Matthieu Giraud

The non-canonical NF-κB pathway, in which NFKB2 plays a central role, is critical for lymphoid organ development and immune homeostasis. Pathogenic NFKB2 variants have been strongly associated with a broad spectrum of human pathologies, notably immunodysregulation syndromes, characterized by recurrent infections, autoantibody production and endocrine abnormalities. The mechanisms underlying the resulting immune defects remain incompletely understood. In the present study, we characterized the immune phenotype of a novel rat model harboring a Nfkb2 non-sense mutation located in the c- terminal region of the gene (c.2602T>A; p.Tyr867*, Nfkb2 ^p.Tyr867* ). This variant has been identified in patients with a familial form of common variable immunodeficiency (CVID) presenting with autoimmunity manifestations. Using this model, we demonstrate that mutated NFKB2 resulted in profound alterations of the thymic microenvironment, including reduced K5 ⁺ medullary thymic epithelial cells and decreased AIRE expression, accompanied by altered thymocyte development and impaired thymic regulatory T populations. Peripheral lymphoid organization was also profoundly affected, with absence of lymph nodes, defects in the splenic marginal zone, and alterations of CD4 ⁺ and CD8 ⁺ T-cell homeostasis, including impaired Treg populations and reduced ICOS expression on CD4 ⁺ T cells. The B-cell compartment was markedly altered, with impaired B-cell maturation, characterized by accumulation of IgM ⁺ IgD ⁻ transitional cells and a marked reduction of IgD ⁺ mature B cells. These abnormalities were associated with impaired humoral immunity and complete absence of circulating immunoglobulins. Collectively, our findings define a systemic combined immunodeficiency phenotype in the Nfkb2-mutated rat, highlighting its utility for studying the role of non-canonical NF-kB signaling in immune system organization and adaptative immunity.

Promiscuous expression of tissue-specific antigens (TSAs) by medullary thymic epithelial cells (mTECs) underpins central T-cell tolerance, relying on NF-κB-driven mTEC maturation and induction of the autoimmune regulator AIRE. However, a substantial fraction of TSAs is AIRE-independent, implying additional regulators that remain only partially identified. Using cross-species single-cell chromatin accessibility profiling of human and mouse TECs, we find that HIVEP motifs are among the most accessible regulatory elements across the mTEC lineage. Among HIVEP paralogs, HIVEP3 is robustly expressed in mature mTECs across human, mouse and rat; given that rat immunity more closely mirrors human physiology than the mouse, we generated a CRISPR Hivep3 -knockout rat line. Hivep3 deletion impairs mTEC maturation, reduces Aire expression and reshapes the TSA repertoire that underlies negative selection and Treg induction, as Hivep3 constrains canonical NF-κB1 and sustains non-canonical NF-κB2, the principal mediator of mTEC maturation. Beyond mTECs, Hivep3 deficiency downregulates Foxn1 target genes in cortical TECs and impairs positive selection. In the periphery, Hivep3 -KO rats show reduced splenic CD4⁺ T-cells, a shift from naïve towards effector-and central-memory phenotypes, increased regulatory T-cells, and a sustained Th1-biased serum profile (elevated IFN-γ, reduced IL-17A) with concurrent systemic metabolic perturbations across all ages. With age, this culminates in chronic inflammation, with multi-organ CD3⁺ T-cell infiltration accompanied by inflammatory lesions. Together, these findings establish HIVEP3 as a previously unrecognised regulator of thymic epithelial function and central tolerance, whose deficiency leads to systemic T-cell-mediated chronic inflammation.

