Joseph P. Sanderson

Invariant natural killer T cells recognize lipid self-antigen induced by microbial danger signals

Invariant natural killer T cells (iNKT cells) have a prominent role during infection and other inflammatory processes, and these cells can be activated through their T cell antigen receptors by microbial lipid antigens. However, increasing evidence shows that they are also activated in situations in which foreign lipid antigens would not be present, which suggests a role for lipid self antigen. We found that an abundant endogenous lipid, β-D-glucopyranosylceramide (β-GlcCer), was a potent iNKT cell self antigen in mouse and human and that its activity depended on the composition of the N-acyl chain. Furthermore, β-GlcCer accumulated during infection and in response to Toll-like receptor agonists, contributing to iNKT cell activation. Thus, we propose that recognition of β-GlcCer by the invariant T cell antigen receptor translates innate danger signals into iNKT cell activation.

Sulfamethoxazole and Its Metabolite Nitroso Sulfamethoxazole Stimulate Dendritic Cell Costimulatory Signaling

Different signals in addition to the antigenic signal are required to initiate an immunological reaction. In the context of sulfamethoxazole allergy, the Ag is thought to be derived from its toxic nitroso metabolite, but little is known about the costimulatory signals, including those associated with dendritic cell maturation. In this study, we demonstrate increased CD40 expression, but not CD80, CD83, or CD86, with dendritic cell surfaces exposed to sulfamethoxazole (250–500 μM) and the protein-reactive metabolite nitroso sulfamethoxazole (1–10 μM). Increased CD40 expression was not associated with apoptosis or necrosis, or glutathione depletion. Covalently modified intracellular proteins were detected when sulfamethoxazole was incubated with dendritic cells. Importantly, the enzyme inhibitor 1-aminobenzotriazole prevented the increase in CD40 expression with sulfamethoxazole, but not with nitroso sulfamethoxazole or LPS. The enzymes CYP2C9, CYP2C8, and myeloperoxidase catalyzed the conversion of sulfamethoxazole to sulfamethoxazole hydroxylamine. Myeloperoxidase was expressed at high levels in dendritic cells. Nitroso sulfamethoxazole immunogenicity was inhibited in mice with a blocking anti-CD40L Ab. In addition, when a primary nitroso sulfamethoxazole-specific T cell response using drug-naive human cells was generated, the magnitude of the response was enhanced when cultures were exposed to a stimulatory anti-CD40 Ab. Finally, increased CD40 expression was 5-fold higher on nitroso sulfamethoxazole-treated dendritic cells from an HIV-positive allergic patient compared with volunteers. These data provide evidence of a link between localized metabolism, dendritic cell activation, and drug immunogenicity.

Innate-Like Control of Human iNKT Cell Autoreactivity via the Hypervariable CDR3β Loop

T-cell receptor variability gives rise to a functional hierarchy of human invariant Natural Killer T-cells through a powerful effect on CD1d binding affinity, which is independent of CD1d ligands.

Role of bioactivation in drug-induced hypersensitivity reactions

Drug-induced hypersensitivity reactions are a major problem in both clinical treatment and drug development. This review covers recent developments in our understanding of the pathogenic mechanisms involved, with special focus on the potential role of metabolism and bioactivation in generating a chemical signal for activation of the immune system. The possible role of haptenation and neoantigen formation is discussed, alongside recent findings that challenge this paradigm. Additionally, the essential role of costimulation is examined, as are the potential points whereby costimulation may be driven by reactive metabolites. The relevance of local generation of metabolites in determining the location and character of a reaction is also covered.

