CD3ζ ITAMs enable ligand discrimination and antagonism by inhibiting TCR signaling in response to low-affinity peptides.

The T cell antigen receptor (TCR) contains ten immunoreceptor tyrosine-based activation motif (ITAM) signaling sequences distributed within six CD3 subunits; however, the reason for such structural complexity and multiplicity is unclear. Here we evaluated the effect of inactivating the three CD3ζ chain ITAMs on TCR signaling and T cell effector responses using a conditional 'switch' mouse model. Unexpectedly, we found that T cells expressing TCRs containing inactivated (non-signaling) CD3ζ ITAMs (6F-CD3ζ) exhibited reduced ability to discriminate between low- and high-affinity ligands, resulting in enhanced signaling and cytokine responses to low-affinity ligands because of a previously undetected inhibitory function of CD3ζ ITAMs. Also, 6F-CD3ζ TCRs were refractory to antagonism, as predicted by a new in silico adaptive kinetic proofreading model that revises the role of ITAM multiplicity in TCR signaling. Finally, T cells expressing 6F-CD3ζ displayed enhanced cytolytic activity against solid tumors expressing low-affinity ligands, identifying a new counterintuitive approach to TCR-mediated cancer immunotherapy.

Themis2 regulates natural killer cell memory function and formation.

Immunological memory is a hallmark of the adaptive immune system. Although natural killer (NK) cells are innate immune cells important for the immediate host defence, they can differentiate into memory NK cells. The molecular mechanisms controlling this differentiation are yet to be fully elucidated. Here we identify the scaffold protein Themis2 as a critical regulator of memory NK cell differentiation and function. Themis2-deficient NK cells expressing Ly49H, an activating NK receptor for the mouse cytomegalovirus (MCMV) antigen m157, show enhanced differentiation into memory NK cells and augment host protection against MCMV infection. Themis2 inhibits the effector function of NK cells after stimulation of Ly49H and multiple activating NK receptors, though not specific to memory NK cells. Mechanistically, Themis2 suppresses Ly49H signalling by attenuating ZAP70/Syk phosphorylation, and it also translocates to the nucleus, where it promotes Zfp740-mediated repression to regulate the persistence of memory NK cells. Zfp740 deficiency increases the number of memory NK cells and enhances the effector function of memory NK cells, which further supports the relevance of the Themis2-Zfp740 pathway. In conclusion, our study shows that Themis2 quantitatively and qualitatively regulates NK cell memory formation.

THEMIS increases TCR signaling in CD4+CD8+ thymocytes by inhibiting the activity of the tyrosine phosphatase SHP1.

The T cell lineage-restricted protein THEMIS plays a critical role in T cell development at the positive selection stage. In the SHP1 activation model, THEMIS is proposed to enhance the activity of the tyrosine phosphatase SHP1 (encoded by Ptpn6), thereby dampening T cell antigen receptor (TCR) signaling and preventing the inappropriate negative selection of CD4+CD8+ thymocytes by positively selecting ligands. In contrast, in the SHP1 inhibition model, THEMIS is proposed to suppress SHP1 activity, rendering CD4+CD8+ thymocytes more sensitive to TCR signaling initiated by low-affinity ligands to promote positive selection. We sought to resolve the controversy regarding the molecular function of THEMIS. We found that the defect in positive selection in Themis-/- thymocytes was ameliorated by pharmacologic inhibition of SHP1 or by deletion of Ptpn6 and was exacerbated by SHP1 overexpression. Moreover, overexpression of SHP1 phenocopied the Themis-/- developmental defect, whereas deletion of Ptpn6, Ptpn11 (encoding SHP2), or both did not result in a phenotype resembling that of Themis deficiency. Last, we found that thymocyte negative selection was not enhanced but was instead impaired in the absence of THEMIS. Together, these results provide evidence favoring the SHP1 inhibition model, supporting a mechanism whereby THEMIS functions to enhance the sensitivity of CD4+CD8+ thymocytes to TCR signaling, enabling positive selection by low-affinity, self-ligand-TCR interactions.

