Combined immunodeficiency caused by pathogenic variants in the ZAP70 C-terminal SH2 domain.

Introduction: ZAP-70, a protein tyrosine kinase recruited to the T cell receptor (TCR), initiates a TCR signaling cascade upon antigen stimulation. Mutations in the ZAP70 gene cause a combined immunodeficiency characterized by low or absent CD8+ T cells and nonfunctional CD4+ T cells. Most deleterious missense ZAP70 mutations in patients are located in the kinase domain but the impact of mutations in the SH2 domains, regulating ZAP-70 recruitment to the TCR, are not well understood. Methods: Genetic analyses were performed on four patients with CD8 lymphopenia and a high resolution melting screening for ZAP70 mutations was developed. The impact of SH2 domain mutations was evaluated by biochemical and functional analyses as well as by protein modeling. Results and discussion: Genetic characterization of an infant who presented with pneumocystis pneumonia, mycobacterial infection, and an absence of CD8 T cells revealed a novel homozygous mutation in the C-terminal SH2 domain (SH2-C) of the ZAP70 gene (c.C343T, p.R170C). A distantly related second patient was found to be compound heterozygous for the R170C variant and a 13bp deletion in the ZAP70 kinase domain. While the R170C mutant was highly expressed, there was an absence of TCR-induced proliferation, associated with significantly attenuated TCR-induced ZAP-70 phosphorylation and a lack of binding of ZAP-70 to TCR-ζ. Moreover, a homozygous ZAP-70 R192W variant was identified in 2 siblings with combined immunodeficiency and CD8 lymphopenia, confirming the pathogenicity of this mutation. Structural modeling of this region revealed the critical nature of the arginines at positions 170 and 192, in concert with R190, forming a binding pocket for the phosphorylated TCR-ζ chain. Deleterious mutations in the SH2-C domain result in attenuated ZAP-70 function and clinical manifestations of immunodeficiency.

Regulatory T cell differentiation is controlled by αKG-induced alterations in mitochondrial metabolism and lipid homeostasis.

Suppressive regulatory T cell (Treg) differentiation is controlled by diverse immunometabolic signaling pathways and intracellular metabolites. Here we show that cell-permeable α-ketoglutarate (αKG) alters the DNA methylation profile of naive CD4 T cells activated under Treg polarizing conditions, markedly attenuating FoxP3+ Treg differentiation and increasing inflammatory cytokines. Adoptive transfer of these T cells into tumor-bearing mice results in enhanced tumor infiltration, decreased FoxP3 expression, and delayed tumor growth. Mechanistically, αKG leads to an energetic state that is reprogrammed toward a mitochondrial metabolism, with increased oxidative phosphorylation and expression of mitochondrial complex enzymes. Furthermore, carbons from ectopic αKG are directly utilized in the generation of fatty acids, associated with lipidome remodeling and increased triacylglyceride stores. Notably, inhibition of either mitochondrial complex II or DGAT2-mediated triacylglyceride synthesis restores Treg differentiation and decreases the αKG-induced inflammatory phenotype. Thus, we identify a crosstalk between αKG, mitochondrial metabolism and triacylglyceride synthesis that controls Treg fate.

Clinical and Product Features Associated with Outcome of DLBCL Patients to CD19-Targeted CAR T-Cell Therapy.

CD19-directed CAR T-cells have been remarkably successful in treating patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) and transformed follicular lymphoma (t-FL). In this cohort study, we treated 60 patients with axicabtagene ciloleucel or tisagenlecleucel. Complete and partial metabolic responses (CMR/PMR) were obtained in 40% and 23% of patients, respectively. After 6.9 months of median follow-up, median progression-free survival (mPFS) and overall survival (mOS) were estimated at 3.1 and 12.3 months, respectively. Statistical analyses revealed that CMR, PFS, and OS were all significantly associated with age-adjusted international prognostic index (aaIPI, p < 0.05). T-cell subset phenotypes in the apheresis product tended to correlate with PFS. Within the final product, increased percentages of both CD4 and CD8 CAR+ effector memory cells (p = 0.02 and 0.01) were significantly associated with CMR. Furthermore, higher CMR/PMR rates were observed in patients with a higher maximal in vivo expansion of CAR T-cells (p = 0.05) and lower expression of the LAG3 and Tim3 markers of exhaustion phenotype (p = 0.01 and p = 0.04). Thus, we find that aaIPI at the time of infusion, phenotype of the CAR T product, in vivo CAR T-cell expansion, and low levels of LAG3/Tim3 are associated with the efficacy of CAR T-cell therapy in DLBCL patients.