The thymus educates thymocytes through a selection process mediated by thymic epithelial cells (TECs). Recent advances have made the generation of T lymphocytes from induced pluripotent stem cells (iPSc) a promising therapeutic strategy. However, current approaches often fail to replicate the thymic niche, leading to impaired T cell generation. Here we address the production of functional mature iPSc-derived TECs supporting in vitro T cell generation. We optimize thymic lineage differentiation through an unbiased multifactorial experimental design. By modulating specific signaling pathways, we generate progenitors that mature into medullary and cortical TECs. Co-culture with primary hematopoietic progenitors in a 3D thymic organoid setup induces their differentiation into CD4 ⁺ and CD8 ⁺ T cells. Importantly, thymic organoids support multilineage differentiation, with dendritic cell populations also emerging. Thus, the presented thymic organoid model provides a practical platform for studying thymic cellular interactions and thymopoiesis in vitro, and opens further research perspectives towards cell-based therapies.

It has been more than 20 years since the AIRE gene was discovered. The mutations in the AIRE gene cause a rare and life-threatening autoimmune disease with severe manifestations against a variety of organs. Since the identification of the AIRE gene in 1997, more than two decades of investigations have revealed key insights into the role of AIRE and its mode of action. These studies have shown that AIRE uniquely induces the expression of thousands of tissue-restricted self-antigens in the thymus. These self-antigens are presented to developing T cells, resulting in the deletion of the self-reactive T cells and the generation of regulatory T cells. Thus, AIRE is a master guardian in establishing and maintaining central immune tolerance.

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the properties of its extracellular matrix (ECM). Thymic epithelial cells (TECs) are crucial in thymopoiesis, nurturing and selecting developing T cells by filtering self-reactive clones. TECs have been empirically demonstrated to be particularly sensitive to physical and chemical clues supplied by the ECM and classical monolayer cell culture leads to a quick loss of functionality until their death. Because of this delicate maintenance combined with relative rarity, and despite the high stakes in modeling thymus biology in vitro, models able to faithfully mimic the TEC niche at scale and over time are still lacking. Here, we describe the formation of a multicellular human thymic organoid model, in which the TEC compartment is derived from human induced pluripotent stem cells (iPSC) and reaggregated with primary early thymocyte progenitors in a three-dimensional (3D) fibrin-based hydrogel. This model answers current needs for a scalable culture system that reproduces the thymic microenvironment ex vivo and demonstrates functionality, i.e., the ability to produce T cells and to support thymus organoid growth over several weeks. Thus, we propose a practical in vitro model of thymus functionality through iPSC-derived organoids that would benefit research on TEC biology and T cell generation ex vivo.

Editorial: Thymic function at single cell resolution

The thymus is a primary lymphoid organ playing a crucial role in immune tolerance, by educating thymocytes through a selection mediated by thymic epithelial cells (TEC). Recent advances in gene editing and immunotherapies have made in vitro generation of iPSC-derived T cells a crucial issue with promising therapeutic applications. Current state-of-the-art approaches often fail to accurately replicate the thymic niche, resulting in impaired T cell generation, particularly for CD4+ T cells. Here we address the production of functional mature iPSc-derived TECs that are able to support in vitro T cell generation. We designed a protocol allowing iPSc differentiation into thymic epithelial progenitors (TEP) through an unbiased multifactorial method based on optimal experimental design. Modulation of signaling pathways known to regulate embryonic thymus organogenesis resulted in the obtention of TEPs expressing typical thymic markers. We achieved TEP maturation into medullary and cortical TECs by setting up a hydrogel-based 3D culture supplemented in RANK ligand, resulting in the formation of thymic organoids. To assess the functionality of the generated TECs, primary hematopoietic progenitors were co-cultured in the organoids and showed significantly improved maturation in single-positive CD4 or CD8 T cells. Remarkably, our thymic organoid model shows multilineage differentiation potential, with generation of distinct dendritic cells populations in addition to the T lineages. Thus, generation of functional TECs and thymic organoids offers a practical platform for the study of thymic cellular interactions and paves the road to future cellular therapies by producing mature thymic immune cells of clinical interest.

