Helper T cell immunity in humans with inherited CD4 deficiency.

CD4+ T cells are vital for host defense and immune regulation. However, the fundamental role of CD4 itself remains enigmatic. We report seven patients aged 5-61 years from five families of four ancestries with autosomal recessive CD4 deficiency and a range of infections, including recalcitrant warts and Whipple's disease. All patients are homozygous for rare deleterious CD4 variants impacting expression of the canonical CD4 isoform. A shorter expressed isoform that interacts with LCK, but not HLA class II, is affected by only one variant. All patients lack CD4+ T cells and have increased numbers of TCRαß+CD4-CD8- T cells, which phenotypically and transcriptionally resemble conventional Th cells. Finally, patient CD4-CD8- αß T cells exhibit intact responses to HLA class II-restricted antigens and promote B cell differentiation in vitro. Thus, compensatory development of Th cells enables patients with inherited CD4 deficiency to acquire effective cellular and humoral immunity against an unexpectedly large range of pathogens. Nevertheless, CD4 is indispensable for protective immunity against at least human papillomaviruses and Trophyrema whipplei.

Pyruvate dehydrogenase complex integrates the metabolome and epigenome in CD8+ memory T cell differentiation in vitro.

Modulation of metabolic flux through pyruvate dehydrogenase complex (PDC) plays an important role in T cell activation and differentiation. PDC sits at the transition between glycolysis and the tricarboxylic acid cycle and is a major producer of acetyl-CoA, marking it as a potential metabolic and epigenetic node To understand the role of pyruvate dehydrogenase complex in T cell differentiation, we generated mice deficient in T cell pyruvate dehydrogenase E1A (Pdha) subunit using a CD4-cre recombinase-based strategy. Herein, we show that genetic ablation of PDC activity in T cells (TPdh-/-) leads to marked perturbations in glycolysis, the tricarboxylic acid cycle, and OXPHOS. TPdh-/- T cells became dependent upon substrate level phosphorylation via glycolysis, secondary to depressed OXPHOS. Due to the block of PDC activity, histone acetylation was also reduced, including H3K27, a critical site for CD8+ TM differentiation. Transcriptional and functional profiling revealed abnormal CD8+ TM differentiation in vitro. Collectively, our data indicate that PDC integrates the metabolome and epigenome in CD8+ memory T cell differentiation. Targeting this metabolic and epigenetic node can have widespread ramifications on cellular function.

Dynamic chromatin accessibility licenses STAT5- and STAT6-dependent innate-like function of TH9 cells to promote allergic inflammation.

Allergic diseases are a major global health issue. Interleukin (IL)-9-producing helper T (TH9) cells promote allergic inflammation, yet TH9 cell effector functions are incompletely understood because their lineage instability makes them challenging to study. Here we found that resting TH9 cells produced IL-9 independently of T cell receptor (TCR) restimulation, due to STAT5- and STAT6-dependent bystander activation. This mechanism was seen in circulating cells from allergic patients and was restricted to recently activated cells. STAT5-dependent Il9/IL9 regulatory elements underwent remodeling over time, inactivating the locus. A broader 'allergic TH9' transcriptomic and epigenomic program was also unstable. In vivo, TH9 cells induced airway inflammation via TCR-independent, STAT-dependent mechanisms. In allergic patients, TH9 cell expansion was associated with responsiveness to JAK inhibitors. These findings suggest that TH9 cell instability is a negative checkpoint on bystander activation that breaks down in allergy and that JAK inhibitors should be considered for allergic patients with TH9 cell expansion.

Pyruvate dehydrogenase complex integrates the metabolome and epigenome in memory T cell differentiation in vitro.

