The transcription factor LRF promotes integrin ß7 expression by and gut homing of CD8αα+ intraepithelial lymphocyte precursors.

While T cell receptor (TCR) αß+CD8α+CD8ß- 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 TCRαß+CD8αα+ IELs and their CD8ß-expressing counterparts, despite giving rise to thymus and spleen CD8αß+ 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 α4ß7. Single-cell RNA-sequencing found that LRF was necessary for the expression of genes characteristic of the most mature IELps, including Itgb7, encoding the ß7 subunit of α4ß7. 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.

An Integrated Epigenomic and Transcriptomic Map of Mouse and Human αß T Cell Development.

αß 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 αß thymocytes have hampered analyses of their intrathymic differentiation. Here, combining single-cell RNA and ATAC (chromatin accessibility) sequencing, we identified mouse and human αß 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 αß T cell development.

The Emergence and Functional Fitness of Memory CD4+ T Cells Require the Transcription Factor Thpok.

Memory CD4+ T cells mediate long-term immunity, and their generation is a key objective of vaccination strategies. However, the transcriptional circuitry controlling the emergence of memory cells from early CD4+ antigen-responders remains poorly understood. Here, using single-cell RNA-seq to study the transcriptome of virus-specific CD4+ T cells, we identified a gene signature that distinguishes potential memory precursors from effector cells. We found that both that signature and the emergence of memory CD4+ T cells required the transcription factor Thpok. We further demonstrated that Thpok cell-intrinsically protected memory cells from a dysfunctional, effector-like transcriptional program, similar to but distinct from the exhaustion pattern of cells responding to chronic infection. Mechanistically, Thpok- bound genes encoding the transcription factors Blimp1 and Runx3 and acted by antagonizing their expression. Thus, a Thpok-dependent circuitry promotes both memory CD4+ T cells' differentiation and functional fitness, two previously unconnected critical attributes of adaptive immunity.

A Thpok-Directed Transcriptional Circuitry Promotes Bcl6 and Maf Expression to Orchestrate T Follicular Helper Differentiation.

The generation of high-affinity neutralizing antibodies, the objective of most vaccine strategies, occurs in B cells within germinal centers (GCs) and requires rate-limiting "help" from follicular helper CD4+ T (Tfh) cells. Although Tfh differentiation is an attribute of MHC II-restricted CD4+ T cells, the transcription factors driving Tfh differentiation, notably Bcl6, are not restricted to CD4+ T cells. Here, we identified a requirement for the CD4+-specific transcription factor Thpok during Tfh cell differentiation, GC formation, and antibody maturation. Thpok promoted Bcl6 expression and bound to a Thpok-responsive region in the first intron of Bcl6. Thpok also promoted the expression of Bcl6-independent genes, including the transcription factor Maf, which cooperated with Bcl6 to mediate the effect of Thpok on Tfh cell differentiation. Our findings identify a transcriptional program that links the CD4+ lineage with Tfh differentiation, a limiting factor for efficient B cell responses, and suggest avenues to optimize vaccine generation.

A ThPOK-LRF transcriptional node maintains the integrity and effector potential of post-thymic CD4+ T cells.

The transcription factor ThPOK promotes CD4(+) T cell differentiation in the thymus. Here, using a mouse strain that allows post-thymic gene deletion, we show that ThPOK maintains CD4(+) T lineage integrity and couples effector differentiation to environmental cues after antigenic stimulation. ThPOK preserved the integrity and amplitude of effector responses and was required for proper differentiation of types 1 and 2 helper T cells in vivo by restraining the expression and function of Runx3, a nuclear factor crucial for cytotoxic T cell differentiation. The transcription factor LRF acts redundantly with ThPOK to prevent the transdifferentiation of mature CD4(+) T cells into CD8(+) T cells. As such, the ThPOK-LRF transcriptional module was essential for CD4(+) T cell integrity and responses.

Hic et Runx: new insights into T cell tissue residency.