An IDH1-vitamin C crosstalk drives human erythroid development by inhibiting pro-oxidant mitochondrial metabolism.

The metabolic changes controlling the stepwise differentiation of hematopoietic stem and progenitor cells (HSPCs) to mature erythrocytes are poorly understood. Here, we show that HSPC development to an erythroid-committed proerythroblast results in augmented glutaminolysis, generating alpha-ketoglutarate (αKG) and driving mitochondrial oxidative phosphorylation (OXPHOS). However, sequential late-stage erythropoiesis is dependent on decreasing αKG-driven OXPHOS, and we find that isocitrate dehydrogenase 1 (IDH1) plays a central role in this process. IDH1 downregulation augments mitochondrial oxidation of αKG and inhibits reticulocyte generation. Furthermore, IDH1 knockdown results in the generation of multinucleated erythroblasts, a morphological abnormality characteristic of myelodysplastic syndrome and congenital dyserythropoietic anemia. We identify vitamin C homeostasis as a critical regulator of ineffective erythropoiesis; oxidized ascorbate increases mitochondrial superoxide and significantly exacerbates the abnormal erythroblast phenotype of IDH1-downregulated progenitors, whereas vitamin C, scavenging reactive oxygen species (ROS) and reprogramming mitochondrial metabolism, rescues erythropoiesis. Thus, an IDH1-vitamin C crosstalk controls terminal steps of human erythroid differentiation.

Entry of glucose- and glutamine-derived carbons into the citric acid cycle supports early steps of HIV-1 infection in CD4 T cells.

The susceptibility of CD4 T cells to human immunodeficiency virus 1 (HIV-1) infection is regulated by glucose and glutamine metabolism, but the relative contributions of these nutrients to infection are not known. Here we show that glutaminolysis is the major pathway fuelling the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) in T-cell receptor-stimulated naïve, as well as memory CD4, subsets and is required for optimal HIV-1 infection. Under conditions of attenuated glutaminolysis, the α-ketoglutarate (α-KG) TCA rescues early steps in infection; exogenous α-KG promotes HIV-1 reverse transcription, rendering both naïve and memory cells more sensitive to infection. Blocking the glycolytic flux of pyruvate to lactate results in altered glucose carbon allocation to TCA and pentose phosphate pathway intermediates, an increase in OXPHOS and augmented HIV-1 reverse transcription. Moreover, HIV-1 infection is significantly higher in CD4 T cells selected on the basis of high mitochondrial biomass and OXPHOS activity. Therefore, the OXPHOS/aerobic glycolysis balance is a major regulator of HIV-1 infection in CD4 T lymphocytes.

Resveratrol stimulates the metabolic reprogramming of human CD4+ T cells to enhance effector function.

The polyphenol resveratrol activates the deacetylase Sirt1, resulting in various antioxidant, chemoprotectant, neuroprotective, cardioprotective, and anti-inflammatory properties. We found that at high concentrations of resveratrol, human CD4+ T cells showed defective antigen receptor signaling and arrest at the G1 stage of the cell cycle, whereas at low concentrations, cells were readily activated and exhibited enhanced Sirt1 deacetylase activity. Nevertheless, low-dose resveratrol rapidly stimulated genotoxic stress in the T cells, which resulted in engagement of a DNA damage response pathway that depended on the kinase ATR [ataxia telangiectasia-mutated (ATM) and Rad3-related], but not ATM, and subsequently in premitotic cell cycle arrest. The concomitant activation of p53 was coupled to the expression of gene products that regulate cell metabolism, leading to a metabolic reprogramming that was characterized by decreased glycolysis, increased glutamine consumption, and a shift to oxidative phosphorylation. These alterations in the bioenergetic homeostasis of CD4+ T cells resulted in enhanced effector function, with both naïve and memory CD4+ T cells secreting increased amounts of the inflammatory cytokine interferon-γ. Thus, our data highlight the wide range of metabolic adaptations that CD4+ T lymphocytes undergo in response to genomic stress.