Epigenetic regulation of T cell development.

Epigenetic regulators are pivotal factors that influence and control T cell development. Recent findings continue to reveal additional elements of epigenetic modifications that play significant and crucial roles at different stages of T cell development. Through gaining a better understanding of the various epigenetic factors that influence the formation and survival of maturing T cells, new therapies can potentially be developed to combat diseases caused by dysregulated epigenetic chromatin modifications. In this review, we summarize the recent studies which shed light on the epigenetic regulation of T cell development especially at the critical stage of ß-selection.

A Simplified Approach to Evaluating T Cell Development by Flow Cytometry.

T cell development is a complex multistep process that requires the coordinated activation of distinct signaling responses and the regulated progression of developing cells (thymocytes) through key stages of maturation. Although sophisticated techniques such as fetal thymus organ culture, in vitro thymocyte culture, and multiparameter flow cytometry-based cell sorting are now widely employed to evaluate thymocyte maturation by experienced laboratories, defects in T cell development can usually be identified with relatively simple flow cytometry screening methods. Here, we provide a basic protocol for assessment of T cell development that will enable laboratories with access to a multi-laser flow cytometer to screen mouse strains, including those generated from embryonic stem cells with targeted gene mutations, for thymocyte maturation defects.

Metabolic regulation of T cell development.

T cell development in the thymus is tightly controlled by complex regulatory mechanisms at multiple checkpoints. Currently, many studies have focused on the transcriptional and posttranslational control of the intrathymic journey of T-cell precursors. However, over the last few years, compelling evidence has highlighted cell metabolism as a critical regulator in this process. Different thymocyte subsets are directed by distinct metabolic pathways and signaling networks to match the specific functional requirements of the stage. Here, we epitomize these metabolic alterations during the development of a T cell and review several recent works that provide insights into equilibrating metabolic quiescence and activation programs. Ultimately, understanding the interplay between cellular metabolism and T cell developmental programs may offer an opportunity to selectively regulate T cell subset functions and to provide potential novel therapeutic approaches to modulate autoimmunity.

THEMIS enhances the magnitude of normal and neuroinflammatory type 1 immune responses by promoting TCR-independent signals.

Signals that determine the differentiation of naïve CD4+ T helper (TH) cells into specific effector cell subsets are primarily stimulated by cytokines, but additional signals are required to adjust the magnitude of TH cell responses and set the balance between effective immunity and immunological tolerance. By inducing the post-thymic deletion of the T cell lineage signaling protein THEMIS, we showed that THEMIS promoted the development of optimal type 1 immune responses to foreign antigens but stimulated signals that favored encephalitogenic responses to self-neuroantigens. THEMIS was required to stimulate the expression of the gene encoding the transcriptional regulator T-BET and the production of the cytokine interferon-γ (IFN-γ), and it enhanced the ability of encephalitogenic CD4+ T cells to migrate into the central nervous system. Consistently, analysis of THEMIS expression in polarized CD4+ T cells showed that THEMIS was selectively increased in abundance in TH1 cells. The stimulation of predifferentiated effector CD4+ T cells with antigen-presenting cells revealed a stimulatory function for THEMIS on type 1 cytokine responses, similar to those observed ex vivo after immunization. In contrast, THEMIS exerted opposing effects on naïve CD4+ T cells in vitro by inhibiting the T cell receptor (TCR)-mediated signals that lead to TH1 cell responses. These data suggest that THEMIS exerts TCR-independent functions in effector T cells, which increase the magnitude of normal and pathogenic TH1 cell-mediated responses.

NuRD complex recruitment to Thpok mediates CD4+ T cell lineage differentiation.