Tissue-resident memory T cells (Trm), which typically do not enter the blood or lymphatic circulation at steady-state, are considered crucial for controlling pathogen entry at skin and mucosal barriers. Two recent studies (Fonseca et al. and Crowl et al.) shed light on the mechanisms of Trm cell differentiation.

The NF-κB regulator Bcl-3 restricts terminal differentiation and promotes memory cell formation of CD8+ T cells during viral infection.

Bcl-3 is an atypical member of the IκB family that acts in the nucleus to modulate transcription of many NF-κB targets in a highly context-dependent manner. Accordingly, complete Bcl-3-/- mice have diverse defects in both innate and adaptive immune responses; however, direct effects of Bcl-3 action in individual immune cell types have not been clearly defined. Here, we document a cell-autonomous role for Bcl-3 in CD8+ T cell differentiation during the response to lymphocytic choriomeningitis virus infection. Single-cell RNA-seq and flow cytometric analysis of virus-specific Bcl3-/- CD8+ T cells revealed that differentiation was skewed towards terminal effector cells at the expense of memory precursor effector cells (MPECs). Accordingly, Bcl3-/- CD8+ T cells exhibited reduced memory cell formation and a defective recall response. Conversely, Bcl-3-overexpression in transgenic CD8+ T cells enhanced MPEC formation but reduced effector cell differentiation. Together, our results establish Bcl-3 as an autonomous determinant of memory/terminal effector cell balance during CD8+ T cell differentiation in response to acute viral infection. Our results provide proof-of-principle for targeting Bcl-3 pharmacologically to optimize adaptive immune responses to infectious agents, cancer cells, vaccines and other stimuli that induce CD8+ T cell differentiation.

Control of the development of CD8αα+ intestinal intraepithelial lymphocytes by TGF-ß.

The molecular mechanisms that direct the development of TCRαß+CD8αα+ intestinal intraepithelial lymphocytes (IELs) are not thoroughly understood. Here we show that transforming growth factor-ß (TGF-ß) controls the development of TCRαß+CD8αα+ IELs. Mice with either a null mutation in the gene encoding TGF-ß1 or T cell-specific deletion of TGF-ß receptor I lacked TCRαß+CD8αα+ IELs, whereas mice with transgenic overexpression of TGF-ß1 had a larger population of TCRαß+CD8αα+ IELs. We observed defective development of the TCRαß+CD8αα+ IEL thymic precursors (CD4⁻CD8⁻TCRαß+CD5+) in the absence of TGF-ß. In addition, we found that TGF-ß signaling induced CD8α expression in TCRαß+CD8αα+ IEL thymic precursors and induced and maintained CD8α expression in peripheral populations of T cells. Our data demonstrate a previously unrecognized role for TGF-ß in the development of TCRαß+CD8αα+ IELs and the expression of CD8α in T cells.

Decision checkpoints in the thymus.

The development of T cells in the thymus involves several differentiation and proliferation events, during which hematopoietic precursors give rise to T cells ready to respond to antigen stimulation and undergo effector differentiation. This review addresses signaling and transcriptional checkpoints that control the intrathymic journey of T cell precursors. We focus on the divergence of alphabeta and gammadelta lineage cells and the elaboration of the alphabeta T cell repertoire, with special emphasis on the emergence of transcriptional programs that direct lineage decisions.

Themis, a T cell-specific protein important for late thymocyte development.

During positive selection, thymocytes transition through a stage during which T cell antigen receptor (TCR) signaling controls CD4-versus-CD8 lineage 'choice' and subsequent maturation. Here we describe a previously unknown T cell-specific protein, Themis, that serves a distinct function during this stage. In Themis(-/-) mice, thymocyte selection was impaired and the number of transitional CD4(+)CD8(int) thymocytes as well as CD4(+) or CD8(+) single-positive thymocytes was lower. Notably, although we detected no overt TCR-proximal signaling deficiencies, Themis(-/-) CD4(+)CD8(int) thymocytes showed developmental defects consistent with attenuated signaling that were reversible by TCR stimulation. Our results identify Themis as a critical component of the T cell developmental program and suggest that Themis functions to sustain and/or integrate signals required for proper lineage commitment and maturation.

