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How CD4 + lineage gene expression is initiated in differentiating thymocytes remains poorly understood. Here, we show that the paralog transcription factors Zfp281 and Zfp148 control both this process and cytokine expression by T helper cell type 2 (T H 2) effector cells. Genetic, single-cell, and spatial transcriptomic analyses showed that these factors promote the intrathymic CD4 + T cell differentiation of class II major histocompatibility complex (MHC II)-restricted thymocytes, including expression of the CD4 + lineage-committing factor Thpok. In peripheral T cells, Zfp281 and Zfp148 promoted chromatin opening at and expression of T H 2 cytokine genes but not of the T H 2 lineage-determining transcription factor Gata3. We found that Zfp281 interacts with Gata3 and is recruited to Gata3 genomic binding sites at loci encoding Thpok and T H 2 cytokines. Thus, Zfp148 and Zfp281 collaborate with Gata3 to promote CD4 + T cell development and T H 2 cell responses.

CD4 + T helper 17 (T H 17) cells protect barrier tissues but also trigger autoimmunity. The mechanisms behind these opposing processes remain unclear. Here, we found that the transcription factor EGR2 controlled the transcriptional program of pathogenic T H 17 cells in the central nervous system (CNS) but not that of protective T H 17 cells at barrier sites. EGR2 was significantly elevated in myelin-reactive CD4 + T cells from patients with multiple sclerosis and mice with autoimmune neuroinflammation. The EGR2 transcriptional program was intricately woven within the T H 17 cell transcriptional regulatory network and showed high interconnectivity with core T H 17 cell-specific transcription factors. Mechanistically, EGR2 enhanced T H 17 cell differentiation and myeloid cell recruitment to the CNS by upregulating pathogenesis-associated genes and myelomonocytic chemokines. T cell-specific deletion of Egr2 attenuated neuroinflammation without compromising the host's ability to control infections. Our study shows that EGR2 regulates tissue-specific and disease-specific functions in pathogenic T H 17 cells in the CNS., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Many analytical or cell culture procedures require homogeneous starting cell populations that cannot be obtained directly from organ dissection. Here, we describe two enrichment procedures to achieve this goal and discuss their respective advantages in specific experimental contexts., (© 2023. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Intra-thymic injection is a powerful tool for adoptive transfer of cells, cellular tag reagents for tracking recent thymic emigrants (RTEs), or other substances directly into the thymus. The traditional approach developed decades ago requires an invasive surgery to open the thoracic cavity and visualize the thymus. Subsequently, a technique was developed requiring only a small skin incision needed to identify the precise injection site. Nevertheless, both techniques require surgical intervention, and this can lead to elevated animal stress levels and pain which necessitates analgesic medication administration. Here we describe a less invasive technique allowing in situ visualization and transfer of cell suspensions or substances into the thymus via an ultrasound-guided intra-thymic injection approach., (© 2023. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

While T cell receptor (TCR) αβ + CD8α + CD8β - intraepithelial lymphocytes (CD8αα + IELs) differentiate from thymic IEL precursors (IELps) and contribute to gut homeostasis, the transcriptional control of their development remains poorly understood. In the present study we showed that mouse thymocytes deficient for the transcription factor leukemia/lymphoma-related factor (LRF) failed to generate TCRαβ + CD8αα + IELs and their CD8β-expressing counterparts, despite giving rise to thymus and spleen CD8αβ + T cells. LRF-deficient IELps failed to migrate to the intestine and to protect against T cell-induced colitis, and had impaired expression of the gut-homing integrin α4β7. Single-cell RNA-sequencing found that LRF was necessary for the expression of genes characteristic of the most mature IELps, including Itgb7, encoding the β7 subunit of α4β7. Chromatin immunoprecipitation and gene-regulatory network analyses both defined Itgb7 as an LRF target. Our study identifies LRF as an essential transcriptional regulator of IELp maturation in the thymus and subsequent migration to the intestinal epithelium., (© 2022. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

During the immune response, CD4+ T cells differentiate into distinct effector subtypes, including follicular helper T (Tfh) cells that help B cells, and into memory cells. Tfh and memory cells are required for long-term immunity; both depend on the transcription factor Bcl6, raising the question whether they differentiate through similar mechanisms. Here, using single-cell RNA and ATAC sequencing, we show that virus-responding CD4+ T cells lacking both Bcl6 and Blimp1 can differentiate into cells with transcriptomic, chromatin accessibility, and functional attributes of memory cells but not of Tfh cells. Thus, Bcl6 promotes memory cell differentiation primarily through its repression of Blimp1. These findings demonstrate that distinct mechanisms underpin the differentiation of memory and Tfh CD4+ cells and define the Bcl6-Blimp1 axis as a potential target for promoting long-term memory T cell differentiation., Competing Interests: Disclosures: The authors declare no competing interests exist., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)

Analysis of the transcriptional profiles of developing thymocytes has shown that T lineage commitment is associated with loss of stem cell and early progenitor gene signatures and the acquisition of T cell gene signatures. Less well understood are the epigenetic alterations that accompany or enable these transcriptional changes. Here, we show that the histone demethylase Lsd1 (Kdm1a) performs a key role in extinguishing stem/progenitor transcriptional programs in addition to key repressive gene programs during thymocyte maturation. Deletion of Lsd1 caused a block in late T cell development and resulted in overexpression of interferon response genes as well as genes regulated by the Gfi1, Bcl6, and, most prominently, Bcl11b transcriptional repressors in CD4+CD8+ thymocytes. Transcriptional overexpression in Lsd1-deficient thymocytes was not always associated with increased H3K4 trimethylation at gene promoters, indicating that Lsd1 indirectly affects the expression of many genes. Together, these results identify a critical function for Lsd1 in the epigenetic regulation of multiple repressive gene signatures during T cell development., Competing Interests: Disclosures: The authors declare no competing interests exist., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)