Although BTB-zinc finger (BTB-ZF) transcription factors control the differentiation of multiple hematopoietic and immune lineages, how they function is poorly understood. The BTB-ZF factor Thpok controls intrathymic CD4+ T cell development and the expression of most CD4+ and CD8+ lineage genes. Here, we identify the nucleosome remodeling and deacetylase (NuRD) complex as a critical Thpok cofactor. Using mass spectrometry and coimmunoprecipitation in primary T cells, we show that Thpok binds NuRD components independently of DNA association. We locate three amino acid residues within the Thpok BTB domain that are required for both NuRD binding and Thpok functions. Conversely, a chimeric protein merging the NuRD component Mta2 to a BTB-less version of Thpok supports CD4+ T cell development, indicating that NuRD recruitment recapitulates the functions of the Thpok BTB domain. We found that NuRD mediates Thpok repression of CD8+ lineage genes, including the transcription factor Runx3, but is dispensable for Cd4 expression. We show that these functions cannot be performed by the BTB domain of the Thpok-related factor Bcl6, which fails to bind NuRD. Thus, cofactor binding critically contributes to the functional specificity of BTB-ZF factors, which control the differentiation of most hematopoietic subsets.

CD5 Controls Gut Immunity by Shaping the Cytokine Profile of Intestinal T Cells.

CD5 is constitutively expressed on all T cells and is a negative regulator of lymphocyte function. However, the full extent of CD5 function in immunity remains unclear. CD5 deficiency impacts thymic selection and extra-thymic regulatory T cell generation, yet CD5 knockout was reported to cause no immune pathology. Here we show that CD5 is a key modulator of gut immunity. We generated mice with inducible CD5 knockdown (KD) in the autoimmune-prone nonobese diabetic (NOD) background. CD5 deficiency caused T cell-dependent wasting disease driven by chronic gut immune dysregulation. CD5 inhibition also exacerbated acute experimental colitis. Mechanistically, loss of CD5 increased phospho-Stat3 levels, leading to elevated IL-17A secretion. Our data reveal a new facet of CD5 function in shaping the T cell cytokine profile.

New Insights into Epigenetic Regulation of T Cell Differentiation.

Immature CD4- CD8- thymocytes progress through several developmental steps in the thymus, ultimately emerging as mature CD4+ (helper) or CD8+ (cytotoxic) T cells. Activation of naïve CD4+ and CD8+ T cells in the presence of specific cytokines results in the induction of transcriptional programs that result in their differentiation into effector or memory cells and in the case of CD4+ T cells, the adoption of distinct T-helper fates. Previous studies have shown that histone modification and DNA methylation play important roles in each of these events. More recently, the roles of specific epigenetic regulators in T cell differentiation have been clarified. The identification of the epigenetic modifications and modifiers that control mature T cell differentiation and specification has also provided further insights into how dysregulation of these processes can lead to cancer or autoimmune diseases. In this review, we summarize recent findings that have provided new insights into epigenetic regulation of T cell differentiation in both mice and humans.

The histone demethylase Lsd1 regulates multiple repressive gene programs during T cell development.

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.

New insights into TCR ß-selection.

T cell receptor (TCR) ß-selection (herein referred to as ß-selection) is a pivotal checkpoint in mammalian T cell development when immature CD4-CD8- T-cells (thymocytes) express pre-TCR following successful Tcrb gene rearrangement. At this stage, αß T cell lineage commitment and allelic exclusion to restrict one ß-chain per cell take place and thymocytes undergo a proliferative burst. ß-selection is known to be crucially dependent upon synchronized Notch and pre-TCR signaling; however, other necessary inputs have been identified over the past decade, expanding our knowledge and understanding of the ß-selection process. In this review, we discuss recent mechanistic findings that have enabled a more detailed decoding of the molecular dynamics of the ß-selection checkpoint and have helped to elucidate its role in early T cell development.

CD5 dynamically calibrates basal NF-κB signaling in T cells during thymic development and peripheral activation.