The thymus is a primary lymphoid organ essential for the induction of central immune tolerance. Maturing T cells undergo several steps of expansion and selection mediated by thymic epithelial cells (TECs). In APECED and other congenital pathologies, a deficiency in genes that regulate TEC development or their ability to select non auto-reactive thymocytes results in a defective immune balance, and consequently in a general autoimmune syndrome. Restoration of thymic function is thus crucial for the emergence of curative treatments. The last decade has seen remarkable progress in both gene editing and pluripotent stem cell differentiation, with the emergence of CRISPR-based gene correction, the trivialization of reprogramming of somatic cells to induced pluripotent stem cells (iPSc) and their subsequent differentiation into multiple cellular fates. The combination of these two approaches has paved the way to the generation of genetically corrected thymic organoids and their use to control thymic genetic pathologies affecting self-tolerance. Here we review the recent advances in differentiation of iPSc into TECs and the ability of the latter to support a proper and efficient maturation of thymocytes into functional and non-autoreactive T cells. A special focus is given on thymus organogenesis and pathway modulation during iPSc differentiation, on the impact of the 2/3D structure on the generated TECs, and on perspectives for therapeutic strategies in APECED based on patient-derived iPSc corrected for AIRE gene mutations.

Thymically-derived Foxp3⁺ regulatory T cells (T_reg) critically control immunological tolerance. These cells are generated in the medulla through high affinity interactions with medullary thymic epithelial cells (mTEC) expressing the Autoimmune regulator (Aire). Recent advances have revealed that thymic T_reg contain not only developing but also recirculating cells from the periphery. Although Aire is implicated in the generation of Foxp3⁺ T_reg, its role in the biology of recirculating T_reg remains elusive. Here, we show that Aire regulates the suppressive signature of recirculating T_reg independently of the remodeling of the medullary 3D organization throughout life where T_reg reside. Accordingly, the adoptive transfer of peripheral Foxp3⁺ T_reg in Aire^KO recipients led to an impaired suppressive signature upon their entry into the thymus. Furthermore, recirculating T_reg from Aire^KO mice failed to attenuate the severity of multiorgan autoimmunity, demonstrating that their suppressive function is altered. Using bone marrow chimeras, we reveal that mTEC-specific expression of Aire controls the suppressive signature of recirculating T_reg. Finally, mature mTEC lacking Aire were inefficient in stimulating peripheral T_reg both in polyclonal and antigen-specific co-culture assays. Overall, this study demonstrates that Aire confers to mTEC the ability to restimulate recirculating T_reg, unravelling a novel function for this master regulator in T_reg biology.

Interactions of developing T cells with Aire⁺ medullary thymic epithelial cells expressing high levels of MHCII molecules (mTEC^hi) are critical for the induction of central tolerance in the thymus. In turn, thymocytes regulate the cellularity of Aire⁺ mTEC^hi. However, it remains unknown whether thymocytes control the precursors of Aire⁺ mTEC^hi that are contained in mTEC^lo cells or other mTEC^lo subsets that have recently been delineated by single-cell transcriptomic analyses. Here, using three distinct transgenic mouse models, in which antigen presentation between mTECs and CD4⁺ thymocytes is perturbed, we show by high-throughput RNA-seq that self-reactive CD4⁺ thymocytes induce key transcriptional regulators in mTEC^lo and control the composition of mTEC^lo subsets, including Aire⁺ mTEC^hi precursors, post-Aire and tuft-like mTECs. Furthermore, these interactions upregulate the expression of tissue-restricted self-antigens, cytokines, chemokines, and adhesion molecules important for T-cell development. This gene activation program induced in mTEC^lo is combined with a global increase of the active H3K4me3 histone mark. Finally, we demonstrate that these self-reactive interactions between CD4⁺ thymocytes and mTECs critically prevent multiorgan autoimmunity. Our genome-wide study thus reveals that self-reactive CD4⁺ thymocytes control multiple unsuspected facets from immature stages of mTECs, which determines their heterogeneity.