How autoreactive thymocytes differentiate into regulatory versus effector CD4+ T cells after avoiding clonal deletion.

Thymocytes bearing autoreactive T cell receptors (TCRs) are agonist-signaled by TCR/co-stimulatory molecules to either undergo clonal deletion or to differentiate into specialized regulatory T (Treg) or effector T (Teff) CD4+ cells. How these different fates are achieved during development remains poorly understood. We now document that deletion and differentiation are agonist-signaled at different times during thymic selection and that Treg and Teff cells both arise after clonal deletion as alternative lineage fates of agonist-signaled CD4+CD25+ precursors. Disruption of agonist signaling induces CD4+CD25+ precursors to initiate Foxp3 expression and become Treg cells, whereas persistent agonist signaling induces CD4+CD25+ precursors to become IL-2+ Teff cells. Notably, we discovered that transforming growth factor-ß induces Foxp3 expression and promotes Treg cell development by disrupting weaker agonist signals and that Foxp3 expression is not induced by IL-2 except under non-physiological in vivo conditions. Thus, TCR signaling disruption versus persistence is a general mechanism of lineage fate determination in the thymus that directs development of agonist-signaled autoreactive thymocytes.

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.

Complete tumor resection reverses neutrophilia-associated suppression of systemic anti-tumor immunity.

OBJECTIVES: Tumor infiltrating neutrophils suppress T cell function, but whether neutrophils in circulation contribute to systemic immunosuppression is unclear. We aimed to study whether peripheral neutrophils that accumulate with tumor progression contribute to systemic immunosuppression, and if observed suppression of systemic anti-tumor immunity could be reversed with complete surgical tumor removal. MATERIALS AND METHODS: Syngeneic murine oral cancers were established in immunocompetent mice. Proteomic and functional immune assays were used to study plasma cytokine concentration, peripheral immune frequencies, and systemic anti-tumor immunity with and without complete primary tumor resection. RESULTS: Ly6G+ neutrophilic cells, but not other myeloid cell types, accumulated in the periphery of mice with progressing tumors. This accumulation positively associated with plasma G-CSF concentration. Circulating neutrophils were functionally immunosuppressive. Complete surgical tumor removal reversed the observed neutrophilia, with neutrophil frequencies returning to baseline in 21 days. Multiple independent functional assays revealed enhanced systemic anti-tumor immunity in mice following tumor resection compared to tumor-bearing mice, and the observed enhanced systemic immunity could be reproduced with selective neutrophil depletion. CONCLUSIONS: Complete primary tumor resection can reverse neutrophilia that develops during tumor progression and result in enhanced systemic anti-tumor immunity. Primary tumor removal relieves neutrophil-driven systemic immunosuppression and may itself contribute to the clinical benefit observed with neoadjuvant immunotherapy.

Egress of resident memory T cells from tissue with neoadjuvant immunotherapy: Implications for systemic anti-tumor immunity.

INTRODUCTION: Resident memory T (TRM) cells are embedded in peripheral tissue and capable of acting as sentinels that can respond quickly to repeat pathogen exposure as part of an endogenous anti-microbial immune response. Recent evidence suggests that chronic antigen exposure and other microenvironment cues may promote the development of TRM cells within solid tumors as well, and that this TRM phenotype can sequester tumor-specific T cells into tumors and out of circulation resulting in limited systemic antitumor immunity. Here, we perform a review of the published English literature and describe tissue-specific mediators of TRM cell differentiation in states of infection and malignancy with special focus on the role of TGF-ß and how targeting TGF-ß signaling could be used as a therapeutical approach to promote tumor systemic immunity. DISCUSSION: The presence of TRM cells with antigen specificity to neoepitopes in tumors associates with positive clinical prognosis and greater responsiveness to immunotherapy. Recent evidence indicates that solid tumors may act as reservoirs for tumor specific TRM cells and limit their circulation - possibly resulting in impaired systemic antitumor immunity. TRM cells utilize specific mechanisms to egress from peripheral tissues into circulation and other peripheral sites, and emerging evidence indicates that immunotherapeutic approaches may initiate these processes and increase systemic antitumor immunity. CONCLUSIONS: Reversing tumor sequestration of tumor-specific T cells prior to surgical removal or radiation of tumor may increase systemic antitumor immunity. This finding may underlie the improved recurrence free survival observed with neoadjuvant immunotherapy in clinical trials.

Phenotypic plasticity and reduced tissue retention of exhausted tumor-infiltrating T cells following neoadjuvant immunotherapy in head and neck cancer.

