Benzofuran sulfonates and small self-lipid antigens activate type II NKT cells via CD1d

Natural killer T (NKT) cells detect lipids presented by CD1d. Most studies focus on type I NKT cells that express semi-invariant αβ T cell receptors (TCR) and recognize α-galactosylceramides. However, CD1d also presents structurally distinct lipids to NKT cells expressing diverse TCRs (type II NKT cells), but our knowledge of the antigens for type II NKT cells is limited. An early study identified a nonlipidic NKT cell agonist, phenyl pentamethyldihydrobenzofuransulfonate (PPBF), which is notable for its similarity to common sulfa drugs, but its mechanism of NKT cell activation remained unknown. Here, we demonstrate that a range of pentamethylbenzofuransulfonates (PBFs), including PPBF, activate polyclonal type II NKT cells from human donors. Whereas these sulfa drug–like molecules might have acted pharmacologically on cells, here we demonstrate direct contact between TCRs and PBF-treated CD1d complexes. Further, PBF-treated CD1d tetramers identified type II NKT cell populations expressing αβTCRs and γδTCRs, including those with variable and joining region gene usage (TRAV12-1–TRAJ6) that was conserved across donors. By trapping a CD1d–type II NKT TCR complex for direct mass-spectrometric analysis, we detected molecules that allow the binding of CD1d to TCRs, finding that both selected PBF family members and short-chain sphingomyelin lipids are present in these complexes. Furthermore, the combination of PPBF and short-chain sphingomyelin enhances CD1d tetramer staining of PPBF-reactive T cell lines over either molecule alone. This study demonstrates that nonlipidic small molecules, which resemble sulfa drugs implicated in systemic hypersensitivity and drug allergy reactions, are targeted by a polyclonal population of type II NKT cells in a CD1d-restricted manner.

Natural killer T (NKT) cells are defined as T cells that are restricted to the lipid antigen-presenting molecule, CD1d. The most extensively studied are type I NKT cells, which typically express an invariant T cell receptor (TCR)-α chain consisting of TRAV10–TRAJ18 in humans (TRAV11–TRAJ18 in mice) paired with a constrained repertoire of TCR-β chains, enriched for TRBV25 in humans (TRBV13, 29, 1 in mice) (reviewed in ref. 1). Type I NKT cells are defined by their strong responses to α-galactosylceramide (α-GalCer) and structurally related hexosylceramides presented by CD1d, while in contrast, type II NKT cells are defined as CD1d-restricted T cells that express diverse TCRs and do not recognize α-GalCer (reviewed in refs. 1 and 2). Very little is known about the chemical identity of antigens for type II NKT cells; however, some studies suggest that these cells are abundant in humans (3–5), and, by virtue of their greater TCR diversity, they can interact with a broader range of antigens compared to type I NKT cells (2, 6–14).In 2004, a nonlipidic molecule, phenyl pentamethyldihydrobenzofuransulfonate (PPBF), was described that stimulated a human TRAV10⁻ (type II) NKT cell line (clone ABd) in a CD1d-dependent manner (15). These observations were notable because PPBF resembles various sulfonamide drugs: furosemide (diuretic), sulfasalazine (disease-modifying antirheumatic), and celecoxib (anti-inflammatory) as well as “sulfa” antibiotics such as sulfonamide, sulfapyridine, sulfamethoxazole, sulfadiazine, and sulfadoxine (16). These drugs can cause systemic delayed-type hypersensitivity reactions, which are thought to be mediated by T cells (17–20). Most of our limited understanding of drug hypersensitivity reactions comes from work focusing on Human Leukocyte Antigen (HLA)-restricted conventional T cells, which has led to the proposal of four main mechanisms for small-drug immune activity as reviewed in ref. 21: 1) hapten/prohapten formation, whereby the drug reacts with self-Ags to generate a neo-product that undergoes processing and presentation to T cells; 2) noncovalent/labile pharmacological interaction with immune receptors on the cell surface; 3) superantigen mediating direct linkage of TCRs and Ag-presenting molecules; and 4) anchor site occupation by small molecules in Ag-presenting molecules inducing an altered self-Ag repertoire (22). Whether the CD1d-NKT cell axis is implicated in drug hypersensitivity remains unclear (23). Whereas most antigens in the CD1d system are lipids that use their alkyl chains to bind to CD1d, PPBF is a polycyclic small molecule and so would have to act through an atypical display mechanism. As previous attempts to stain the ABd clone with CD1d-PPBF tetramers were unsuccessful, the atypical drug-like structure of PPBF raised the possibility of direct pharmacological action on T cells rather than the presentation of CD1d-PPBF complexes to TCRs. However, the mechanism of PPBF-mediated type II NKT cell activation remains undefined (15).Here, using TCR-transduced cell lines, CD1d tetramers treated with PPBF, and new analogs in the pentamethylbenzofuransulfonate (PBF) family, we discovered that several molecules stimulate polyclonal NKT cells. Using CD1d tetramers treated with a newly identified and more potent analog of PPBF, we identified populations of type II NKT cells that comprise a polyclonal repertoire of both αβ and γδ T cells, including those with conserved TCR sequences. This enigmatic nature of T cell responses to PBF molecules was resolved using TCR trap technology (24, 25). Mass-spectrometric analysis of all molecules present in CD1d-PBF-TCR complexes indicates that CD1d binds PBFs and small self-lipids that promote CD1d-TCR binding. These data support a model of type II NKT cell recognition of small sulfa drug–like compounds in association with CD1d and flag a possible mechanism in which such cells may be involved in sulfa drug hypersensitivity.