Clonal selection in the human Vδ1 T cell repertoire indicates γδ TCR-dependent adaptive immune surveillance

γδ T cells are considered to be innate-like lymphocytes that respond rapidly to stress without clonal selection and differentiation. Here we use next-generation sequencing to probe how this paradigm relates to human Vδ2neg T cells, implicated in responses to viral infection and cancer. The prevalent Vδ1 T cell receptor (TCR) repertoire is private and initially unfocused in cord blood, typically becoming strongly focused on a few high-frequency clonotypes by adulthood. Clonal expansions have differentiated from a naive to effector phenotype associated with CD27 downregulation, retaining proliferative capacity and TCR sensitivity, displaying increased cytotoxic markers and altered homing capabilities, and remaining relatively stable over time. Contrastingly, Vδ2+ T cells express semi-invariant TCRs, which are present at birth and shared between individuals. Human Vδ1+ T cells have therefore evolved a distinct biology from the Vδ2+ subset, involving a central, personalized role for the γδ TCR in directing a highly adaptive yet unconventional form of immune surveillance

Maturation-Dependent Licensing of Naive T Cells for Rapid TNF Production

The peripheral naïve T cell pool is comprised of a heterogeneous population of cells at various stages of development, which is a process that begins in the thymus and is completed after a post-thymic maturation phase in the periphery. One hallmark of naïve T cells in secondary lymphoid organs is their unique ability to produce TNF rapidly after activation and prior to acquiring other effector functions. To determine how maturation influences the licensing of naïve T cells to produce TNF, we compared cytokine profiles of CD4+ and CD8+ single positive (SP) thymocytes, recent thymic emigrants (RTEs) and mature-naïve (MN) T cells during TCR activation. SP thymocytes exhibited a poor ability to produce TNF when compared to splenic T cells despite expressing similar TCR levels and possessing comparable activation kinetics (upregulation of CD25 and CD69). Provision of optimal antigen presenting cells from the spleen did not fully enable SP thymocytes to produce TNF, suggesting an intrinsic defect in their ability to produce TNF efficiently. Using a thymocyte adoptive transfer model, we demonstrate that the ability of T cells to produce TNF increases progressively with time in the periphery as a function of their maturation state. RTEs that were identified in NG-BAC transgenic mice by the expression of GFP showed a significantly enhanced ability to express TNF relative to SP thymocytes but not to the extent of fully MN T cells. Together, these findings suggest that TNF expression by naïve T cells is regulated via a gradual licensing process that requires functional maturation in peripheral lymphoid organs

Abnormal Changes in NKT Cells, the IGF-1 Axis, and Liver Pathology in an Animal Model of ALS

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing fatal neurodegenerative disorder characterized by the selective death of motor neurons (MN) in the spinal cord, and is associated with local neuroinflammation. Circulating CD4+ T cells are required for controlling the local detrimental inflammation in neurodegenerative diseases, and for supporting neuronal survival, including that of MN. T-cell deficiency increases neuronal loss, while boosting T cell levels reduces it. Here, we show that in the mutant superoxide dismutase 1 G93A (mSOD1) mouse model of ALS, the levels of natural killer T (NKT) cells increased dramatically, and T-cell distribution was altered both in lymphoid organs and in the spinal cord relative to wild-type mice. The most significant elevation of NKT cells was observed in the liver, concomitant with organ atrophy. Hepatic expression levels of insulin-like growth factor (IGF)-1 decreased, while the expression of IGF binding protein (IGFBP)-1 was augmented by more than 20-fold in mSOD1 mice relative to wild-type animals. Moreover, hepatic lymphocytes of pre-symptomatic mSOD1 mice were found to secrete significantly higher levels of cytokines when stimulated with an NKT ligand, ex-vivo. Immunomodulation of NKT cells using an analogue of α-galactosyl ceramide (α-GalCer), in a specific regimen, diminished the number of these cells in the periphery, and induced recruitment of T cells into the affected spinal cord, leading to a modest but significant prolongation of life span of mSOD1 mice. These results identify NKT cells as potential players in ALS, and the liver as an additional site of major pathology in this disease, thereby emphasizing that ALS is not only a non-cell autonomous, but a non-tissue autonomous disease, as well. Moreover, the results suggest potential new therapeutic targets such as the liver for immunomodulatory intervention for modifying the disease, in addition to MN-based neuroprotection and systemic treatments aimed at reducing oxidative stress

A natural killer T (NKT) cell developmental pathway involving a thymus-dependent NK1.1(-) CD4(+) CD1d-dependent precursor stage

The development of CD1d-dependent natural killer T (NKT) cells is poorly understood. We have used both CD1d/alpha-galactosylceramide (CD1d/alphaGC) tetramers and anti-NK1.1 to investigate NKT cell development in vitro and in vivo. Confirming the thymus-dependence of these cells, we show that CD1d/alphaGC tetramer-binding NKT cells, including NK1.1(+) and NK1.1(-) subsets, develop in fetal thymus organ culture (FTOC) and are completely absent in nude mice. Ontogenically, CD1d/alphaGC tetramer-binding NKT cells first appear in the thymus, at day 5 after birth, as CD4(+)CD8(-)NK1.1(-)cells. NK1.1(+) NKT cells, including CD4(+) and CD4(-)CD8(-) subsets, appeared at days 7-8 but remained a minor subset until at least 3 wk of age. Using intrathymic transfer experiments, CD4(+)NK1.1(-) NKT cells gave rise to NK1.1(+) NKT cells (including CD4(+) and CD4(-) subsets), but not vice-versa. This maturation step was not required for NKT cells to migrate to other tissues, as NK1.1(-) NKT cells were detected in liver and spleen as early as day 8 after birth, and the majority of NKT cells among recent thymic emigrants (RTE) were NK1.1(-). Further elucidation of this NKT cell developmental pathway should prove to be invaluable for studying the mechanisms that regulate the development of these cells