Neoadjuvant immunotherapies (NITs) have led to clinical benefits in several cancers. Characterization of the molecular mechanisms underlying responses to NIT may lead to improved treatment strategies. Here we show that exhausted, tumor-infiltrating CD8+ T (Tex) cells display local and systemic responses to concurrent neoadjuvant TGF-ß and PD-L1 blockade. NIT induces a significant and selective increase in circulating Tex cells associated with reduced intratumoral expression of the tissue-retention marker CD103. TGF-ß-driven CD103 expression on CD8+ T cells is reversed following TGF-ß neutralization in vitro, implicating TGF-ß in T cell tissue retention and impaired systemic immunity. Transcriptional changes implicate T cell receptor signaling and glutamine metabolism as important determinants of enhanced or reduced Tex treatment response, respectively. Our analysis illustrates physiological and metabolic changes underlying T cell responses to NIT, highlighting the interplay between immunosuppression, tissue retention, and systemic anti-tumor immunity and suggest antagonism of T cell tissue retention as a promising neoadjuvant treatment strategy.

MHC-independent αßT cells: Lessons learned about thymic selection and MHC-restriction.

Understanding the generation of an MHC-restricted T cell repertoire is the cornerstone of modern T cell immunology. The unique ability of αßT cells to only recognize peptide antigens presented by MHC molecules but not conformational antigens is referred to as MHC restriction. How MHC restriction is imposed on a very large T cell receptor (TCR) repertoire is still heavily debated. We recently proposed the selection model, which posits that newly re-arranged TCRs can structurally recognize a wide variety of antigens, ranging from peptides presented by MHC molecules to native proteins like cell surface markers. However, on a molecular level, the sequestration of the essential tyrosine kinase Lck by the coreceptors CD4 and CD8 allows only MHC-restricted TCRs to signal. In the absence of Lck sequestration, MHC-independent TCRs can signal and instruct the generation of mature αßT cells that can recognize native protein ligands. The selection model thus explains how only MHC-restricted TCRs can signal and survive thymic selection. In this review, we will discuss the genetic evidence that led to our selection model. We will summarize the selection mechanism and structural properties of MHC-independent TCRs and further discuss the various non-MHC ligands we have identified.

Influence of an Audiovisual Resource on the Preoperative Anxiety of Adult Endodontic Patients: A Randomized Controlled Clinical Trial.

INTRODUCTION: The expectation of undergoing endodontic treatment can cause anxiety in patients. Anxiety is described as a transient emotional state closely related to pain, fear, and imbalance of the organism. The clinician commonly must use some type of tool to alleviate the patient's preoperative anxiety before treatment can be applied. The aim of this study was to evaluate the influence of an audiovisual resource on the preoperative anxiety of adult patients undergoing endodontic treatment. METHODS: One hundred sixty endodontic patients were randomly divided into experimental and control groups (n = 80) and then assessed at 2 preoperative time points separated by a 10-minute interval. After the first assessment, the patients in the experimental group watched a video of their own choice obtained from the Internet to provide them with a relaxing experience. In both groups and at both time points, the assessments consisted of collecting the patients' vital signs (diastolic blood pressure, systolic blood pressure, and heart rate) and data regarding their subjective perception of anxiety using a visual analog scale. RESULTS: There were no significant differences between the groups regarding the vital sign variation observed between the 2 assessment time points. However, the variation in the scores obtained on the visual analog scale was significantly greater in the experimental group (P < .05), indicating a greater reduction in the level of preoperative anxiety in this group. CONCLUSIONS: The preoperative use of an audiovisual resource was associated with a decrease in the perception of anxiety by patients undergoing endodontic treatment.

Phosphate Transporter Profiles in Murine and Human Thymi Identify Thymocytes at Distinct Stages of Differentiation.

Thymocyte differentiation is dependent on the availability and transport of metabolites in the thymus niche. As expression of metabolite transporters is a rate-limiting step in nutrient utilization, cell surface transporter levels generally reflect the cell's metabolic state. The GLUT1 glucose transporter is upregulated on actively dividing thymocytes, identifying thymocytes with an increased metabolism. However, it is not clear whether transporters of essential elements such as phosphate are modulated during thymocyte differentiation. While PiT1 and PiT2 are both phosphate transporters in the SLC20 family, we show here that they exhibit distinct expression profiles on both murine and human thymocytes. PiT2 expression distinguishes thymocytes with high metabolic activity, identifying immature murine double negative (CD4-CD8-) DN3b and DN4 thymocyte blasts as well as immature single positive (ISP) CD8 thymocytes. Notably, the absence of PiT2 expression on RAG2-deficient thymocytes, blocked at the DN3a stage, strongly suggests that high PiT2 expression is restricted to thymocytes having undergone a productive TCRß rearrangement at the DN3a/DN3b transition. Similarly, in the human thymus, PiT2 was upregulated on early post-ß selection CD4+ISP and TCRαß-CD4hiDP thymocytes co-expressing the CD71 transferrin receptor, a marker of metabolic activity. In marked contrast, expression of the PiT1 phosphate importer was detected on mature CD3+ murine and human thymocytes. Notably, PiT1 expression on CD3+DN thymocytes was identified as a biomarker of an aging thymus, increasing from 8.4 ± 1.5% to 42.4 ± 9.4% by 1 year of age (p < 0.0001). We identified these cells as TCRγδ and, most significantly, NKT, representing 77 ± 9% of PiT1+DN thymocytes by 1 year of age (p < 0.001). Thus, metabolic activity and thymic aging are associated with distinct expression profiles of the PiT1 and PiT2 phosphate transporters.

