Regenerative capacity of adult cortical thymic epithelial cells

Abstract

Involution of the thymus is accompanied by a decline in the number of thymic epithelial cells (TECs) and a severely restricted peripheral repertoire of T-cell specificities. TECs are essential for T-cell differentiation; they originate from a bipotent progenitor that gives rise to cells of cortical (cTEC) and medullary (mTEC) phenotypes, via compartment-specific progenitors. Upon acute selective near-total ablation during embryogenesis, regeneration of TECs fails, suggesting that losses from the pool of TEC progenitors are not compensated. However, it is unclear whether this is also true for the compartment-specific progenitors. The decline of cTECs is a prominent feature of thymic involution. Because cTECs support early stages of T-cell development and hence determine the overall lymphopoietic capacity of the thymus, it is possible that the lack of sustained regenerative capacity of cTEC progenitor cells underlies the process of thymic involution. Here, we examine this hypothesis by cell-type-specific conditional ablation of cTECs. Expression of the human diphtheria toxin receptor (hDTR) gene under the regulatory influence of the chemokine receptor Ccx-ckr1 gene renders cTECs sensitive to the cytotoxic effects of diphtheria toxin (DT). As expected, DT treatment of preadolescent and adult mice led to a dramatic loss of cTECs, accompanied by a rapid demise of immature thymocytes. Unexpectedly, however, the cTEC compartment regenerated after cessation of treatment, accompanied by the restoration of T-cell development. These findings provide the basis for the development of targeted interventions unlocking the latent regenerative potential of cTECs to counter thymic involution.

Fig. 1.

cTEC specific cytoablation. (A) Schematic representation of the Ccx-ckr1:DTR transgene. (B) Flow cytometric isolation of stromal components. Neural-crest derived mesenchymal cells (MEC), EpCam⁻, CD45⁻, CD31⁻, Ly51⁺ marker (Center); endothelial cells (EC), EpCam⁻CD45⁻CD31⁺Ly51⁻ (Center); cTECs, EpCam⁺CD45⁻Ly51⁺UEA-1⁻ (Right); and mTECs, EpCam⁺CD45⁻Ly51⁻UEA1⁺ (Right). (C) Expression levels of the endogenous Ccx-ckr1 gene, the Ccx-ckr1:eGFP knock-in allele, and the Ccx-ckr1:hDTR transgene in Ccx-ckr1^eGFP/+ control (ctrl) and Ccx-ckr1^eGFP/+;Ccx-ckr1:DTR transgenic (tg) mice. Results of quantitative RT-PCR normalized to the expression levels of Hprt are expressed relative to the levels of Ccx-ckr1:hDTR transgenic cTECs (dashed line). (D) Absolute numbers of TEC subsets (Left) and MECs and ECs (Right) of untreated and diphtheria toxin (DT)-treated nontransgenic (ctrl) or hDTR-transgenic mice (tg); animals received a single injection of DT (30 ng/g body weight, i.p.) and were analyzed 1 d (P8) thereafter. Statistical analysis was performed using Fisher´s t test (***P < 0.001).

Fig. 2.

Loss of cTECs abrogates T-cell differentiation. (A) Representative cryosections of thymic tissue from mice treated as in Fig. 1D. Sections were stained with antibodies for an mTEC marker (cytokeratin 5, K5, green fluorescence) and a cTEC marker (cytokeratin 8, K8, red fluorescence). At this point the thymic cortex still contains many lymphocytes (Inset), as revealed by intense nuclear DNA staining (DAPI, blue fluorescence). (Scale bar, 100 μm.) (B) Representative cryosections as in A stained with antibodies for mesenchymal cells (ERTR7, blue fluorescence) and K5. (Scale bar, 100 μm.) Other designations in A and B as in legend to Fig. 1. (C–F) Differential effect on thymocytes 1, 3, and 7 d after DT treatment. (C) DN2–3 (Lin⁻CD25⁺). (D) DP thymocytes (CD4⁺CD8⁺). (E) CD4⁺ single positive. (F) CD8⁺ single positive. Cell numbers are expressed relative to control mice (mean ± SD) (for absolute numbers see Dataset S1); the number of animals in each group is given in parentheses Above bars). Statistical analysis was performed using Fisher´s t test (***P < 0.001).

Fig. 3.

Regenerative capacity of cTECs after near-total ablation in young mice. (A) Phenotypic characterization of TECs (CD45⁻Epcam⁺) at various time points after DT treatment at P7; the presence of cTECs and mTECs was determined by flow cytometry. (B) Representative cryosections of thymic tissue at various time points after DT treatment at P7 stained with antibodies for an mTEC marker (cytokeratin 5, green fluorescence) and a cTEC marker (cytokeratin 8, red fluorescence). (Scale bar, 100 μm.) (C) Temporal changes in thymocyte subsets after DT treatment. DN2–3 (Lin⁻CD25⁺); DP thymocytes (CD4⁺CD8⁺); CD4⁺ single positive; CD8⁺ single positive; cell numbers are expressed relative to age-matched control mice (mean ± SD) (for absolute numbers see Dataset S1); the number of animals in each group is given in parentheses Above bars).

Fig. 4.

Regenerative capacity of adult cTECs. (A and B) Recovery of DP thymocytes in adult female (A) and male (B) mice at various time points after DT treatment at 2–3 mo; dpi, days after toxin injection. Each dot represents one animal. (C) Castration of male mice 1 d after DT treatment improves DP recovery; sham, sham operation. (D) Virilization of female mice by 5α-DHT prevents cTEC recovery; DT-treated adult female mice additionally received a slow-release pellet (p, placebo; DHT, 5α-dihydroxytestosterone). For C and D, the absolute number of DP thymocytes was determined 3 wk after DT treatment (mean values; each symbol represents one animal). Statistical analysis was performed using Fisher´s t test (***P < 0.001; **P < 0.01).