Immature T cells undergo a process of positive selection in the thymus when their new T cell receptor (TCR) engages and signals in response to self-peptides. As the T cell matures, a slew of negative regulatory molecules, including the inhibitory surface glycoprotein CD5, are up-regulated in proportion to the strength of the self-peptide signal. Together these regulators dampen TCR-proximal signaling and help avoid any subsequent peripheral activation of T cells by self-peptides. Paradoxically, antigen-specific T cells initially expressing more CD5 (CD5hi) have been found to better persist as effector/memory cells after a peripheral challenge. The molecular mechanisms underlying such a duality in CD5 function is not clear. We found that CD5 alters the basal activity of the NF-κB signaling in resting peripheral T cells. When CD5 was conditionally ablated, T cells were unable to maintain higher expression of the cytoplasmic NF-κB inhibitor IκBα. Consistent with this, resting CD5hi T cells expressed more of the NF-κB p65 protein than CD5lo cells, without significant increases in transcript levels, in the absence of TCR signals. This posttranslationally stabilized cellular NF-κB depot potentially confers a survival advantage to CD5hi T cells over CD5lo ones. Taken together, these data suggest a two-step model whereby the strength of self-peptide-induced TCR signal lead to the up-regulation of CD5, which subsequently maintains a proportional reserve of NF-κB in peripheral T cells poised for responding to agonistic antigen-driven T cell activation.

CD5 signalosome coordinates antagonist TCR signals to control the generation of Treg cells induced by foreign antigens.

CD5 is characterized as an inhibitory coreceptor with an important regulatory role during T cell development. The molecular mechanism by which CD5 operates has been puzzling and its function in mature T cells suggests promoting rather than repressing effects on immune responses. Here, we combined quantitative mass spectrometry and genetic studies to analyze the components and the activity of the CD5 signaling machinery in primary T cells. We found that T cell receptor (TCR) engagement induces the selective phosphorylation of CD5 tyrosine 429, which serves as a docking site for proteins with adaptor functions (c-Cbl, CIN85, CRKL), connecting CD5 to positive (PI3K) and negative (UBASH3A, SHIP1) regulators of TCR signaling. c-CBL acts as a coordinator in this complex enabling CD5 to synchronize positive and negative feedbacks on TCR signaling through the other components. Disruption of CD5 signalosome in mutant mice reveals that it modulates TCR signal outputs to selectively repress the transactivation of Foxp3 and limit the inopportune induction of peripherally induced regulatory T cells during immune responses against foreign antigen. Our findings bring insights into the paradigm of coreceptor signaling, suggesting that, in addition to providing dualistic enhancing or dampening inputs, coreceptors can engage concomitant stimulatory and inhibitory signaling events, which act together to promote specific functional outcomes.

Ldb1 is required for Lmo2 oncogene-induced thymocyte self-renewal and T-cell acute lymphoblastic leukemia.

Prolonged or enhanced expression of the proto-oncogene Lmo2 is associated with a severe form of T-cell acute lymphoblastic leukemia (T-ALL), designated early T-cell precursor ALL, which is characterized by the aberrant self-renewal and subsequent oncogenic transformation of immature thymocytes. It has been suggested that Lmo2 exerts these effects by functioning as component of a multi-subunit transcription complex that includes the ubiquitous adapter Ldb1 along with b-HLH and/or GATA family transcription factors; however, direct experimental evidence for this mechanism is lacking. In this study, we investigated the importance of Ldb1 for Lmo2-induced T-ALL by conditional deletion of Ldb1 in thymocytes in an Lmo2 transgenic mouse model of T-ALL. Our results identify a critical requirement for Ldb1 in Lmo2-induced thymocyte self-renewal and thymocyte radiation resistance and for the transition of preleukemic thymocytes to overt T-ALL. Moreover, Ldb1 was also required for acquisition of the aberrant preleukemic ETP gene expression signature in immature Lmo2 transgenic thymocytes. Co-binding of Ldb1 and Lmo2 was detected at the promoters of key upregulated T-ALL driver genes (Hhex, Lyl1, and Nfe2) in preleukemic Lmo2 transgenic thymocytes, and binding of both Ldb1 and Lmo2 at these sites was reduced following Cre-mediated deletion of Ldb1. Together, these results identify a key role for Ldb1, a nonproto-oncogene, in T-ALL and support a model in which Lmo2-induced T-ALL results from failure to downregulate Ldb1/Lmo2-nucleated transcription complexes which normally function to enforce self-renewal in bone marrow hematopoietic progenitors.