Molecular constraints on CDR3 for thymic selection of MHC-restricted TCRs from a random pre-selection repertoire.

The αß T cell receptor (TCR) repertoire on mature T cells is selected in the thymus, but the basis for thymic selection of MHC-restricted TCRs from a randomly generated pre-selection repertoire is not known. Here we perform comparative repertoire sequence analyses of pre-selection and post-selection TCR from multiple MHC-sufficient and MHC-deficient mouse strains, and find that MHC-restricted and MHC-independent TCRs are primarily distinguished by features in their non-germline CDR3 regions, with many pre-selection CDR3 sequences not compatible with MHC-binding. Thymic selection of MHC-independent TCR is largely unconstrained, but the selection of MHC-specific TCR is restricted by both CDR3 length and specific amino acid usage. MHC-restriction disfavors TCR with CDR3 longer than 13 amino acids, limits positively charged and hydrophobic amino acids in CDR3ß, and clonally deletes TCRs with cysteines in their CDR3 peptide-binding regions. Together, these MHC-imposed structural constraints form the basis to shape VDJ recombination sequences into MHC-restricted repertoires.

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.

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.

Influence of coronal restoration and root canal filling quality on periapical status: clinical and radiographic evaluation.

INTRODUCTION: The purpose of this study was to evaluate the relationship between periapical status and the quality of coronal restoration and of root canal filling, assessed both clinically and radiographically, in a cohort of Brazilian patients. METHODS: A total of 523 teeth from 337 patients submitted to endodontic treatment were clinically and radiographically reexamined 2-10 years postoperatively. Restoration and root canal filling quality were classified according to modified criteria from Tronstad et al. Periapical status was evaluated according to periapical index scores. Mann-Whitney and Friedman tests were used to conduct the descriptive analysis. Correlations were analyzed by using simple and multivariate logistic regression analysis. RESULTS: No significant difference was observed between the rates of apical periodontitis for adequate or inadequate coronal restorations assessed clinically (12.8% versus 19.4%), whereas these rates were significantly different when the restoration quality was assessed radiographically (11.6% versus 28.7%, P < .001). The rates of apical periodontitis in teeth with inadequate root canal filling, with or without adequate restoration, were significantly higher than in teeth with adequate canal filling, with or without adequate restoration (38.6% and 48.4% versus 6.5% and 14.6%, respectively; P < .0001). CONCLUSIONS: Using either a radiographic or clinical assessment alone was not a reliable method to ascertain whether restoration quality could be correlated with postoperative periapical status. Poor root canal filling quality was a prognostic determinant of endodontic treatment failure, whereas coronal restoration quality had a lesser impact on the outcome of the endodontic treatment.

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.

Metabolic pathways as regulators of HIV infection.

PURPOSE OF REVIEW: Activation of the immune system only occurs when stimulated cells generate sufficient energy to support their growth and proliferation. Moreover, efficient HIV-1 infection requires that CD4(+) T cells meet the energy demands involved in completing the different steps of the virus life cycle. In this review, we highlight recent studies revealing the importance of nutrient fuels, nucleotide metabolism and the oxygen microenvironment in regulating HIV-1 infection, T-cell differentiation and the generation of HIV-1-specific immune responses. RECENT FINDINGS: Glucose uptake via the Glut1 glucose transporter is required for efficient HIV-1 infection of CD4(+) lymphocytes. Other nutrients can also be used as sources of energy and their utilization conditions the differentiation of CD4(+) T cells to distinct effector fates. The conversion of ATP to adenosine inhibits HIV-specific effector cells and the hydrolysis of dNTPs by SAMHD1 restricts infection. Furthermore, oxygen concentration modulates metabolic status, thereby altering T-cell differentiation and potential to mediate a specific immune response. SUMMARY: The availability and use of energy resources in fluctuating environments regulate T-cell function and susceptibility to HIV-1 infection. Identification of the targets coordinating the selected metabolic pathways will advance new strategic avenues for controlling HIV-1 disease progression.