The transcription factors Thpok and LRF are necessary and partly redundant for T helper cell differentiation.

T helper (Th) cells are critical for defenses against infection and recognize peptides bound to class II major histocompatibility complex (MHC II) molecules. Although transcription factors have been identified that direct Th cells into specific effector fates, whether a "master" regulator controls the developmental program common to all Th cells remains unclear. Here, we showed that the two transcription factors Thpok and LRF share this function. Although disruption of both factors did not prevent the generation of MHC II-specific T cells, these cells failed to express Th cell genes or undergo Th cell differentiation in vivo. In contrast, T cells lacking Thpok, which only displayed LRF-dependent functions, contributed to multiple effector responses, both in vitro and in vivo, with the notable exception of Th2 cell responses that control extracellular parasites. These findings identify the Thpok-LRF pair as a core node of Th cell differentiation and function.

Distinct functions for the transcription factors GATA-3 and ThPOK during intrathymic differentiation of CD4(+) T cells.

The transcription factors GATA-3 and ThPOK are required for intrathymic differentiation of CD4(+) T cells, but their precise functions in this process remain unclear. Here we show that, contrary to previous findings, Gata3 disruption blocked differentiation into the CD4(+) T cell lineage before commitment to the CD4(+) lineage and in some contexts permitted the 'redirection' of major histocompatibility complex class II-restricted thymocytes into the CD8(+) lineage. GATA-3 promoted ThPOK expression and bound to a region of the locus encoding ThPOK established as being critical for ThPOK expression. Finally, ThPOK promoted differentiation into the CD4(+) lineage in a way dependent on GATA-3 but inhibited differentiation into the CD8(+) lineage independently of GATA-3. We propose that GATA-3 acts as a specification factor for the CD4(+) lineage 'upstream' of the ThPOK-controlled CD4(+) commitment checkpoint.

RUNX transcription factor-mediated association of Cd4 and Cd8 enables coordinate gene regulation.

T cell fate is associated with mutually exclusive expression of CD4 or CD8 in helper and cytotoxic T cells, respectively. How expression of one locus is temporally coordinated with repression of the other has been a long-standing enigma, though we know RUNX transcription factors activate the Cd8 locus, silence the Cd4 locus, and repress the Zbtb7b locus (encoding the transcription factor ThPOK), which is required for CD4 expression. Here we found that nuclear organization was altered by interplay among members of this transcription factor circuitry: RUNX binding mediated association of Cd4 and Cd8 whereas ThPOK binding kept the loci apart. Moreover, targeted deletions within Cd4 modulated CD8 expression and pericentromeric repositioning of Cd8. Communication between Cd4 and Cd8 thus appears to enable long-range epigenetic regulation to ensure that expression of one excludes the other in mature CD4 or CD8 single-positive (SP) cells.

A STAT3-dependent transcriptional circuitry inhibits cytotoxic gene expression in T cells.

CD8+ T cells are preprogrammed for cytotoxic differentiation in the thymus as they acquire expression of the transcription factor Runx3. However, a subset of effector CD8+ T cells (Tc17) produce IL-17 and fail to express cytotoxic genes. Here, we show that the transcription factors directing IL-17 production, STAT3 and RORγt, inhibit cytotoxicity despite persistent Runx3 expression. Cytotoxic gene repression did not require the transcription factor Thpok, which in CD4+ T cells restrains Runx3 functions and cytotoxicity; and STAT3 restrained cytotoxic gene expression in CD8+ T cells responding to viral infection in vivo. STAT3-induced RORγt represses cytotoxic genes by inhibiting the functions but not the expression of the "cytotoxic" transcription factors T-bet and Eomesodermin. Thus, the transcriptional circuitry directing IL-17 expression inhibits cytotoxic functions. However, by allowing expression of activators of the cytotoxic program, this inhibitory mechanism contributes to the instability of IL-17-producing T cells.

Pleiotropic Functions of H3K27Me3 Demethylases in Immune Cell Differentiation.

