A Novel TCR Transgenic Model Reveals That Negative Selection Involves an Immediate, Bim-Dependent Pathway and a Delayed, Bim-Independent Pathway

A complete understanding of negative selection has been elusive due to the rapid apoptosis and clearance of thymocytes in vivo. We report a TCR transgenic model in which expression of the TCR during differentiation occurs only after V(D)J-like recombination. TCR expression from this transgene closely mimics expression of the endogenous TCRα locus allowing for development that is similar to wild type thymocytes. This model allowed us to characterize the phenotypic changes that occurred after TCR-mediated signaling in self-reactive thymocytes prior to their deletion in a highly physiological setting. Self-reactive thymocytes were identified as being immature, activated and CD4loCD8lo. These cells had upregulated markers of negative selection and were apoptotic. Elimination of Bim reduced the apoptosis of self-reactive thymocytes, but it did not rescue their differentiation and the cells remained at the immature CD4loCD8lo stage of development. These cells upregulate Nur77 and do not contribute to the peripheral T cell repertoire in vivo. Remarkably, development past the CD4loCD8lo stage was possible once the cells were removed from the negatively selecting thymic environment. In vitro development of these cells occurred despite their maintenance of high intracellular levels of Nur77. Therefore, in vivo, negatively selected Bim-deficient thymocytes are eliminated after prolonged developmental arrest via a Bim-independent pathway that is dependent on the thymic microenvironment. These data newly reveal a layering of immediate, Bim-dependent, and delayed Bim-independent pathways that both contribute to elimination of self-reactive thymocytes in vivo

DKK1 Mediated Inhibition of Wnt Signaling in Postnatal Mice Leads to Loss of TEC Progenitors and Thymic Degeneration

Thymic epithelial cell (TEC) microenvironments are essential for the recruitment of T cell precursors from the bone marrow, as well as the subsequent expansion and selection of thymocytes resulting in a mature self-tolerant T cell repertoire. The molecular mechanisms, which control both the initial development and subsequent maintenance of these critical microenvironments, are poorly defined. Wnt signaling has been shown to be important to the development of several epithelial tissues and organs. Regulation of Wnt signaling has also been shown to impact both early thymocyte and thymic epithelial development. However, early blocks in thymic organogenesis or death of the mice have prevented analysis of a role of canonical Wnt signaling in the maintenance of TECs in the postnatal thymus.Here we demonstrate that tetracycline-regulated expression of the canonical Wnt inhibitor DKK1 in TECs localized in both the cortex and medulla of adult mice, results in rapid thymic degeneration characterized by a loss of DeltaNP63(+) Foxn1(+) and Aire(+) TECs, loss of K5K8DP TECs thought to represent or contain an immature TEC progenitor, decreased TEC proliferation and the development of cystic structures, similar to an aged thymus. Removal of DKK1 from DKK1-involuted mice results in full recovery, suggesting that canonical Wnt signaling is required for the differentiation or proliferation of TEC populations needed for maintenance of properly organized adult thymic epithelial microenvironments.Taken together, the results of this study demonstrate that canonical Wnt signaling within TECs is required for the maintenance of epithelial microenvironments in the postnatal thymus, possibly through effects on TEC progenitor/stem cell populations. Downstream targets of Wnt signaling, which are responsible for maintenance of these TEC progenitors may provide useful targets for therapies aimed at counteracting age associated thymic involution or the premature thymic degeneration associated with cancer therapy and bone marrow transplants

The Wnt Signaling Antagonist Kremen1 is Required for Development of Thymic Architecture

