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.
Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) is a rare life-threatening autoimmune disease that attacks multiple organs and has its onset in childhood. It is an inherited ...condition caused by a variety of mutations in the autoimmune regulator (
gene that encodes a protein whose function has been uncovered by the generation and study of
-KO mice. These provided invaluable insights into the link between
expression in medullary thymic epithelial cells (mTECs), and the broad spectrum of self-antigens that these cells express and present to the developing thymocytes. However, these murine models poorly recapitulate all phenotypic aspects of human APECED. Unlike
-KO mice, the recently generated
-KO rat model presents visual features, organ lymphocytic infiltrations and production of autoantibodies that resemble those observed in APECED patients, making the rat model a main research asset. In addition,
models of AIRE-dependent self-antigen expression in primary mTECs have been successfully set up. Thymus organoids based on pluripotent stem cell-derived TECs from APECED patients are also emerging, and constitute a promising tool to engineer
-corrected mTECs and restore the generation of regulatory T cells. Eventually, these new models will undoubtedly lead to main advances in the identification and assessment of specific and efficient new therapeutic strategies aiming to restore immunological tolerance in APECED patients.
Interactions of developing T cells with Aire
medullary thymic epithelial cells expressing high levels of MHCII molecules (mTEC
) are critical for the induction of central tolerance in the thymus. In ...turn, thymocytes regulate the cellularity of Aire
mTEC
. However, it remains unknown whether thymocytes control the precursors of Aire
mTEC
that are contained in mTEC
cells or other mTEC
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
and control the composition of mTEC
subsets, including Aire
mTEC
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
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.
Aire allows medullary thymic epithelial cells (mTECs) to express and present a large number of self‐antigens for central tolerance. Although mTECs express a high diversity of self‐antigen splice ...isoforms, the extent and regulation of alternative splicing events (ASEs) in their transcripts, notably in those induced by Aire, is unknown. In contrast to Aire‐neutral genes, we find that transcripts of Aire‐sensitive genes show only a low number of ASEs in mTECs, with about a quarter present in peripheral tissues excluded from the thymus. We identify Raver2, as a splicing‐related factor overexpressed in mTECs and dependent on H3K36me3 marks, that promotes ASEs in transcripts of Aire‐neutral genes, leaving Aire‐sensitive ones unaffected. H3K36me3 profiling reveals its depletion at Aire‐sensitive genes and supports a mechanism that is preceding Aire expression leading to transcripts of Aire‐sensitive genes with low ASEs that escape Raver2‐induced alternative splicing. The lack of ASEs in Aire‐induced transcripts would result in an incomplete Aire‐dependent negative selection of autoreactive T cells, thus highlighting the need of complementary tolerance mechanisms to prevent activation of these cells in the periphery.
SYNOPSIS
Genes in the thymic epithelium sensitive to Aire expression are characterized by low levels of H3K36me3 marks and show few alternative splicing events (ASEs) in their transcripts, as they escape the splicing‐related factor Raver2.
Aire‐sensitive genes in mTECs are devoid of H3K36me3 and escape Raver2.
Aire‐neutral genes show high levels of H3K36m3 and many ASEs in their transcripts.
In peripheral tissues Aire‐sensitive genes are enriched for H3K36me3 and show higher rates of alternative splicing.
Aire‐induced transcripts show a fraction of all ASEs, suggesting an incomplete Aire‐dependent negative selection.
Genes in the thymic epithelium sensitive to Aire expression are characterized by low levels of H3K36me3 marks and show few alternative splicing events (ASEs) in their transcripts, as they escape the splicing‐related factor Raver2.
Thirteen states associated with the Southern Extension and Research Activities Information Exchange Group‐6 (SERA‐IEG‐6) agreed to share their soil test based P and K rate recommendations for nine ...major crops. The objectives were to compare fertilizer P and K rate recommendations, to look for opportunities to rationalize similar recommendations across state lines, and to examine challenges to the development of a cooperative regional approach to P and K recommendations. Mehlich‐3 (eight states), Mehlich‐1 (five states), or Lancaster (one state) extractions were the basis of plant available soil P (STP) and K (STK) assessment. Fertilizer recommendation philosophies (sufficiency, build and maintain, and/or hybrid) variation among the states might be the main reason behind such discrepancies. Although a few similarities in P and K rate recommendations were found, the different philosophies, numerical presentations, and extraction procedures drove important recommendation differences. Widespread adoption of the Mehlich‐3 extraction procedure has not reduced variation in fertilizer P and K rate recommendations among the states. Instead, for states using Mehlich 3, soil test critical concentrations ranged from 30 to 75 mg P kg–1 and 60 to 175 mg K kg–1 for corn (Zea mays L.) grain and warm‐season grass hay production. The adoption of uniform soil testing terminology, sample collection guidelines, extraction methods, and interpretations across common physiographic regions, soils, and state lines remains a challenge. Differences arise because of the different soil orders and properties, climate conditions, and resulting crop responses to added P and K fertilizers. Such differences in soil‐test‐based fertilizer P and K recommendations are state specific and highlight needs to examine the soil testing and recommendation process, make soil test results end‐user friendly, and, when appropriate, standardize fundamental information used in the soil testing guidelines.
Core Ideas
Soil test‐based P and K fertilizer recommendations vary widely across the southern U.S. states.
Mehlich‐3 and Mehlich‐1 extractions were the basis of plant available soil P and K assessment.
Differences in P and K rate recommendations exists even with a consistent set of extractants.
Recommendations differences are attributed to philosophical and soil properties differences.
More consistent P and K recommendations would enhance the creditability of soil testing.
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