Cell surface Glut1 levels distinguish human CD4 and CD8 T lymphocyte subsets with distinct effector functions.

CD4 and CD8 T lymphocyte activation requires the generation of sufficient energy to support new biosynthetic demands. Following T cell receptor (TCR) engagement, these requirements are met by an increased glycolysis, due, at least in part, to induction of the Glut1 glucose transporter. As Glut1 is upregulated on tumor cells in response to hypoxia, we assessed whether surface Glut1 levels regulate the antigen responsiveness of human T lymphocytes in both hypoxic and atmospheric oxygen conditions. Notably, Glut1 upregulation in response to TCR stimulation was significantly higher in T lymphocytes activated under hypoxic as compared to atmospheric oxygen conditions. Furthermore, TCR-stimulated human T lymphocytes sorted on the basis of Glut1-Lo and Glut1-Hi profiles maintained distinct characteristics, irrespective of the oxygen tension. While T cells activated in hypoxia divided less than those activated in atmospheric oxygen, Glut1-Hi lymphocytes exhibited increased effector phenotype acquisition, augmented proliferation, and an inverted CD4/CD8 ratio in both oxygen conditions. Moreover, Glut1-Hi T lymphocytes exhibited a significantly enhanced ability to produce IFN-γ and this secretion potential was completely dependent on continued glycolysis. Thus, Glut1 surface levels identify human T lymphocytes with distinct effector functions in both hypoxic and atmospheric oxygen tensions.

Glutamine-dependent α-ketoglutarate production regulates the balance between T helper 1 cell and regulatory T cell generation.

T cell activation requires that the cell meet increased energetic and biosynthetic demands. We showed that exogenous nutrient availability regulated the differentiation of naïve CD4(+) T cells into distinct subsets. Activation of naïve CD4(+) T cells under conditions of glutamine deprivation resulted in their differentiation into Foxp3(+) (forkhead box P3-positive) regulatory T (Treg) cells, which had suppressor function in vivo. Moreover, glutamine-deprived CD4(+) T cells that were activated in the presence of cytokines that normally induce the generation of T helper 1 (TH1) cells instead differentiated into Foxp3(+) Treg cells. We found that α-ketoglutarate (αKG), the glutamine-derived metabolite that enters into the mitochondrial citric acid cycle, acted as a metabolic regulator of CD4(+) T cell differentiation. Activation of glutamine-deprived naïve CD4(+) T cells in the presence of a cell-permeable αKG analog increased the expression of the gene encoding the TH1 cell-associated transcription factor Tbet and resulted in their differentiation into TH1 cells, concomitant with stimulation of mammalian target of rapamycin complex 1 (mTORC1) signaling. Together, these data suggest that a decrease in the intracellular amount of αKG, caused by the limited availability of extracellular glutamine, shifts the balance between the generation of TH1 and Treg cells toward that of a Treg phenotype.

Glucose and glutamine metabolism regulate human hematopoietic stem cell lineage specification.

The metabolic state of quiescent hematopoietic stem cells (HSCs) is an important regulator of self-renewal, but it is unclear whether or how metabolic parameters contribute to HSC lineage specification and commitment. Here, we show that the commitment of human and murine HSCs to the erythroid lineage is dependent upon glutamine metabolism. HSCs require the ASCT2 glutamine transporter and active glutamine metabolism for erythroid specification. Blocking this pathway diverts EPO-stimulated HSCs to differentiate into myelomonocytic fates, altering in vivo HSC responses and erythroid commitment under stress conditions such as hemolytic anemia. Mechanistically, erythroid specification of HSCs requires glutamine-dependent de novo nucleotide biosynthesis. Exogenous nucleosides rescue erythroid commitment of human HSCs under conditions of limited glutamine catabolism, and glucose-stimulated nucleotide biosynthesis further enhances erythroid specification. Thus, the availability of glutamine and glucose to provide fuel for nucleotide biosynthesis regulates HSC lineage commitment under conditions of metabolic stress.

CTP synthase 1 deficiency in humans reveals its central role in lymphocyte proliferation.