CD1d protein structure determines species-selective antigenicity of isoglobotrihexosylceramide (iGb3) to invariant NKT cells

Isoglobotrihexosylceramide (iGb3) has been identified as a potent CD1d‐presented self‐antigen for mouse invariant natural killer T (iNKT) cells. The role of iGb3 in humans remains unresolved, however, as there have been conflicting reports about iGb3‐dependent human iNKT‐cell activation, and humans lack iGb3 synthase, a key enzyme for iGb3 synthesis. Given the importance of human immune responses, we conducted a human–mouse cross‐species analysis of iNKT‐cell activation by iGb3‐CD1d. Here we show that human and mouse iNKT cells were both able to recognise iGb3 presented by mouse CD1d (mCD1d), but not human CD1d (hCD1d), as iGb3‐hCD1d was unable to support cognate interactions with the iNKT‐cell TCRs tested in this study. The structural basis for this discrepancy was identified as a single amino acid variation between hCD1d and mCD1d, a glycine‐to‐tryptophan modification within the α2‐helix that prevents flattening of the iGb3 headgroup upon TCR ligation. Mutation of the human residue, Trp153, to the mouse ortholog, Gly155, therefore allowed iGb3‐hCD1d to stimulate human iNKT cells. In conclusion, our data indicate that iGb3 is unlikely to be a major antigen in human iNKT‐cell biology.

Structural and Functional Changes of the Invariant NKT Clonal Repertoire in Early Rheumatoid Arthritis.

Invariant NKT cells (iNKT) are potent immunoregulatory T cells that recognize CD1d via a semi-invariant TCR (iNKT-TCR). Despite the knowledge of a defective iNKT pool in several autoimmune conditions, including rheumatoid arthritis (RA), a clear understanding of the intrinsic mechanisms, including qualitative and structural changes of the human iNKT repertoire at the earlier stages of autoimmune disease, is lacking. In this study, we compared the structure and function of the iNKT repertoire in early RA patients with age- and gender-matched controls. We analyzed the phenotype and function of the ex vivo iNKT repertoire as well as CD1d Ag presentation, combined with analyses of a large panel of ex vivo sorted iNKT clones. We show that circulating iNKTs were reduced in early RA, and their frequency was inversely correlated to disease activity score 28. Proliferative iNKT responses were defective in early RA, independent of CD1d function. Functional iNKT alterations were associated with a skewed iNKT-TCR repertoire with a selective reduction of high-affinity iNKT clones in early RA. Furthermore, high-affinity iNKTs in early RA exhibited an altered functional Th profile with Th1- or Th2-like phenotype, in treatment-naive and treated patients, respectively, compared with Th0-like Th profiles exhibited by high-affinity iNKTs in controls. To our knowledge, this is the first study to provide a mechanism for the intrinsic qualitative defects of the circulating iNKT clonal repertoire in early RA, demonstrating defects of iNKTs bearing high-affinity TCRs. These defects may contribute to immune dysregulation, and our findings could be exploited for future therapeutic intervention.

Natural variations at position 93 of the invariant Vα24-Jα18 α chain of human iNKT-cell TCRs strongly impact on CD1d binding

Human invariant natural killer T (NKT) cell TCRs bind to CD1d via an “invariant” Vα24‐Jα18 chain (iNKTα) paired to semi‐invariant Vβ11 chains (iNKTβ). Single‐amino acid variations at position 93 (p93) of iNKTα, immediately upstream of the “invariant” CDR3α region, have been reported in a substantial proportion of human iNKT‐cell clones (4–30%). Although p93, a serine in most human iNKT‐cell TCRs, makes no contact with CD1d, it could affect CD1d binding by altering the conformation of the crucial CDR3α loop. By generating recombinant refolded iNKT‐cell TCRs, we show that natural single‐nucleotide variations in iNKTα, translating to serine, threonine, asparagine or isoleucine at p93, exert a powerful effect on CD1d binding, with up to 28‐fold differences in affinity between these variants. This effect was observed with CD1d loaded with either the artificial α‐galactosylceramide antigens KRN7000 or OCH, or the endogenous glycolipid β‐galactosylceramide, and its importance for autoreactive recognition of endogenous lipids was demonstrated by the binding of variant iNKT‐cell TCR tetramers to cell surface expressed CD1d. The serine‐containing variant showed the strongest CD1d binding, offering an explanation for its predominance in vivo. Complementary molecular dynamics modeling studies were consistent with an impact of p93 on the conformation of the CDR3α loop.

