Bruno Kyewski

The basic two-step terminal differentiation model of the medullary thymic epithelial cell (mTEC) lineage from immature MHC class II (MHCII)^lo to mature MHCII^hi mTECs has recently been extended to include a third stage, namely the post-Aire MHCII^lo subset as identified by lineage-tracing models. However, a suitable surface marker distinguishing the phenotypically overlapping pre- from the post-Aire MHCII^lo stage has been lacking. In this study, we introduce the lectin Tetragonolobus purpureas agglutinin (TPA) as a novel cell surface marker that allows for such delineation. Based on our data, we derived the following sequence of mTEC differentiation: TPA^loMHCII^lo → TPA^loMHCII^hi → TPA^hiMHCII^hi → TPA^hiMHCII^lo. Surprisingly, in the steady-state postnatal thymus TPA^loMHCII^lo pre-Aire rather than terminally differentiated post-Aire TPA^hiMHCII^lo mTECs were marked for apoptosis at an exceptionally high rate of ∼70%. Hence, only the minor cycling fraction of the MHCII^lo subset (lo mTECs points to a “quality control” step during early mTEC differentiation.

The origin of the thymic epithelium, i.e. the cortical (cTEC) and medullary (mTEC) epithelial cells, from bipotent stem cells through TEC progenitors and lineage‐specific progeny still remains poorly understood. We sought to obtain an unbiased view of the incipient emergence of TEC subsets by following embryonic TEC development based on co‐expression of EpCAM, CD80 and MHC class II (MHCII) on non‐hematopoietic (CD45⁻) thymic stromal cells in wild‐type BL6 mice. Using a combination of ex vivo analysis, Re‐aggregate Thymic Organ Culture (RTOC) reconstitution assays and mathematical modeling, we traced emergent lineage commitment in murine embryonic TECs. Both experimental and mathematical datasets supported the following developmental sequence: MHCII⁻CD80⁻ → MHCII^loCD80⁻ → MHCII^hiCD80⁻ → MHCII^hiCD80^hi TECs, whereby MHCII^hiCD80⁻ and MHCII^hiCD80^hi TECs bear features of cTECs and mTECs respectively. These emergent MHCII^hiCD80⁻ cTECs directly generate mature MHCII^hiCD80^hi mTECs in vivo and in vitro, thus supporting the asynchronous model of TEC lineage commitment.

The first signs of autoimmune activation leading to β-cell destruction in type 1 diabetes (T1D) appear during the first months of life. Thus, the perinatal period offers a suitable time window for disease prevention. Moreover, thymic selection of autoreactive T cells is most active during this period, providing a therapeutic opportunity not exploited to date. We therefore devised a strategy by which the T1D-triggering antigen preproinsulin fused with the immunoglobulin (Ig)G Fc fragment (PPI-Fc) is delivered to fetuses through the neonatal Fc receptor (FcRn) pathway, which physiologically transfers maternal IgGs through the placenta. PPI-Fc administered to pregnant PPIB15–23 T-cell receptor–transgenic mice efficiently accumulated in fetuses through the placental FcRn and protected them from subsequent diabetes development. Protection relied on ferrying of PPI-Fc to the thymus by migratory dendritic cells and resulted in a rise in thymic-derived CD4+ regulatory T cells expressing transforming growth factor-β and in increased effector CD8+ T cells displaying impaired cytotoxicity. Moreover, polyclonal splenocytes from nonobese diabetic (NOD) mice transplacentally treated with PPI-Fc were less diabetogenic upon transfer into NOD.scid recipients. Transplacental antigen vaccination provides a novel strategy for early T1D prevention and, further, is applicable to other immune-mediated conditions.

Immunity to tumor differentiation antigens, such as melanoma antigen recognized by T cells 1 (MART‐1), has been comprehensively studied. Intriguingly, CD8⁺ T cells specific for the MART‐1_26(27)‐35 epitope in the context of HLA‐A0201 are about 100 times more abundant compared with T cells specific for other tumor‐associated antigens. Moreover, MART‐1‐specific CD8⁺ T cells show a highly biased usage of the Vα‐region gene TRAV12–2. Here, we provide independent support for this notion, by showing that the combinatorial pairing of different TCRα‐ and TCRβ‐ chains derived from HLA‐A2–MART‐1_26–35‐specific CD8⁺ T‐cell clones is unusually permissive in conferring MART‐1 specificity, provided the CDR1α TRAV12–2 region is used. Whether TCR bias alone accounts for the unusual abundance of HLA‐A2–MART‐1_26–35‐specific CD8⁺ T cells has remained conjectural. Here, we provide an alternative explanation: misinitiated transcription of the MART‐1 gene resulting in truncated mRNA isoforms leads to lack of promiscuous transcription of the MART‐1_26–35 epitope in human medullary thymic epithelial cells and, consequently, evasion of central self‐tolerance toward this epitope. Thus, biased TCR usage and leaky central tolerance might act in an independent and additive manner to confer high frequency of MART‐1_26–35‐specific CD8⁺ T cells.

Significance: The ability of the immune system to distinguish self from foreign (“self-tolerance”) is largely established in the thymus, a primary lymphoid organ where T cells develop. Intriguingly, T cells encounter most tissue-specific constituents already in the thymus, thus imposing a broad scope of tolerance before T cells circulate through the body. This preemption of the “immunological self” is afforded by the “promiscuous” expression of numerous tissue-specific antigens in medullary thymic epithelial cells. Here, we identified principles by which promiscuous gene expression at the single-cell level adds up to the full diversity of self-antigens displayed at the population level.

The establishment and maintenance of central tolerance depends to a large extent on the ability of medullary thymic epithelial cells to express a variety of tissue‐restricted antigens, the so‐called promiscuous gene expression (pGE). Autoimmune regulator (Aire) is to date the best characterised transcriptional regulator known to at least partially coordinate pGE. There is accruing evidence that the expression of Aire‐dependent and ‐independent genes is modulated by higher order chromatin configuration, epigenetic modifications and post‐transcriptional control. Given the involvement of microRNAs (miRNAs) as potent post‐transcriptional modulators of gene expression, we investigated their role in the regulation of pGE in purified mouse and human thymic epithelial cells (TECs). Microarray profiling of TEC subpopulations revealed evolutionarily conserved cell type and differentiation‐specific miRNA signatures with a subset of miRNAs being significantly upregulated during terminal medullary thymic epithelial cell differentiation. The differential regulation of this subset of miRNAs was correlated with Aire expression and some of these miRNAs were misexpressed in the Aire knockout thymus. In turn, the specific absence of miRNAs in TECs resulted in a progressive reduction of Aire expression and pGE, affecting both Aire‐dependent and ‐independent genes. In contrast, the absence of miR‐29a only affected the Aire‐dependent gene pool. These findings reveal a mutual interdependence of miRNA and Aire.