Lymphocyte functions triggered by antigen recognition and co-stimulation signals are associated with a rapid and intense cell division, and hence with metabolism adaptation. The nucleotide cytidine 5' triphosphate (CTP) is a precursor required for the metabolism of DNA, RNA and phospholipids. CTP originates from two sources: a salvage pathway and a de novo synthesis pathway that depends on two enzymes, the CTP synthases (or synthetases) 1 and 2 (CTPS1 with CTPS2); the respective roles of these two enzymes are not known. CTP synthase activity is a potentially important step for DNA synthesis in lymphocytes. Here we report the identification of a loss-of-function homozygous mutation (rs145092287) in CTPS1 in humans that causes a novel and life-threatening immunodeficiency, characterized by an impaired capacity of activated T and B cells to proliferate in response to antigen receptor-mediated activation. In contrast, proximal and distal T-cell receptor (TCR) signalling events and responses were only weakly affected by the absence of CTPS1. Activated CTPS1-deficient cells had decreased levels of CTP. Normal T-cell proliferation was restored in CTPS1-deficient cells by expressing wild-type CTPS1 or by addition of exogenous CTP or its nucleoside precursor, cytidine. CTPS1 expression was found to be low in resting T cells, but rapidly upregulated following TCR activation. These results highlight a key and specific role of CTPS1 in the immune system by its capacity to sustain the proliferation of activated lymphocytes during the immune response. CTPS1 may therefore represent a therapeutic target of immunosuppressive drugs that could specifically dampen lymphocyte activation.

Glut1-mediated glucose transport regulates HIV infection.

Cell cycle entry is commonly considered to positively regulate HIV-1 infection of CD4 T cells, raising the question as to how quiescent lymphocytes, representing a large portion of the viral reservoir, are infected in vivo. Factors such as the homeostatic cytokine IL-7 have been shown to render quiescent T cells permissive to HIV-1 infection, presumably by transiently stimulating their entry into the cell cycle. However, we show here that at physiological oxygen (O(2)) levels (2-5% O(2) tension in lymphoid organs), IL-7 stimulation generates an environment permissive to HIV-1 infection, despite a significantly attenuated level of cell cycle entry. We identify the IL-7-induced increase in Glut1 expression, resulting in augmented glucose uptake, as a key factor in rendering these T lymphocytes susceptible to HIV-1 infection. HIV-1 infection of human T cells is abrogated either by impairment of Glut1 signal transduction or by siRNA-mediated Glut1 down-regulation. Consistent with this, we show that the susceptibility of human thymocyte subsets to HIV-1 infection correlates with Glut1 expression; single-round infection is markedly higher in the Glut1-expressing double-positive thymocyte population than in any of the Glut1-negative subsets. Thus, our studies reveal the Glut1-mediated metabolic pathway as a critical regulator of HIV-1 infection in human CD4 T cells and thymocytes.

In vivo and ex vivo gene transfer in thymocytes and thymocyte precursors.

The thymus provides a specialized environment allowing the differentiation of T lymphocytes from bone marrow-derived progenitor cells. We and others have demonstrated that gene transfer into distinct thymocyte populations can be obtained, both in vivo and ex vivo, using lentiviral vectors. Here, we describe techniques for intrathymic lentiviral transduction in mice, using a surgical approach wherein the thoracic cavity is exposed as well as a significantly less invasive strategy wherein virions are directly injected through the skin. Moreover, thymocyte differentiation from murine and human progenitors is now feasible in vitro, under conditions wherein the Notch and IL-7 signaling pathways are activated. We describe methods allowing transduction of murine and human progenitors and their subsequent differentiation into more mature thymocytes. Conditions for lentiviral gene transfer into more differentiated human thymocyte subsets are also presented. Optimization of technologies for HIV-based gene transfer into murine and human thymocyte progenitors will advance strategies aimed at modulating T-cell differentiation and function in-vivo; approaches potentially targeting patients with genetic and acquired immunodeficiencies as well as immune-sensitive tumors. Furthermore, this technology will foster the progression of basic research aimed at elucidating molecular aspects of T-cell differentiation in mice and humans.

Cell surface expression of the bovine leukemia virus-binding receptor on B and T lymphocytes is induced by receptor engagement.