Background: Modulation of metabolic flux through pyruvate dehydrogenase complex (PDC) plays an important role in T cell activation and differentiation. PDC sits at the transition between glycolysis and the tricarboxylic acid cycle and is a major producer of acetyl-CoA, marking it as a potential metabolic and epigenetic node. Methods: To understand the role of pyruvate dehydrogenase complex in T cell differentiation, we generated mice deficient in T cell pyruvate dehydrogenase E1A (Pdha) subunit using a CD4-cre recombinase-based strategy. To control for the contribution of exogenous metabolites in vivo, we conducted our T cell functional studies in vitro. T cells were differentiated into memory and effector T cells using standardized protocols. Cells were analyzed using stable isotopic tracing studies, metabolomics, RNAseq, ATACseq, ChIPseq and histone proteomics. Results: Herein, we show that genetic ablation of PDC activity in T cells (TPdh-/-) leads to marked perturbations in glycolysis, the tricarboxylic acid cycle, and OXPHOS. Due to depressed OXPHOS, TPdh-/-T cells became dependent upon substrate level phosphorylation via glycolysis. Due to the block of PDC activity, histone acetylation was reduced, as were most other types of post translational modifications. Transcriptional and functional profiling revealed abnormal CD8+ memory T cell differentiation in vitro. Conclusions: Collectively, our data indicate that PDC integrates the metabolome and epigenome in memory T cell differentiation. Targeting this metabolic and epigenetic node can have widespread ramifications on cellular function.

In vivo CRISPR screens reveal a HIF-1α-mTOR-network regulates T follicular helper versus Th1 cells.

T follicular helper (Tfh) cells provide signals to initiate and maintain the germinal center (GC) reaction and are crucial for the generation of robust, long-lived antibody responses, but how the GC microenvironment affects Tfh cells is not well understood. Here we develop an in vivo T cell-intrinsic CRISPR-knockout screen to evaluate Tfh and Th1 cells in an acute viral infection model to identify regulators of Tfh cells in their physiological setting. Using a screen of druggable-targets, alongside genetic, transcriptomic and cellular analyses, we identify a function of HIF-1α in suppressing mTORC1-mediated and Myc-related pathways, and provide evidence that VHL-mediated degradation of HIF-1α is required for Tfh development; an expanded in vivo CRISPR screen reveals multiple components of these pathways that regulate Tfh versus Th1 cells, including signaling molecules, cell-cycle regulators, nutrient transporters, metabolic enzymes and autophagy mediators. Collectively, our data serve as a resource for studying Tfh versus Th1 decisions, and implicate the VHL-HIF-1α axis in fine-tuning Tfh generation.

PI3Kδ coordinates transcriptional, chromatin, and metabolic changes to promote effector CD8+ T cells at the expense of central memory.

Patients with activated phosphatidylinositol 3-kinase delta (PI3Kδ) syndrome (APDS) present with sinopulmonary infections, lymphadenopathy, and cytomegalvirus (CMV) and/or Epstein-Barr virus (EBV) viremia, yet why patients fail to clear certain chronic viral infections remains incompletely understood. Using patient samples and a mouse model (Pik3cdE1020K/+ mice), we demonstrate that, upon activation, Pik3cdE1020K/+ CD8+ T cells exhibit exaggerated features of effector populations both in vitro and after viral infection that are associated with increased Fas-mediated apoptosis due to sustained FoxO1 phosphorylation and Fasl derepression, enhanced mTORC1 and c-Myc signatures, metabolic perturbations, and an altered chromatin landscape. Conversely, Pik3cdE1020K/+ CD8+ cells fail to sustain expression of proteins critical for central memory, including TCF1. Strikingly, activated Pik3cdE1020K/+ CD8+ cells exhibit altered transcriptional and epigenetic circuits characterized by pronounced interleukin-2 (IL-2)/STAT5 signatures and heightened IL-2 responses that prevent differentiation to memory-like cells in IL-15. Our data position PI3Kδ as integrating multiple signaling nodes that promote CD8+ T cell effector differentiation, providing insight into phenotypes of patients with APDS.

T and B-cell signaling in activated PI3K delta syndrome: From immunodeficiency to autoimmunity.

