A multipotent precursor in the thymus maps to the branching point of the T versus B lineage decision

Hematopoietic precursors continuously colonize the thymus where they give rise mainly to T cells, but also to B and dendritic cells. The lineage relationship between these three cell types is unclear, and it remains to be determined if precursors in the thymus are multipotent, oligopotent, or lineage restricted. Resolution of this question necessitates the determination of the clonal differentiation potential of the most immature precursors in the thymus. Using a CC chemokine receptor 9–enhanced green fluorescent protein knock-in allele like a surface marker of unknown function, we identify a multipotent precursor present in bone marrow, blood, and thymus. Single cells of this precursor give rise to T, B, and dendritic cells. A more differentiated stage of this multipotent precursor in the thymus has lost the capacity to generate B but not T, dendritic, and myeloid cells. Thus, the newly identified precursor maps to the branching point of the T versus B lineage decision in the hematopoietic lineage hierarchy

Thymic Medullary Epithelial Cell Differentiation, Thymocyte Emigration, and the Control of Autoimmunity Require Lympho–Epithelial Cross Talk via LTβR

Thymocytes depend on the interaction with thymic epithelial cells for the generation of a diverse, nonautoreactive T cell repertoire. In turn, thymic epithelial cells acquire their three-dimensional cellular organization via instructive signals from developing thymocytes. The nature of these signals has been elusive so far. We show that thymocytes and medullary epithelial cells (MECs) communicate via the lymphotoxin β receptor (LTβR) signaling axis. Normal differentiation of thymic MECs requires LTβR ligand on thymocytes and LTβR together with nuclear factor–κB-inducing kinase (Nik) in thymic epithelial cells. Impaired lympho–epithelial cross talk in the absence of the LTβR causes aberrant differentiation and reduced numbers of thymic MECs, leads to the retention of mature T lymphocytes, and is associated with autoimmune phenomena, suggesting an unexpected role for LTβR signaling in central tolerance induction

(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 ). 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.<p><b>Copyright information:</b></p><p>Taken from "The stream of precursors that colonizes the thymus proceeds selectively through the early T lineage precursor stage of T cell development"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1187-1199.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373849.</p><p></p

The LSK population was analyzed by FACS in CCR9/ //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.<p><b>Copyright information:</b></p><p>Taken from "The stream of precursors that colonizes the thymus proceeds selectively through the early T lineage precursor stage of T cell development"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1187-1199.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373849.</p><p></p