Drugs as Haptens, Antigens, and Immunogens

It is known that drugs and other small molecular weight compounds can activate the immune system. In this review we discuss the known and proposed mechanisms by which such compounds can act as hapt

Characterization of drug-specific lymphocyte responses in a patient with drug-induced liver injury

To the Editor: Drug-induced liver injury takes many forms and can mimic naturally occurring liver disease. In 1997,Maria andVictorino described lymphocyte responses to drugs in more than 50% of patients with drug-induced liver injury. More recently, histologic examination of inflamed liver tissue from a patient exposed to sulfasalazine revealed an infiltration of granzyme B–secreting T lymphocytes. Importantly, the phenotype and function of T cells frompatientswith drug-induced liver injury have not been studied. In the present investigationwe used lymphocytes from a patient with trimethoprim-induced liver injury as part of a generalized hypersensitivity reaction (drug rash with eosinophilia and systemic symptoms) to define the frequency of circulating drugspecific T cells and to characterize the cellular response in terms of phenotype and function. The patient was admitted to the hospital in the fourth week after onset of trimethoprim treatment for acnewith fever, desquamating rash, and pruritus. Initial results on admission were as follows: white cell count of 24.93 10/mL with eosinophilia of 13% and increased liver function test results (see Table E1 in this article’s Online Repository at www. jacionline.org). She was later transferred to a specialized liver unit at Queen Elizabeth Hospital in Birmingham, where her liver test results continued to deteriorate. Blood samples were taken for the immunologic investigations on referral and 3, 12, and 24 months after recovery. Details of the methods used are available in the Methods section of this article’s Online repository at www.jacionline.org. Ex vivo lymphocytes isolated from the patient, but not shortand long-term drug-exposed control subjects, were shown to readily proliferate and secrete cytokines in vitro after challenge with trimethoprim (Fig 1, A) to confirm that the reaction had an immunologic cause. The frequency of trimethoprim-specific T cells measured by generating libraries of amplified CD4 and CD8 T cells from peripheral blood were estimated to be 75/10 cells (95% CI, 51-110/10 cells) and 31/10 cells (95% CI, 17-56/10 cells), respectively (Fig 1, B). Cells were expanded and proliferation and IFN-g/IL-13 levels were measured to confirm that the amplified cultures contained T cells responsive against trimethoprim (Fig 1, C and D). Similar experiments were conducted with lymphocytes from trimethoprim-exposed control subjects and patients with trimethoprim-mediated skin reactions (with no liver injury, n 5 7), but it was not possible to estimate a drug-specific precursor frequency. Beeler et al recently demonstrated that the frequency of drug-specific T cells in patients with cutaneous reactions was 100 to 4000/10 cells. Although themethodological approach in the 2 studies differs, the data suggest that reaction severity, organ selectivity, or both are not directly related to the frequency of antigen-specific T cells. Forty-five CD4 and CD8 clones expressing different Vb receptors were generated to define the cellular pathophysiology of the reaction to trimethoprim. Proliferative responses to a number of clones were detected with trimethoprim at concentrations as low as 1 mg/mL, indicating that these clones might be preferentially activated in vivo. Cytotoxicity measured with a classical Cr release assay and by monitoring transient increases in CD107a (lysosomal-associated membrane protein 1) expression was detected with both CD4 and CD8 T cells after trimethoprim exposure (see Fig E1 in this article’s Online Repository at www.jacionline.org). Thus, as described previously with T cells isolated from epicutaneous test reactions, both populations of T cells might play a role in the development of clinical signs. Trimethoprim-specific T-cell clones secreted a restricted cytokine profile (Fig 2, A, and see Table E2 in this article’s Online Repository at www.jacionline.org); IL-13, IFN-g, and TNF-a were detected after trimethoprim stimulation. Clones seemed to corelease IFN-g and TNF-a (R 5 0.73), and an association between the levels of these cytokines and cytolytic activity was observed. Cytotoxicity was seen with CD4 and CD8 clones secreting IFN-g and TNF-a at concentrations of greater than 500 pg/mL. Picard et al recently characterized activated IFNg/TNF-a CD8 T lymphocytes, which were largely directed against herpes viruses in hypersensitive patients with systemic symptoms. Furthermore, the authors suggested that reactivation of virus-specific T cells contributes to the development of clinical signs of hypersensitivity. Although screening for the underlying infection or infections was beyond the scope of the current study, our data describing drug-responsive cytotoxic CD4 T cells clearly highlight the need for further mechanistic studies to unequivocally define the disease’s pathogenesis. A particular area of interest was why the T-cell response observed in this patient targeted the liver. For lymphocytes to exert a cytotoxic response in a particular organ, they must be recruited from the circulation. The expression of specific chemokines produced in response to inflammatory stress and their subsequent interaction with chemokine receptors on the surface of lymphocytes is known to promote T-cell migration through the endothelium and into tissues. T cells express distinct chemokine receptors that direct cellular migration. T cells infiltrating skin express high levels of cutaneous lymphocyte antigen and the chemokine receptors CCR4 and CCR10, and indeed, drugspecific T cells from patients with cutaneous drug-induced hypersensitivity reactions express high levels of these receptors. Although the expression of chemokine receptors on liver-homing T cells is less well defined, CXCR3, CCR5, CCR9, and CXCR6 have been found on T cells isolated from patients with various forms of liver disease. In the current study CD11aCD27 (memory and terminally differentiated) trimethoprim-specific CD4 and CD8 clones expressed high levels of CXCR3 and CCR9 but only low levels of skin-homing receptors, which might explain the relatively mild cutaneous symptoms (Fig 2, B, and see Table E3 in this article’s Online Repository at www.jacionline. org). CXCR3-expressing T cells are a common feature of inflammatory conditions in the liver, whereas CCR9 is expressed on T cells that predominantly migrate to the small intestine and enter the liver after activation because of hepatic expression of the CCR9 chemokine CCL25 (thymus-expressed chemokine). In line with this observation, it is possible that an undiagnosed disease or infection of the gut might have played a role in priming dendritic cells to imprint trimethoprim-specific T cells with a profile of homing receptors that directed the tissue injury. CXCR6, which interacts with the chemokine expressed on liver endothelium CXCL16 (Bonzo), was also detected on the majority of