Phosphatidylinositol 3 kinases (PI3K) are a family of lipid kinases that are activated by a variety of cell-surface receptors, and regulate a wide range of downstream readouts affecting cellular metabolism, growth, survival, differentiation, adhesion, and migration. The importance of these lipid kinases in lymphocyte signaling has recently been highlighted by genetic analyses, including the recognition that both activating and inactivating mutations of the catalytic subunit of PI3Kδ, p110δ, lead to human primary immunodeficiencies. In this article, we discuss how studies on the human genetic disorder "Activated PI3K-delta syndrome" and mouse models of this disease (Pik3cdE1020K/+ mice) have provided fundamental insight into pathways regulated by PI3Kδ in T and B cells and their contribution to lymphocyte function and disease, including responses to commensal bacteria and the development of autoimmunity and tumors. We highlight critical roles of PI3Kδ in T follicular helper cells and the orchestration of the germinal center reaction, as well as in CD8+ T-cell function. We further  present data demonstrating the ability of the AKT-resistant FOXO1AAA mutant to rescue IgG1 class switching defects in Pik3cdE1020K/+ B cells, as well as data supporting a role for PI3Kδ in promoting multiple T-helper effector cell lineages.

A mouse model of Proteus syndrome.

Proteus syndrome is a mosaic, progressive overgrowth disorder caused by a somatic activating variant c.49G > A p.(E17K) in AKT1. The presentation in affected individuals is variable, with a diversity of tissues demonstrating abnormalities. Common manifestations include skin and bony overgrowth, vascular malformations (VMs), cysts and benign tumors. We used two methods to create mouse models that had endogenously-regulated mosaic expression of the Proteus syndrome variant. Variant allele fractions (VAFs) ranged from 0% to 50% across numerous tissues in 44 Proteus syndrome mice. Mice were phenotypically heterogeneous with lesions rarely observed before 12 months of age. VMs were the most frequent finding with a total of 69 found in 29 of 44 Proteus syndrome mice. Twenty-eight cysts and ectasia, frequently biliary, were seen in 22 of 44 Proteus syndrome mice. Varying levels of mammary hyperplasia were seen in 10 of 16 female Proteus syndrome mice with other localized regions of hyperplasia and stromal expansion noted in several additional animals. Interestingly, 27 of 31 Proteus syndrome animals had non-zero blood VAF that is in contrast to the human disorder where it is rarely seen in peripheral blood. Identification of variant-positive cells by green fluorescent protein (GFP) staining in chimeric Proteus syndrome mice showed that in some lesions, hyperplastic cells were predominantly GFP/Akt1E17K-positive and showed increased pAKT signal compared to GFP-negative cells. However, hyperplastic mammary epithelium was a mixture of GFP/Akt1E17K-positive and negative cells with some GFP/Akt1E17K-negative cells also having increased pAKT signal suggesting that the variant-positive cells can induce lesion formation in a non-cell autonomous manner.

Hyperactivated PI3Kδ promotes self and commensal reactivity at the expense of optimal humoral immunity.

Gain-of-function mutations in the gene encoding the phosphatidylinositol-3-OH kinase catalytic subunit p110δ (PI3Kδ) result in a human primary immunodeficiency characterized by lymphoproliferation, respiratory infections and inefficient responses to vaccines. However, what promotes these immunological disturbances at the cellular and molecular level remains unknown. We generated a mouse model that recapitulated major features of this disease and used this model and patient samples to probe how hyperactive PI3Kδ fosters aberrant humoral immunity. We found that mutant PI3Kδ led to co-stimulatory receptor ICOS-independent increases in the abundance of follicular helper T cells (TFH cells) and germinal-center (GC) B cells, disorganized GCs and poor class-switched antigen-specific responses to immunization, associated with altered regulation of the transcription factor FOXO1 and pro-apoptotic and anti-apoptotic members of the BCL-2 family. Notably, aberrant responses were accompanied by increased reactivity to gut bacteria and a broad increase in autoantibodies that were dependent on stimulation by commensal microbes. Our findings suggest that proper regulation of PI3Kδ is critical for ensuring optimal host-protective humoral immunity despite tonic stimulation from the commensal microbiome.