Wnt signaling has been reported to regulate thymocyte proliferation and selection at several stages during T cell ontogeny, as well as the expression of FoxN1 in thymic epithelial cells (TECs). Kremen1 (Krm1) is a negative regulator of the canonical Wnt signaling pathway, and functions together with the secreted Wnt inhibitor Dickkopf (Dkk) by competing for the lipoprotein receptor-related protein (LRP)-6 co-receptor for Wnts. Here krm1 knockout mice were used to examine krm1 expression in the thymus and its function in thymocyte and TEC development. krm1 expression was detected in both cortical and medullary TEC subsets, as well as in immature thymocyte subsets, beginning at the CD25+CD44+ (DN2) stage and continuing until the CD4+CD8+(DP) stage. Neonatal mice show elevated expression of krm1 in all TEC subsets. krm1− / − mice exhibit a severe defect in thymic cortical architecture, including large epithelial free regions. Much of the epithelial component remains at an immature Keratin 5+ (K5) Keratin 8+(K8) stage, with a loss of defined cortical and medullary regions. A TOPFlash assay revealed a 2-fold increase in canonical Wnt signaling in TEC lines derived from krm1− / − mice, when compared with krm1+ / + derived TEC lines. Fluorescence activated cell sorting (FACS) analysis of dissociated thymus revealed a reduced frequency of both cortical (BP1+EpCAM+) and medullary (UEA-1+ EpCAMhi) epithelial subsets, within the krm1− / − thymus. Surprisingly, no change in thymus size, total thymocyte number or the frequency of thymocyte subsets was detected in krm1− / − mice. However, our data suggest that a loss of Krm1 leads to a severe defect in thymic architecture. Taken together, this study revealed a new role for Krm1 in proper development of thymic epithelium

Label Retention Identifies a Multipotent Mesenchymal Stem Cell-Like Population in the Postnatal Thymus

Thymic microenvironments are essential for the proper development and selection of T cells critical for a functional and self-tolerant adaptive immune response. While significant turnover occurs, it is unclear whether populations of adult stem cells contribute to the maintenance of postnatal thymic epithelial microenvironments. Here, the slow cycling characteristic of stem cells and their property of label-retention were used to identify a K5-expressing thymic stromal cell population capable of generating clonal cell lines that retain the capacity to differentiate into a number of mesenchymal lineages including adipocytes, chondrocytes and osteoblasts suggesting a mesenchymal stem cell-like phenotype. Using cell surface analysis both culture expanded LRCs and clonal thymic mesenchymal cell lines were found to express Sca1, PDGFRα, PDGFRβ,CD29, CD44, CD49F, and CD90 similar to MSCs. Sorted GFP-expressing stroma, that give rise to TMSC lines, contribute to thymic architecture when reaggregated with fetal stroma and transplanted under the kidney capsule of nude mice. Together these results show that the postnatal thymus contains a population of mesenchymal stem cells that can be maintained in culture and suggests they may contribute to the maintenance of functional thymic microenvironments

Label Retention Identifies a Multipotent Mesenchymal Stem Cell-Like Population in the Postnatal Thymus

<div><p>Thymic microenvironments are essential for the proper development and selection of T cells critical for a functional and self-tolerant adaptive immune response. While significant turnover occurs, it is unclear whether populations of adult stem cells contribute to the maintenance of postnatal thymic epithelial microenvironments. Here, the slow cycling characteristic of stem cells and their property of label-retention were used to identify a K5-expressing thymic stromal cell population capable of generating clonal cell lines that retain the capacity to differentiate into a number of mesenchymal lineages including adipocytes, chondrocytes and osteoblasts suggesting a mesenchymal stem cell-like phenotype. Using cell surface analysis both culture expanded LRCs and clonal thymic mesenchymal cell lines were found to express Sca1, PDGFRα, PDGFRβ,CD29, CD44, CD49F, and CD90 similar to MSCs. Sorted GFP-expressing stroma, that give rise to TMSC lines, contribute to thymic architecture when reaggregated with fetal stroma and transplanted under the kidney capsule of nude mice. Together these results show that the postnatal thymus contains a population of mesenchymal stem cells that can be maintained in culture and suggests they may contribute to the maintenance of functional thymic microenvironments. </p> </div

Changes in H2BGFP expression within TECs in K5tTA;TetO-H2BGFP transgenic mice following a time course of Dox feeding.

<p>A. Thymic sections prepared at the initiation of Dox feeding and from 2-6 weeks after the start of Dox feeding showing the reduction in H2BGFP expressing TECs. Sections were stained with anti-K8 (red) and anti-K5 antibodies (blue) to allow localization of the H2BGFP expressing nuclei within TECs. B. Representative FACS analysis of CD45^- dissociated thymic stroma showing the frequency of EpCAM⁺ H2BGFP⁺ cells at time 0, 2, 4 and 6 weeks after the initiation of Dox feeding to inhibit H2BGFP expression in K5⁺TECs. Gates from left to right in each panel show the frequency of EpCAM⁺H2BGFP^-, EpCAM⁺H2BGFP^lo and EpCAM⁺GFP^hi TECs at each time point. C. Graph shows the mean number of EpCAM⁺H2BGFP^hi label-retaining cells and the number of EpCAM⁺H2BGFP^lo cycling cells per thymus at 0, 2, 4, 6 and 12 weeks after the inhibition of H2BGFP expression through Dox feeding. Error bars are +/- the standard deviation of the mean. Results are representative of 3 independent experiments with 5 mice at each time point/experiment. D. Graph depicts the mean number of CD45^- EpCAM⁺ TECs/thymus at 0, 2, 4, 6 and 12 weeks after the inhibition of H2BGFP expression through Dox feeding. Error bars are +/- the standard deviation of the mean.</p