Bovine leukemia virus (BLV), one of the most common infectious viruses of cattle, is endemic in many herds. Approximately 30-40% of adult cows in the United States are infected by this oncogenic C-type retrovirus and 1-5% of animals will eventually develop a malignant lymphoma. BLV, like the human and simian T cell leukemia viruses, is a deltaretrovirus but, in contrast with the latter, the BLV receptor remains unidentified. In this study, we demonstrate that the amino-terminal 182 residues of the BLV envelope glycoprotein surface unit encompasses the receptor-binding domain. A bona fide interaction of this receptor-binding domain with the BLV receptor was demonstrated by specific interference with BLV, but not human T cell leukemia virus, envelope glycoprotein-mediated binding. We generated a rabbit Ig Fc-tagged BLV receptor-binding domain construct and ascertained that the ligand binds the BLV receptor on target cells from multiple species. Using this tool, we determined that the BLV-binding receptor is expressed on differentiating pro/pre-B cells in mouse bone marrow. However, the receptor was not detected on mature/quiescent B cells but was induced upon B cell activation. Activation of human B and T lymphocytes also induced surface BLV-binding receptor expression and required de novo protein synthesis. Receptor levels were down-regulated as activated lymphocytes returned to quiescence. In the human thymus, BLV-binding receptor expression was specifically detected on thymocytes responding to the IL-7 cytokine. Thus, expression of the BLV-binding receptor is a marker of enhanced metabolic activity in B cells, T cells, and thymocytes.

Lentiviral transduction of immune cells.

Gene transfer into mammalian cells has been of crucial importance for studies determining the role of specific genes in the differentiation and cell fate of various hematopoietic lineages. Until recently, the majority of these studies were performed in transformed cell lines due to difficulties in achieving levels of transfection of greater than 1-3% in primary hematopoietic cells. Vectors based on retrovirus and lentivirus backbones have revolutionized our ability to transfer genes into primary hematopoietic cells. These vectors have allowed extensive ex vivo and in vivo studies following introduction of a gene of interest and have been used clinically in individuals suffering from cancers, infections, and genetic diseases. Ex vivo lentiviral gene transfer can result in efficient transduction of progenitor cells (>80%) that can then be further differentiated into immune lineage cells including T, B, dendritic, or natural killer cells. Alternatively, differentiated immune cells can themselves be transduced ex vivo with lentiviral vectors. Here, we discuss optimization of technologies for human immunodeficiency virus (HIV)-based gene transfer into murine and human progenitor and immune cell lineages.

Erythrocyte Glut1 triggers dehydroascorbic acid uptake in mammals unable to synthesize vitamin C.

Of all cells, human erythrocytes express the highest level of the Glut1 glucose transporter. However, the regulation and function of Glut1 during erythropoiesis are not known. Here, we report that glucose transport actually decreases during human erythropoiesis despite a >3-log increase in Glut1 transcripts. In contrast, Glut1-mediated transport of L-dehydroascorbic acid (DHA), an oxidized form of ascorbic acid (AA), is dramatically enhanced. We identified stomatin, an integral erythrocyte membrane protein, as regulating the switch from glucose to DHA transport. Notably though, we found that erythrocyte Glut1 and associated DHA uptake are unique traits of humans and the few other mammals that have lost the ability to synthesize AA from glucose. Accordingly, we show that mice, a species capable of synthesizing AA, express Glut4 but not Glut1 in mature erythrocytes. Thus, erythrocyte-specific coexpression of Glut1 with stomatin constitutes a compensatory mechanism in mammals that are unable to synthesize vitamin C.

Isolated receptor binding domains of HTLV-1 and HTLV-2 envelopes bind Glut-1 on activated CD4+ and CD8+ T cells.