Cytochrome c Oxidase Activity Is a Metabolic Checkpoint that Regulates Cell Fate Decisions During T Cell Activation and Differentiation.

T cells undergo metabolic reprogramming with major changes in cellular energy metabolism during activation. In patients with mitochondrial disease, clinical data were marked by frequent infections and immunodeficiency, prompting us to explore the consequences of oxidative phosphorylation dysfunction in T cells. Since cytochrome c oxidase (COX) is a critical regulator of OXPHOS, we created a mouse model with isolated dysfunction in T cells by targeting a gene, COX10, that produces mitochondrial disease in humans. COX dysfunction resulted in increased apoptosis following activation in vitro and immunodeficiency in vivo. Select T cell effector subsets were particularly affected; this could be traced to their bioenergetic requirements. In summary, the findings presented herein emphasize the role of COX particularly in T cells as a metabolic checkpoint for cell fate decisions following T cell activation, with heterogeneous effects in T cell subsets. In addition, our studies highlight the utility of translational models that recapitulate human mitochondrial disease for understanding immunometabolism.

T Cell Receptor and Co-Stimulatory Signals for Th9 Generation.

In vitro polarization of naïve CD4+ T cells toward distinct T helper lineages is crucial for establishing the factors and features that determine the differentiation, stability, and effector function for each T helper subsets. In this regard, the recently defined Th9 subset has been reported with two essential cytokines requirement for their generation. Generating Th9 cells in vitro from naïve CD4+ T cells requires the combination of TGF-ß and IL-4. However, the amount of IL-9 producing under these minimal conditions is often small. The intent of this chapter is to provide examples to increase the generation of IL-9 producing T cells in vitro by modulating TCR strength and co-stimulation through the TNF family member TL1A. We hope that these methods to efficiently differentiate naïve CD4+ T cells toward IL-9 producing cells will facilitate understanding the differentiation and function of Th9 cells and their pathogenesis in various inflammatory and autoimmune diseases.

CRISPR-Mediated Triple Knockout of SLAMF1, SLAMF5 and SLAMF6 Supports Positive Signaling Roles in NKT Cell Development.

The SLAM family receptors contribute to diverse aspects of lymphocyte biology and signal via the small adaptor molecule SAP. Mutations affecting SAP lead to X-linked lymphoproliferative syndrome Type 1, a severe immunodysregulation characterized by fulminant mononucleosis, dysgammaglobulinemia, and lymphoproliferation/lymphomas. Patients and mice having mutations affecting SAP also lack germinal centers due to a defect in T:B cell interactions and are devoid of invariant NKT (iNKT) cells. However, which and how SLAM family members contribute to these phenotypes remains uncertain. Three SLAM family members: SLAMF1, SLAMF5 and SLAMF6, are highly expressed on T follicular helper cells and germinal center B cells. SLAMF1 and SLAMF6 are also implicated in iNKT development. Although individual receptor knockout mice have limited iNKT and germinal center phenotypes compared to SAP knockout mice, the generation of multi-receptor knockout mice has been challenging, due to the genomic linkage of the genes encoding SLAM family members. Here, we used Cas9/CRISPR-based mutagenesis to generate mutations simultaneously in Slamf1, Slamf5 and Slamf6. Genetic disruption of all three receptors in triple-knockout mice (TKO) did not grossly affect conventional T or B cell development and led to mild defects in germinal center formation post-immunization. However, the TKO worsened defects in iNKT cells development seen in SLAMF6 single gene-targeted mice, supporting data on positive signaling and potential redundancy between these receptors.

Itk is required for Th9 differentiation via TCR-mediated induction of IL-2 and IRF4.

