Dedicated mTEC progenitors stay true, even into adulthood.

Knowledge about the cells giving rise to and maintaining the thymic structure remains limited. In this issue of Immunity, Sekai et al. (2014) identify a postnatal self-renewing unipotential progenitor population capable of generating thymic medullary cells and lay the foundation for research into thymic regeneration.

Induction of T-cell development by Delta-like 4-expressing fibroblasts.

The thymus provides a unique environment for the induction of T-cell lineage commitment and differentiation, which is predicted by specific Notch ligand-receptor interactions on epithelial cells and lymphoid progenitors, respectively. Accordingly, a bone marrow-derived stromal cell line (OP9) ectopically expressing the Notch ligand Delta-like 1 (Dll1) or Dll4 (OP9-DL1 and OP9-DL4, respectively) gains the ability to recapitulate thymus-like function, supporting T-cell differentiation of both mouse and human progenitors. In this study, we extend these findings by demonstrating that, unlike the NIH3T3 cell line, mouse primary fibroblasts made to express Dll4 (mFibro-DL4) acquire the capacity to promote and support T-cell development from hematopoietic stem cells (HSCs) into TCRαß(+), CD4(+) and CD8(+) T-lineage cells. However, mFibro-DL4 cells showed a lower efficiency of T-cell generation than OP9-DL4 cells did. Nevertheless, progenitor T-cells (CD117(+) CD44(+) CD25(+)) generated in HSC/mFibro-DL4 co-cultures, when intravenously transferred into immunodeficient (Rag2(-/-) γc(-/-)) mice, home to the thymus, undergo differentiation, and give rise to mature T-cells that go on to populate the periphery. Surprisingly, primary human fibroblast cells expressing Dll4 showed very low efficiency in supporting human T-lineage differentiation, which could not be improved by blocking myelopoiesis. Nevertheless, mFibro-DL4 cells could support human T-cell lineage differentiation. Our results provide a functional framework for the induction of T-cell development using easily accessible fibroblasts made to express Dll4. These cells, which are amenable for in vitro applications, can be further utilized in the design of individualized systems for T-cell production, with implications for the treatment of immunodeficiencies.

Comparative and functional evaluation of in vitro generated to ex vivo CD8 T cells.

The generation of the cytotoxic CD8 T cell response is dependent on the functional outcomes imposed by the intrathymic constraints of differentiation and self-tolerance. Although thymic function can be partly replicated in vitro using OP9-DL1 cell cultures to yield CD8 αß TCR-bearing cells from hematopoietic progenitor cells, a comprehensive and functional assessment of entirely in vitro generated CD8 T cells derived from bone marrow hematopoietic stem cells has not been established and remains controversial. In this study, we demonstrate that a phenotypic, molecular, and functional signature of in vitro derived CD8 T cells is akin to that of ex vivo CD8 T cells, although several significant differences were also observed. Transfer of in vitro derived CD8 T cells into syngeneic and immunodeficient host mice showed no graft-versus-host response, whereas a robust homeostatic proliferation was observed, respectively. These findings, along with a diverse and broad TCR repertoire expressed by the in vitro derived CD8 T cells, allowed for the successful generation of Ag-specific T cells to be obtained from an entirely in vitro generated CD8 T cell pool. These findings support the use of Ag-specific in vitro derived effector CD8 T cells for immune reconstitution approaches, which would be amenable to further tailoring for their use against viral infections or malignancies.

Direct comparison of Dll1- and Dll4-mediated Notch activation levels shows differential lymphomyeloid lineage commitment outcomes.