SOCS3 is a suppressor of γc cytokine signaling and constrains generation of murine Foxp3+ regulatory T cells.

SOCS3 is a cytosolic inhibitor of cytokine signaling that suppresses the activation of cytokine receptor-associated JAK kinases. Mechanistically, SOCS3 is recruited to a site in the cytokine receptors known as the SOCS3-interaction motif, and then binds JAK molecules to inhibit their kinase activity. The SOCS3-interaction motif is found in receptors of the gp130 cytokine family but mostly absent from other cytokine receptors, including γc. Thus, SOCS3 has been considered a selective suppressor of gp130 family cytokines, but not γc cytokines. Considering that γc signaling induces SOCS3 expression in T cells, here we revisited the role of SOCS3 on γc signaling. Using SOCS3 transgenic mice, we found that increased abundance of SOCS3 not only suppressed signaling of the gp130 family cytokine IL-6, but also signaling of the γc family cytokine IL-7. Consequently, SOCS3 transgenic mice were impaired in IL-7-dependent T cell development in the thymus and the homeostasis of mature T cells in peripheral tissues. Moreover, enforced SOCS3 expression interfered with the generation of Foxp3+ regulatory T cells that requires signaling by the γc family cytokine IL-2. Collectively, we report an underappreciated role for SOCS3 in suppressing γc cytokine signaling, effectively expanding its scope of target cytokines in T cell immunity.

Notch and the pre-TCR coordinate thymocyte proliferation by induction of the SCF subunits Fbxl1 and Fbxl12.

Proliferation is tightly regulated during T cell development, and is limited to immature CD4-CD8- thymocytes. The major proliferative event is initiated at the 'ß-selection' stage following successful rearrangement of Tcrß, and is triggered by and dependent on concurrent signaling by Notch and the pre-T cell receptor (TCR); however, it is unclear how these signals cooperate to promote cell proliferation. Here, we found that ß-selection-associated proliferation required the combined activity of two Skp-cullin-F-box (SCF) ubiquitin ligase complexes that included as substrate recognition subunits the F-box proteins Fbxl1 or Fbxl12. Both SCF complexes targeted the cyclin-dependent kinase inhibitor Cdkn1b for polyubiquitination and proteasomal degradation. We found that Notch signals induced the transcription of Fbxl1, whereas pre-TCR signals induced the transcription of Fbxl12. Thus, concurrent Notch and pre-TCR signaling induced the expression of two genes, Fbxl1 and Fbxl12, whose products functioned identically but additively to promote degradation of Cdkn1b, cell cycle progression, and proliferation of ß-selected thymocytes.

Pax3 cooperates with Ldb1 to direct local chromosome architecture during myogenic lineage specification.

Chromatin looping allows enhancer-bound regulatory factors to influence transcription. Large domains, referred to as topologically associated domains, participate in genome organization. However, the mechanisms underlining interactions within these domains, which control gene expression, are not fully understood. Here we report that activation of embryonic myogenesis is associated with establishment of long-range chromatin interactions centered on Pax3-bound loci. Using mass spectrometry and genomic studies, we identify the ubiquitously expressed LIM-domain binding protein 1 (Ldb1) as the mediator of looping interactions at a subset of Pax3 binding sites. Ldb1 is recruited to Pax3-bound elements independently of CTCF-Cohesin, and is necessary for efficient deposition of H3K4me1 at these sites and chromatin looping. When Ldb1 is deleted in Pax3-expressing cells in vivo, specification of migratory myogenic progenitors is severely impaired. These results highlight Ldb1 requirement for Pax3 myogenic activity and demonstrate how transcription factors can promote formation of sub-topologically associated domain interactions involved in lineage specification.