The trimethylation of histone H3 lysine 27 (H3K27Me3) contributes to gene repression, notably through recruitment of Polycomb complexes, and has long been considered essential to maintain cell identity. Whereas H3K27Me3 was thought to be stable and not catalytically reversible, the discovery of the Utx and Jmjd3 demethylases changed this notion, raising new questions on the role of these enzymes in gene expression and cell differentiation. Recent studies have demonstrated critical roles for Utx and Jmjd3 in the development and function of immune cells, and revealed both demethylase and demethylase-independent activities of these enzymes. I review these finding here, and discuss the current understanding of the mechanisms that underlie the broad, yet highly cell- and gene-specific, impact of these enzymes in vivo.

Control of Regulatory T Cell Differentiation by the Transcription Factors Thpok and LRF.

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.

What Happens in the Thymus Does Not Stay in the Thymus: How T Cells Recycle the CD4+-CD8+ Lineage Commitment Transcriptional Circuitry To Control Their Function.

MHC-restricted CD4(+) and CD8(+) T cells are at the core of most adaptive immune responses. Although these cells carry distinct functions, they arise from a common precursor during thymic differentiation, in a developmental sequence that matches CD4 and CD8 expression and functional potential with MHC restriction. Although the transcriptional control of CD4(+)-CD8(+) lineage choice in the thymus is now better understood, less was known about what maintains the CD4(+) and CD8(+) lineage integrity of mature T cells. In this review, we discuss the mechanisms that establish in the thymus, and maintain in postthymic cells, the separation of these lineages. We focus on recent studies that address the mechanisms of epigenetic control of Cd4 expression and emphasize how maintaining a transcriptional circuitry nucleated around Thpok and Runx proteins, the key architects of CD4(+)-CD8(+) lineage commitment in the thymus, is critical for CD4(+) T cell helper functions.

HMGN proteins modulate chromatin regulatory sites and gene expression during activation of naïve B cells.

The activation of naïve B lymphocyte involves rapid and major changes in chromatin organization and gene expression; however, the complete repertoire of nuclear factors affecting these genomic changes is not known. We report that HMGN proteins, which bind to nucleosomes and affect chromatin structure and function, co-localize with, and maintain the intensity of DNase I hypersensitive sites genome wide, in resting but not in activated B cells. Transcription analyses of resting and activated B cells from wild-type and Hmgn(-/-) mice, show that loss of HMGNs dampens the magnitude of the transcriptional response and alters the pattern of gene expression during the course of B-cell activation; defense response genes are most affected at the onset of activation. Our study provides insights into the biological function of the ubiquitous HMGN chromatin binding proteins and into epigenetic processes that affect the fidelity of the transcriptional response during the activation of B cell lymphocytes.

Transcriptional and epigenetic networks of helper T and innate lymphoid cells.

The discovery of the specification of CD4(+) helper T cells to discrete effector 'lineages' represented a watershed event in conceptualizing mechanisms of host defense and immunoregulation. However, our appreciation for the actual complexity of helper T-cell subsets continues unabated. Just as the Sami language of Scandinavia has 1000 different words for reindeer, immunologists recognize the range of fates available for a CD4(+) T cell is numerous and may be underestimated. Added to the crowded scene for helper T-cell subsets is the continuously growing family of innate lymphoid cells (ILCs), endowed with common effector responses and the previously defined 'master regulators' for CD4(+) helper T-cell subsets are also shared by ILC subsets. Within the context of this extraordinary complexity are concomitant advances in the understanding of transcriptomes and epigenomes. So what do terms like 'lineage commitment' and helper T-cell 'specification' mean in the early 21st century? How do we put all of this together in a coherent conceptual framework? It would be arrogant to assume that we have a sophisticated enough understanding to seriously answer these questions. Instead, we review the current status of the flexibility of helper T-cell responses in relation to their genetic regulatory networks and epigenetic landscapes. Recent data have provided major surprises as to what master regulators can or cannot do, how they interact with other transcription factors and impact global genome-wide changes, and how all these factors come together to influence helper cell function.