In the thymus, Notch signaling is essential for T lymphopoiesis, with Delta-like (Dll)4 uniquely involved in this process. However, using cocultures, either Dll4 or Dll1 were shown to support T lymphopoiesis. To address which Dll is more effective at inducing hematopoietic progenitor cells to give rise to T lineage cells in vitro, we generated OP9 cells expressing a series of incrementally discrete and equivalent levels of Dll1 or Dll4. In keeping with previous findings, OP9 cells expressing high levels of either Dll1 or Dll4 gave rise to T lineage cells with similar efficacy, and prevented the differentiation of B and myeloid-lineage cells. However, at limiting levels, Dll4 maintained its ability to inhibit B lineage choice and induce T lineage commitment and differentiation at lower levels than Dll1. This manifest property of Dll4 is evident despite lower levels of steady-state surface expression than Dll1 on OP9 cells. The heightened effectiveness of Dll4 over Dll1 also corresponded to the induction of Notch target genes, and inhibition of B and myeloid-specific transcription factors. Furthermore, we show that OP9 cells expressing levels of Dll4 equivalent to those present in thymic epithelial cells, as expected, gave rise to T lineage cells, but were also permissive for the differentiation of myeloid cells; whereas, still inhibiting B lymphopoiesis. Our findings show that Dll4 expressed at physiological levels on OP9 cells is functionally distinct from similarly expressed levels of Dll1, illustrating the unique properties of Dll4 in supporting the combined T lineage and specific myeloid-lineage outcomes that underpin its function within the thymus.

Elevated frequencies of self-reactive CD8+ T cells following immunization with a xenoantigen are due to the presence of a heteroclitic CD4+ T-cell helper epitope.

Immunization of mice with human dopachrome tautomerase (hDCT) provides greater protection against melanoma than immunization with the murine homologue (mDCT). We mapped the CD8(+) and CD4(+) T-cell epitopes in both proteins to better understand the mechanisms of the enhanced protection. The dominant CD8(+) T-cell epitopes were fully conserved between both proteins, yet immunization with hDCT produced frequencies of CD8(+) T cells that were 5- to 10-fold higher than immunization with mDCT. This difference was not intrinsic to the two proteins because comparable frequencies of CD8(+) T cells were elicited by both antigens in DCT-deficient mice. Strikingly, only hDCT elicited a significant level of specific CD4(+) T cells in wild-type (WT) mice. The murine protein was not devoid of CD4(+) T-cell epitopes because immunization of DCT-deficient mice with mDCT resulted in robust CD4(+) T-cell immunity directed against two epitopes that were not identified in WT mice. These results suggested that the reduced immunogenicity of mDCT in WT mice may be a function of insufficient CD4(+) T-cell help. To address this possibility, the dominant CD4(+) T-cell epitope from hDCT was introduced into mDCT. Immunization with the mutated mDCT evoked CD8(+) T-cell frequencies and protective immunity comparable with hDCT. These results reveal a novel mechanism by which xenoantigens overcome tolerance. Our data also suggest that immunologic tolerance is more stringent for CD4(+) T cells than CD8(+) T cells, providing a mechanism of peripheral tolerance where autoreactive CD8(+) T cells fail to be activated due to a lack of autoreactive CD4(+) T cells specific for the same antigen.

FOXN1 (GFP/w) reporter hESCs enable identification of integrin-ß4, HLA-DR, and EpCAM as markers of human PSC-derived FOXN1(+) thymic epithelial progenitors.

Thymic epithelial cells (TECs) play a critical role in T cell maturation and tolerance induction. The generation of TECs from in vitro differentiation of human pluripotent stem cells (PSCs) provides a platform on which to study the mechanisms of this interaction and has implications for immune reconstitution. To facilitate analysis of PSC-derived TECs, we generated hESC reporter lines in which sequences encoding GFP were targeted to FOXN1, a gene required for TEC development. Using this FOXN1 (GFP/w) line as a readout, we developed a reproducible protocol for generating FOXN1-GFP(+) thymic endoderm cells. Transcriptional profiling and flow cytometry identified integrin-ß4 (ITGB4, CD104) and HLA-DR as markers that could be used in combination with EpCAM to selectively purify FOXN1(+) TEC progenitors from differentiating cultures of unmanipulated PSCs. Human FOXN1(+) TEC progenitors generated from PSCs facilitate the study of thymus biology and are a valuable resource for future applications in regenerative medicine.