BACKGROUND: We previously identified the glucose transporter Glut-1, a member of the multimembrane-spanning facilitative nutrient transporter family, as a receptor for both HTLV-1 and HTLV-2. However, a recent report concluded that Glut-1 cannot serve as a receptor for HTLV-1 on CD4 T cells: This was based mainly on their inability to detect Glut-1 on this lymphocyte subset using the commercial antibody mAb1418. It was therefore of significant interest to thoroughly assess Glut-1 expression on CD4 and CD8 T cells, and its association with HTLV-1 and -2 envelope binding. RESULTS: As previously reported, ectopic expression of Glut-1 but not Glut-3 resulted in significantly augmented binding of tagged proteins harboring the receptor binding domains of either HTLV-1 or HTLV-2 envelope glycoproteins (H1RBD or H2RBD). Using antibodies raised against the carboxy-terminal peptide of Glut-1, we found that Glut-1 expression was significantly increased in both CD4 and CD8 cells following TCR stimulation. Corresponding increases in the binding of H1RBD as well as H2RBD, not detected on quiescent T cells, were observed following TCR engagement. Furthermore, increased Glut-1 expression was accompanied by a massive augmentation in glucose uptake in TCR-stimulated CD4 and CD8 lymphocytes. Finally, we determined that the apparent contradictory results obtained by Takenouchi et al were due to their monitoring of Glut-1 with a mAb that does not bind cells expressing endogenous Glut-1, including human erythrocytes that harbor 300,000 copies per cell. CONCLUSION: Transfection of Glut-1 directly correlates with the capacities of HTLV-1 and HTLV-2 envelope-derived ligands to bind cells. Moreover, Glut-1 is induced by TCR engagement, resulting in massive increases in glucose uptake and binding of HTLV-1 and -2 envelopes to both CD4 and CD8 T lymphocytes. Therefore, Glut-1 is a primary binding receptor for HTLV-1 and HTLV-2 envelopes on activated CD4 as well as CD8 lymphocytes.

IL-7-induced proliferation of recent thymic emigrants requires activation of the PI3K pathway.

The IL-7 cytokine promotes the survival of a diverse T-cell pool, thereby ensuring an efficient immune response. Moreover, IL-7 induces the proliferation of recent thymic emigrants (RTEs) in neonates. Here, we demonstrate that the survival and proliferative effects of IL-7 on human RTEs can be distinguished on the basis of dose as well as duration of IL-7 administration. A dose of 0.1 ng/mL IL-7 is sufficient to promote viability, whereas cell-cycle entry is observed only at doses higher than 1 ng/mL. Moreover, a short 1-hour exposure to high-dose IL-7 (10 ng/mL) induces long-term survival but continuous IL-7 exposure is necessary for optimal cell-cycle entry and proliferation. We find that distinct signaling intermediates are activated under conditions of IL-7-induced survival and proliferation; STAT5 tyrosine phosphorylation does not correlate with proliferation, whereas up-regulation of the glucose transporter Glut-1 as well as increased glucose uptake are markers of IL-7-induced cell cycle entry. Glut-1 is directly regulated by PI3K and, indeed, inhibiting PI3K activity abrogates IL-7-induced proliferation. Our finding that the survival and proliferation of RTEs are differentially modulated by the dose and kinetics of exogenous IL-7 has important implications for the clinical use of this cytokine.

Intrathymic administration of hematopoietic progenitor cells enhances T cell reconstitution in ZAP-70 severe combined immunodeficiency.

Patients with severe combined immunodeficiency (SCID) present with opportunistic infections that are almost universally fatal in infancy. The mainstay treatment for these patients is allogeneic hematopoietic stem cell (HSC) transplantation, but sustained polyclonal T cell reconstitution is too often unsatisfactory. Although transplantation is conventionally performed by i.v. administration of HSC, we hypothesized that an intrathymic strategy would be superior. Indeed, several progenitor cell populations are incapable of homing to the thymus, the major site of T cell differentiation, and it appears that there are extensive time periods during which the thymus is refractory to progenitor cell import. To test this hypothesis, nonconditioned infant ZAP-70-deficient SCID mice were intrathymically injected with WT bone marrow progenitor cells, a procedure accomplished without surgical intervention. Upon intrathymic HSC injection, there was a more rapid T cell differentiation, with mature thymocytes detected by 4 weeks after transplantation. Intrathymic injection of HSC also resulted in significantly higher numbers of peripheral T cells, increased percentages of naïve T cells, and more diverse T cell receptor repertoires. Moreover, T cell reconstitution after intrathymic transplantation was obtained after injection of 10-fold fewer donor HSC. Thus, this intrathymic transplantation approach may improve the outcome of SCID patients by enhancing T cell reconstitution.