Clonal TMSC lines maintain the capacity to form Adipocytes, Osteoblasts and Chondrocytes <i>in vitro</i>.

<p>A. TMSC7 cell line cultured in MEMα medium supplemented with EGF, FGF and LIF and then stained with Oil Red O; B. Oil Red S staining of TMSC7 cultured in KSFM + 10% FBS + EGF which induced adipogenesis (inset 400X image of Oil Red O staining droplets of lipid); C. Alizarin Red S staining of TMSC7 cultured in control medium; D. Alizarin Red S staining of mineral deposits in TMSC7 cultured in osteogenesis differentiation medium; E. Alcian Blue staining of frozen section of Chondro-nodule following culture of TMSC7 cultured in chondrogenesis conditions. (Mag. A - D, 100X; inset and E 400X) Similar results for all differentiation assays were obtained in a minimum of 3 experiments and with multiple TMSC lines.</p

Localization and characterization of thymic label-retaining cells in K5tTA;TetO-H2BGFP mice.

<p>A. H2BGFP expression in thymic section prior to Dox feeding (100X). B. H2BGFP expression in thymic section after 10-week Dox feeding. C. Overlay of H2BGFP (green), K8 (red) and K5 (blue) expression prior to Dox feeding. White arrows show H2BGFP LRCs concentrated at cortico-medullary junction defined by K5 and K8 staining (inset= K5, H2BGFP overlay 400X showing that H2BGFP is restricted to K5-expressing cells). D. Overlay of H2BGFP, K8 and K5 in thymic section following 10-week Dox chase (inset: 400X of H2BGFP LRCs stained with K5 and K8 at CMJ). E. 400x images of thymic sections showing expression of MTS10, H2BGFP, Ki67 and merge. White arrows define position of H2BGFP^hi LRCs. F. 400X images of thymic sections showing expression of ΔNP63, H2BGFP, Aire and merge. White arrows define position of H2BGFP^hi LRCs. G 400x images of thymic sections showing staining with UEA1 PE (red) and DEC 205 Alexa 647 (pink) together with H2BGFP and Merge. H. 400x images of thymic sections derived from K5tTA;tetO-H2BGFP mice prior to Dox feeding stained with UEA1PE (red) and DEC205 Alexa 647(Pink) together with H2BGFP and merge.</p

<i>In vitro</i> growth potential of H2BGFP LRCs.

<p>Methylene blue stained colonies derived from FACS sorted CD45^- EpCAM^lo MHCII^lo/-Sca1⁺H2BGFP+ LRCs (A) or CD45^-MHCII^intEpCAM^hi Sca1⁺ H2BGFP⁺ stroma (B). C. Merge of H2BGFP expression and phase contrast image of expanded EpCAM^loMHCII^lo/- H2BGFP⁺ LRCs 1 week after sorting, demonstrating dramatic <i>in </i><i>vitro</i> growth. D. Merge of H2BGFP expression and phase contrast image showing limited expansion of EpCAM^hi MHCII^intH2BGFP⁺ LRCs 1 week after sorting. E. H2BGFP expression in same field as C. F. H2BGFP expression in same field as D. These results are representative of 3 independent experiments performed with 10-12 week Dox fed H2BGFP mice. Methylene blue stained colonies derived from FACS sorted CD45^-MHCII^lo/-EpCAM^lo Sca1⁺CD49F^lo CD29⁺ (G) or CD45^-MHCII^intEpCAM^hiSca1⁺CD49F^hiCD29⁺ stroma (H) derived from WT C57BL/6J mice. I. Colony forming potential of sorted LRCs and defined TEC subsets sorted from dissociated thymus derived from postnatal C57BL/6J mice. Error bars show standard deviation of means calculated from 5 independent experiments. P values are derived by comparison of colony forming potential of sets of populations using T test.</p