Developmental pathway of CD4+CD8- medullary thymocytes during mouse ontogeny and its defect in Aire-/- mice - PubMed (original) (raw)

Developmental pathway of CD4+CD8- medullary thymocytes during mouse ontogeny and its defect in Aire-/- mice

Juan Li et al. Proc Natl Acad Sci U S A. 2007.

Abstract

The newly generated single-positive (SP) thymocytes undergo further maturation in the thymic medulla before their emigration to the periphery. The present study was undertaken to validate a developmental program we proposed for CD4SP medullary thymocytes and to explore the mechanisms regulating this process. During mouse ontogeny, the emergence of different subsets of CD4SP thymocytes followed a strict temporal order from SP1 to SP4. Parallel to the transition in surface phenotype, a steady increase in function was observed. As further evidence, purified SP1 cells were able to sequentially give rise to SP2, SP3, and SP4 cells in intrathymic adoptive transfer and in culture. Notably, the development of CD4SP cells in the medulla seemed to be critically dependent on a functionally intact medullary epithelial cell compartment because Relb and Aire deficiency were found to cause severe blockage at the transition from SP3 to SP4. Taken together, this work establishes an ontogenetically and functionally relevant maturation program for CD4SP thymocytes. Precise dissection of this program should facilitate further inquiry into the molecular mechanisms governing normal thymocyte development and its disturbance in pathological conditions.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Phenotypic development of TCR+CD4SP medullary thymocytes during mouse ontogeny. CD8-depleted thymocytes from various time points during mouse ontogeny were stained for CD4, TCRβ, CD69, and 6C10 or Qa-2. The expression of 6C10 versus CD69 and CD69 versus Qa-2 was analyzed in TCRβ+CD4SP medullary thymocytes. The age of the fetus or infant is indicated to the left of the plots. The quadrant markers are placed according to the staining pattern of appropriate isotype controls. Results are representative of three separate experiments.

Fig. 2.

Fig. 2.

Proliferation and cytokine production by distinct subsets of CD4SP thymocytes at discrete time points during ontogeny. Different subsets of CD4SP thymocytes (SP1–SP4) and splenic CD4+ T cells were obtained at each time point as indicated and stimulated with Con A. (A) Cell proliferation was measured by [3H]TdR incorporation assay at day 3. For cytokine production assays, culture supernatant was harvested at day 2 of culture. (B) IL-2 was measured by [3H]TdR incorporation assay by using IL-2-dependent cell line HT-2. (C and D) IL-4 (C) and IFN-γ (D) were measured by ELISA. Data shown are the mean ± SD of two to three repeated experiments. Filled diamond, SP1; ×, SP2; filled triangle, SP3; filled square, SP4; filled circle, splenic CD4 T cell.

Fig. 3.

Fig. 3.

Differentiation of SP1 CD4SP thymocytes in intrathymic cell-adoptive transfer assay. SP1 cells were isolated from CD45.1+ donor mice by cell sorting with a purity of ≥97% (pretransfer). The 1 × 106 cells were then injected into the thymus of CD45.2+ recipient mice. At 24, 48, and 72 h after the adoptive transfer, donor cells in the recipient thymus were analyzed by flow cytometry. (A) Dot plots show 6C10 versus CD69 expression in the whole donor population, and histograms show Qa-2 expression in the 6C10−CD69− fraction. The number of cells recovered at different time points is shown in brackets. (B) Also analyzed is the presence of donor-derived CD45.1+CD4+ T cells in the spleen. Experiments were repeated two to three times with similar results.

Fig. 4.

Fig. 4.

In vitro differentiation of SP1 CD4SP thymocytes. Purified 1.5 × 105 SP1 cells were cultured in medium supplemented with 30 ng/ml IL-7 in the presence or absence of the thymic epithelial cell line mTEC1 or mTEC9. Cells were harvested at days 2 and 4 and analyzed for surface expression of 6C10, CD69, and Qa-2. The number of cells recovered from different cultures is shown in brackets, and the number in each quadrant indicates the percentage of the corresponding subsets. Results are representative of four to five separate experiments.

Fig. 5.

Fig. 5.

Developmental defects of CD4SP thymocytes in _Relb_−/− and _Aire_−/− mice. Thymocytes were harvested from wild-type (WT), _Relb_−/−, and _Aire_−/− mice and analyzed by flow cytometry. Total numbers of CD4SP and CD8SP medullary thymocytes were comparable in each thymus. The dot plots show 6C10 versus CD69 and CD69 versus Qa-2 expression in CD4SP thymocytes. The percentage of cells in each quadrant is indicated. Results are representative of two separate experiments.

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