Normal thymocyte negative selection in TRAIL-deficient mice

The molecular basis of thymocyte negative selection, which plays a critical role in establishing and maintaining immunological tolerance, is not yet resolved. In particular, the importance of the death receptor subgroup of the tumor necrosis factor (TNF)-family has been the subject of many investigations, with equivocal results. A recent report suggested that TRAIL was a critical factor in this process, a result that does not fit well with previous studies that excluded a role for the FADD-caspase 8 pathway, which is essential for TRAIL and Fas ligand (FasL) signaling, in negative selection. We have investigated intrathymic negative selection of TRAIL-deficient thymocytes, using four well-established models, including antibody-mediated TCR/CD3 ligation in vitro, stimulation with endogenous superantigen in vitro and in vivo, and treatment with exogenous superantigen in vitro. We were unable to demonstrate a role for TRAIL signaling in any of these models, suggesting that this pathway is not a critical factor for thymocyte negative selection

Activation of thymic regeneration in mice and humans following androgen blockade

C1 - Journal Articles RefereedThe thymus undergoes age-related atrophy, coincident with increased circulating sex steroids from puberty. The impact of thymic atrophy is most profound in clinical conditions that cause a severe loss in peripheral T cells with the ability to regenerate adequate numbers of naive CD4+ T cells indirectly correlating with patient age. The present study demonstrates that androgen ablation results in the complete regeneration of the aged male mouse thymus, restoration of peripheral T cell phenotype and function and enhanced thymus regeneration following bone marrow transplantation. Importantly, this technique is also applicable to humans, with analysis of elderly males undergoing sex steroid ablation therapy for prostatic carcinoma, demonstrating an increase in circulating T cell numbers, particularly naive (TREC+) T cells. Collectively these studies represent a fundamentally new approach to treating immunodeficiency states in humans

alpha-Glucuronosyl and alpha-glucosyl diacylglycerides, natural killer T cell-activating lipids from bacteria and fungi

Natural killer T cells express T cell receptors (TCRs) that recognize glycolipid antigens in association with the antigen-presenting molecule CD1d. Here, we report the concise chemical synthesis of a range of saturated and unsaturated α-glucosyl and α-glucuronosyl diacylglycerides of bacterial and fungal origins from allyl α-glucoside with Jacobsen kinetic resolution as a key step. These glycolipids are recognized by a classical type I NKT TCR that uses an invariant Vα14-Jα18 TCR α-chain, but also by an atypical NKT TCR that uses a different TCR α-chain (Vα10-Jα50). In both cases, recognition is sensitive to the lipid fine structure, and includes recognition of glycosyl diacylglycerides bearing branched (R- and S-tuberculostearic acid) and unsaturated (oleic and vaccenic) acids. The TCR footprints on CD1d loaded with a mycobacterial α-glucuronosyl diacylglyceride were assessed using mutant CD1d molecules and, while similar to that for α-GalCer recognition by a type I NKT TCR, were more sensitive to mutations when α-glucuronosyl diacylglyceride was the antigen. In summary, we provide an efficient approach for synthesis of a broad class of bacterial and fungal α-glycosyl diacylglyceride antigens and demonstrate that they can be recognised by TCRs derived from type I and atypical NKT cells

Antigen Specificity of Type I NKT Cells Is Governed by TCR beta-Chain Diversity

NKT cells recognize lipid-based Ags presented by CD1d. Type I NKT cells are often referred to as invariant owing to their mostly invariant TCR α-chain usage (Vα14-Jα18 in mice, Vα24-Jα18 in humans). However, these cells have diverse TCR β-chains, including Vβ8, Vβ7, and Vβ2 in mice and Vβ11 in humans, joined to a range of TCR Dβ and Jβ genes. In this study, we demonstrate that TCR β-chain composition can dramatically influence lipid Ag recognition in an Ag-dependent manner. Namely, the glycolipids α-glucosylceramide and isoglobotrihexosylceramide were preferentially recognized by Vβ7(+) NKT cells from mice, whereas the α-galactosylceramide analog OCH, with a truncated sphingosine chain, was preferentially recognized by Vβ8(+) NKT cells from mice. We show that the influence of the TCR β-chain is due to a combination of Vβ-, Jβ-, and CDR3β-encoded residues and that these TCRs can recapitulate the selective Ag reactivity in TCR-transduced cell lines. Similar observations were made with human NKT cells where different CDR3β-encoded residues determined Ag preference. These findings indicate that NKT TCR β-chain diversity results in differential and nonhierarchical Ag recognition by these cells, which implies that some Ags can preferentially activate type I NKT cell subsets