Th9 cells produce interleukin (IL)-9, a cytokine implicated in allergic asthma and autoimmunity. Here we show that Itk, a mediator of T cell receptor signalling required for Th2 immune responses and the development of asthma, is a positive regulator of Th9 differentiation. In a model of allergic lung disease, Itk-deficient mice show reduced pulmonary inflammation and IL-9 production by T cells and innate lymphoid type 2 cells (ILC2), despite normal early induction of ILC2s. In vitro, Itk(-/-) CD4(+) T cells do not produce IL-9 and have reduced levels of IRF4 (Interferon Regulator Factor 4), a critical transcription factor for effector T cell function. Both IL-9 and IRF4 expression are rescued by either IL-2 or constitutively active STAT5, but not NFATc1. STAT5 binds the Irf4 promoter, demonstrating one mechanism by which IL-2 rescues weakly activated T cells. Itk inhibition also reduces IL-9 expression by human T cells, implicating ITK as a key regulator of Th9 induction.

Disruption of in vivo Chronic Lymphocytic Leukemia Tumor-Microenvironment Interactions by Ibrutinib--Findings from an Investigator-Initiated Phase II Study.

PURPOSE: Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental interactions for proliferation and survival that are at least partially mediated through B-cell receptor (BCR) signaling. Ibrutinib, a Bruton tyrosine kinase inhibitor, disrupts BCR signaling and leads to the egress of tumor cells from the microenvironment. Although the on-target effects on CLL cells are well defined, the impact on the microenvironment is less well studied. We therefore sought to characterize the in vivo effects of ibrutinib on the tumor microenvironment. EXPERIMENTAL DESIGN: Patients received single-agent ibrutinib on an investigator-initiated phase II trial. Serial blood and tissue samples were collected pretreatment and during treatment. Changes in cytokine levels, cellular subsets, and microenvironmental interactions were assessed. RESULTS: Serum levels of key chemokines and inflammatory cytokines decreased significantly in patients on ibrutinib. Furthermore, ibrutinib treatment decreased circulating tumor cells and overall T-cell numbers. Most notably, a reduced frequency of the Th17 subset of CD4(+)T cells was observed concurrent with reduced expression of activation markers and PD-1 on T cells. Consistent with direct inhibition of T cells, ibrutinib inhibited Th17 differentiation of murine CD4(+)T cells in vitro Finally, in the bone marrow microenvironment, we found that ibrutinib disaggregated the interactions of macrophages and CLL cells, inhibited secretion of CXCL13, and decreased the chemoattraction of CLL cells. CONCLUSIONS: In conjunction with inhibition of BCR signaling, these changes in the tumor microenvironment likely contribute to the antitumor activity of ibrutinib and may impact the efficacy of immunotherapeutic strategies in patients with CLL. See related commentary by Bachireddy and Wu, p. 1547.

The TNF-family ligand TL1A and its receptor DR3 promote T cell-mediated allergic immunopathology by enhancing differentiation and pathogenicity of IL-9-producing T cells.

The TNF family cytokine TL1A (Tnfsf15) costimulates T cells and type 2 innate lymphocytes (ILC2) through its receptor DR3 (Tnfrsf25). DR3-deficient mice have reduced T cell accumulation at the site of inflammation and reduced ILC2-dependent immune responses in a number of models of autoimmune and allergic diseases. In allergic lung disease models, immunopathology and local Th2 and ILC2 accumulation is reduced in DR3-deficient mice despite normal systemic priming of Th2 responses and generation of T cells secreting IL-13 and IL-4, prompting the question of whether TL1A promotes the development of other T cell subsets that secrete cytokines to drive allergic disease. In this study, we find that TL1A potently promotes generation of murine T cells producing IL-9 (Th9) by signaling through DR3 in a cell-intrinsic manner. TL1A enhances Th9 differentiation through an IL-2 and STAT5-dependent mechanism, unlike the TNF-family member OX40, which promotes Th9 through IL-4 and STAT6. Th9 differentiated in the presence of TL1A are more pathogenic, and endogenous TL1A signaling through DR3 on T cells is required for maximal pathology and IL-9 production in allergic lung inflammation. Taken together, these data identify TL1A-DR3 interactions as a novel pathway that promotes Th9 differentiation and pathogenicity. TL1A may be a potential therapeutic target in diseases dependent on IL-9.

Disruption of Visc-2, a Brain-Expressed Conserved Long Noncoding RNA, Does Not Elicit an Overt Anatomical or Behavioral Phenotype.

