CD27, CD135, CD117, CD127 and CD90. A) Gating strategy for the identification of TNPs in BM and blood. Representative data from 1 out of 4 mice (BM) and pooled cells from 10 mice (blood). B) Relative quantification of MPPs (lin⁻CD27⁺CD135⁺CD127⁻CD117^hi), CLPs (lin⁻CD27⁺CD135⁺CD127⁺CD117^+/low), TNPs (lin⁻CD27⁺CD135⁺CD127⁻CD117^−/lowCD90⁻) and CD90⁺ (lin⁻CD27⁺CD135⁺CD127⁻CD117^−/lowCD90⁺) candidate T cell precursors within lin⁻CD27⁺CD135⁺ cells. Combined data of 4 mice and 20 mice for BM and blood, respectively, from 2 independent experiments. Error bars indicate SEM.</p

TNPs do not efficiently differentiate upon intravenous transfer.

<p>BM derived MPPs, CLPs and TNPs from C57BL/6 mice were sorted and 20,000 cells were intravenously injected into non-irradiated <i>Il7ra</i>-deficient mice expressing CD45.1 or CD45.1 and CD45.2. Donor derived cells were analyzed in thymus and BM by FACS for the expression of CD45.1 and CD45.2 at A) 2 weeks and B) 4 weeks after transfer. One representative experiment out of four and three (2 weeks and 4 weeks, respectively) independent experiments with 2 mice each is shown. C) Combined analysis of frequencies of donor-derived thymocytes of 4 and 3 individual experiments 2 and 4 weeks after transfer, respectively. Error bars indicate SEM. D) CD4 vs. CD8 surface expression of donor-derived thymocytes 2 and 4 weeks after transfer. One representative experiment out of four and three (2 weeks and 4 weeks, respectively) independent experiments with 2 mice each is shown.</p

TNPs have robust T lineage potential.

<p>BM-derived MPPs, CLPs, TNPs and thymic ETPs were sorted from C57BL/6 mice and 500 cells of each population were cultured on OP9-DL1 cells in the presence of 1 ng/ml IL-7 and 5 ng/ml Flt3L. A) Cells were gated as CD4⁻CD8⁻ (DNs) and analyzed for expression of CD44 and CD25 at the indicated time points. One representative experiment out of 2 independent experiments is shown. B) Cells were analyzed for expression of CD4 and CD8 at the indicated time point. C, D) Quantification of data obtained from panel B. E) Relative expansion of cultures based on an input cell number of 500. Combined data of two independent experiments with 6 wells per experiment. Error bars indicate SEM.</p