αβ lineage T cells, most of which are CD4 + or CD8 + and recognize MHC I- or MHC II-presented antigens, are essential for immune responses and develop from CD4 + CD8 + thymocytes. The absence of in vitro models and the heterogeneity of αβ thymocytes have hampered analyses of their intrathymic differentiation. Here, combining single-cell RNA and ATAC (chromatin accessibility) sequencing, we identified mouse and human αβ thymocyte developmental trajectories. We demonstrated asymmetric emergence of CD4 + and CD8 + lineages, matched differentiation programs of agonist-signaled cells to their MHC specificity, and identified correspondences between mouse and human transcriptomic and epigenomic patterns. Through computational analysis of single-cell data and binding sites for the CD4 + -lineage transcription factor Thpok, we inferred transcriptional networks associated with CD4 + - or CD8 + -lineage differentiation, and with expression of Thpok or of the CD8 + -lineage factor Runx3. Our findings provide insight into the mechanisms of CD4 + and CD8 + T cell differentiation and a foundation for mechanistic investigations of αβ T cell development., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier Inc.)

RORγt is well recognized as the lineage-defining transcription factor for T helper 17 (T H 17) cell development. However, the cell-intrinsic mechanisms that negatively regulate T H 17 cell development and autoimmunity remain poorly understood. Here, we demonstrate that the transcriptional repressor REV-ERBα is exclusively expressed in T H 17 cells, competes with RORγt for their shared DNA consensus sequence, and negatively regulates T H 17 cell development via repression of genes traditionally characterized as RORγt dependent, including Il17a. Deletion of REV-ERBα enhanced T H 17-mediated pro-inflammatory cytokine expression, exacerbating experimental autoimmune encephalomyelitis (EAE) and colitis. Treatment with REV-ERB-specific synthetic ligands, which have similar phenotypic properties as RORγ modulators, suppressed T H 17 cell development, was effective in colitis intervention studies, and significantly decreased the onset, severity, and relapse rate in several models of EAE without affecting thymic cellularity. Our results establish that REV-ERBα negatively regulates pro-inflammatory T H 17 responses in vivo and identifies the REV-ERBs as potential targets for the treatment of T H 17-mediated autoimmune diseases., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

To track recent thymic emigrants (RTEs) or study T cell development in the thymus, intra-thymic injection of a cellular tag or precursor cells for various T cell lineages is often desired. However, the traditional surgical approach to expose the thymus for intra-thymic injection is time-consuming and can cause a high level of pain and stress to animals, which might disrupt immune homeostasis, potentially confounding the results. Here, we introduce an ultrasound guided intra-thymic injection procedure, which is non-surgical and minimally invasive to animals. This technique is relatively easy to learn and offers an efficient and accurate tool to track RTEs or perform intra-thymic transfer of various cell types to investigate their differentiation., Competing Interests: Competing interestsThe authors declare no competing financial interests., (Copyright © The Authors; exclusive licensee Bio-protocol LLC.)

The CD4 + lineage-specific transcription factor Thpok is required for intrathymic CD4 + T cell differentiation and, together with its homolog LRF, supports CD4 + T cell helper effector responses. However, it is not known whether these factors are needed for the regulatory T cell (Treg) arm of MHC class II responses. In this study, by inactivating in mice the genes encoding both factors in differentiated Tregs, we show that Thpok and LRF are redundantly required to maintain the size and functions of the postthymic Treg pool. They support IL-2-mediated gene expression and the functions of the Treg-specific factor Foxp3. Accordingly, Treg-specific disruption of Thpok and Lrf causes a lethal inflammatory syndrome similar to that resulting from Treg deficiency. Unlike in conventional T cells, Thpok and LRF functions in Tregs are not mediated by their repression of the transcription factor Runx3. Additionally, we found that Thpok is needed for the differentiation of thymic Treg precursors, an observation in line with the fact that Foxp3 + Tregs are CD4 + cells. Thus, a common Thpok-LRF node supports both helper and regulatory arms of MHC class II responses.

Antigen-specific T cell responses can be visualized using MHC:peptide multimers. In cases where robust T cell controls are not readily available to assess the integrity of multimer reagents prior to analyzing limited sample, the ability to assess the structural integrity of MHC multimers before their use in critical experiments would be useful. We present a method to probe the structural integrity of MHC multimers using antibodies specific for conformational determinants. Beads coated with anti-mouse Ig are incubated with conformation-specific mouse monoclonal antibody and then with fluorescently tagged MHC multimer. The ability of the bead to capture the labeled multimer can be measured semi-quantitatively by flow cytometry. In this manner, the correct folding of MHC multimers can be visualized and batches of multimer can be compared for quality control. Because there are multiple conformational epitopes formed by various molecular interactions among heavy chain, peptide, and β2M, this capture assay can assess the fidelity of each aspect of multimer structure, depending on the availability of antibodies. The described approach could be particularly useful for studies using irreplaceable samples, including patient samples collected in clinical trials.