Although long noncoding RNAs (lncRNAs) are proposed to play essential roles in mammalian neurodevelopment, we know little of their functions from their disruption in vivo. Combining evidence for evolutionary constraint and conserved expression data, we previously identified candidate lncRNAs that might play important and conserved roles in brain function. Here, we demonstrate that the sequence and neuronal transcription of lncRNAs transcribed from the previously uncharacterized Visc locus are conserved across diverse mammals. Consequently, one of these lncRNAs, Visc-2, was selected for targeted deletion in the mouse, and knockout animals were subjected to an extremely detailed anatomical and behavioral characterization. Despite a neurodevelopmental expression pattern of Visc-2 that is highly localized to the cortex and sites of neurogenesis, anomalies in neither cytoarchitecture nor neuroproliferation were identified in knockout mice. In addition, no abnormal motor, sensory, anxiety, or cognitive behavioral phenotypes were observed. These results are important because they contribute to a growing body of evidence that lncRNA loci contribute on average far less to brain and biological functions than protein-coding loci. A high-throughput knockout program focussing on lncRNAs, similar to that currently underway for protein-coding genes, will be required to establish the distribution of their organismal functions.

Impaired T cell function in argininosuccinate synthetase deficiency.

ASS1 is a cytosolic enzyme that plays a role in the conversion of citrulline to arginine. In human and mouse tissues, ASS1 protein is found in several components of the immune system, including the thymus and T cells. However, the role of ASS1 in these tissues remains to be defined. Considerable attention has been focused recently on the role of metabolism in T cell differentiation and function. Based on the expression of ASS1 in the immune system, we hypothesized that ASS1 deficiency would result in T cell defects. To evaluate this question, we characterized immune function in hypomorphic fold/fold mice. Analysis of splenic T cells by flow cytometry showed a marked reduction in T cell numbers with normal expression of activation surface markers. Gene therapy correction of liver ASS1 to enhance survival resulted in a partial recovery of splenic T cells for characterization. In vitro and in vivo studies demonstrated the persistence of the ASS1 enzyme defect in T cells and abnormal T cell differentiation and function. Overall, our work suggests that ASS1 plays a role in T cell function, and deficiency produces primary immune dysfunction. In addition, these data suggest that patients with ASS1 deficiency (citrullinemia type I) may have T cell dysfunction.

Itk-mediated integration of T cell receptor and cytokine signaling regulates the balance between Th17 and regulatory T cells.

A proper balance between Th17 and T regulatory cells (Treg cells) is critical for generating protective immune responses while minimizing autoimmunity. We show that the Tec family kinase Itk (IL2-inducible T cell kinase), a component of T cell receptor (TCR) signaling pathways, influences this balance by regulating cross talk between TCR and cytokine signaling. Under both Th17 and Treg cell differentiation conditions, Itk(-/-) CD4(+) T cells develop higher percentages of functional FoxP3(+) cells, associated with increased sensitivity to IL-2. Itk(-/-) CD4(+) T cells also preferentially develop into Treg cells in vivo. We find that Itk-deficient T cells exhibit reduced TCR-induced phosphorylation of mammalian target of rapamycin (mTOR) targets, accompanied by downstream metabolic alterations. Surprisingly, Itk(-/-) cells also exhibit reduced IL-2-induced mTOR activation, despite increased STAT5 phosphorylation. We demonstrate that in wild-type CD4(+) T cells, TCR stimulation leads to a dose-dependent repression of Pten. However, at low TCR stimulation or in the absence of Itk, Pten is not effectively repressed, thereby uncoupling STAT5 phosphorylation and phosphoinositide-3-kinase (PI3K) pathways. Moreover, Itk-deficient CD4(+) T cells show impaired TCR-mediated induction of Myc and miR-19b, known repressors of Pten. Our results demonstrate that Itk helps orchestrate positive feedback loops integrating multiple T cell signaling pathways, suggesting Itk as a potential target for altering the balance between Th17 and Treg cells.