The Clonal Invariant NKT Cell Repertoire in People with Type 1 Diabetes Is Characterized by a Loss of Clones Expressing High-Affinity TCRs.

Invariant NKT (iNKT) cells in healthy people express iNKT-TCRs with widely varying affinities for CD1d, suggesting different roles for high- and low-affinity iNKT clones in immune regulation. However, the functional implications of this heterogeneity have not yet been determined. Functionally aberrant iNKT responses have been previously demonstrated in different autoimmune diseases, including human type 1 diabetes, but their relationship to changes in the iNKT clonal repertoire have not been addressed. In this study, we directly compared the clonal iNKT repertoire of people with recent onset type 1 diabetes and age- and gender-matched healthy controls with regard to iNKT-TCR affinity and cytokine production. Our results demonstrate a selective loss of clones expressing high-affinity iNKT-TCRs from the iNKT repertoire of people with type 1 diabetes. Furthermore, this bias in the clonal iNKT repertoire in type 1 diabetes was associated with increased GM-CSF, IL-4, and IL-13 cytokine secretion among Ag-stimulated low-affinity iNKT clones. Thus, qualitative changes of the clonal iNKT repertoire with the potential to affect the regulatory function of this highly conserved T cell population are already established at the early stages in type 1 diabetes. These findings may inform future rationales for the development of iNKT-based therapies aiming to restore immune tolerance in type 1 diabetes.