Search

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.

CD1c presents lipid-based antigens to CD1c-restricted T cells which are thought to be a major component of the human T cell pool. The study of CD1c-restricted T cells, however, is hampered by the presence of an abundantly expressed CD1c-binding partner on blood cells distinct to the T cell receptor (TCR), confounding analysis of TCR-mediated CD1c tetramer staining. Here, we identify the CD36 family (CD36, CD36-L1 and CD36-L2) as novel ligands for CD1c, CD1b and CD1d proteins, and show that CD36 is the receptor responsible for non-TCR-mediated CD1c tetramer staining of blood cells. Moreover, CD36-blockade enables tetramer-based identification of CD1c-restricted T cells and clarifies identification of CD1b- and CD1d-restricted T cells. We use this technique to characterise CD1c-restricted T cells ex vivo and show diverse phenotypic features, TCR repertoire and antigen-specific subsets. Accordingly, this work will enable further studies into the biology of CD1 and human CD1-restricted T cells.One Sentence SummaryCD1 molecules bind CD36 family members and blockade of this interaction facilitates the study of CD1-restricted T cells.

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 recognise 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 an 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 pentamethylbenzofuransulfonate (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 cells expressing αβ 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 binding of CD1d to TCRs, finding that both PBF 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 non-lipidic small molecules, that resemble sulfa-drugs implicated in systemic hypersensitivity and drug allergy reactions, activate a polyclonal population of type II NKT cells in a CD1d-restricted manner.Significance StatementWhereas T cells are known to recognize peptide, vitamin B metabolite or lipid antigens, we identify several non-lipidic small molecules, pentamethylbenzofuransulfonates (PBFs), that activate a population of CD1d-restricted NKT cells. This represents a breakthrough in the field of NKT cell biology. This study also reveals a previously unknown population of PBF-reactive NKT cells in healthy individuals with stereotyped receptors that paves the way for future studies of the role of these cells in immunity, including sulfa-drug hypersensitivity.

CD1c presents lipid-based antigens to CD1c-restricted T cells, which are thought to be a major component of the human T cell pool. However, the study of CD1c-restricted T cells is hampered by the presence of an abundantly expressed, non-T cell receptor (TCR) ligand for CD1c on blood cells, confounding analysis of TCR-mediated CD1c tetramer staining. Here, we identified the CD36 family (CD36, SR-B1, and LIMP-2) as ligands for CD1c, CD1b, and CD1d proteins and showed that CD36 is the receptor responsible for non-TCR-mediated CD1c tetramer staining of blood cells. Moreover, CD36 blockade clarified tetramer-based identification of CD1c-restricted T cells and improved identification of CD1b- and CD1d-restricted T cells. We used this technique to characterize CD1c-restricted T cells ex vivo and showed diverse phenotypic features, TCR repertoire, and antigen-specific subsets. Accordingly, this work will enable further studies into the biology of CD1 and human CD1-restricted T cells.

Summary: Vδ2+ T cells play a critical role in immunity to micro-organisms and cancer but exhibit substantial heterogeneity in humans. Here, we demonstrate that CD26 and CD94 define transcriptionally, phenotypically, and functionally distinct Vδ2+ T cell subsets. Despite distinct antigen specificities, CD26hiCD94lo Vδ2+ cells exhibit substantial similarities to CD26hi mucosal-associated invariant T (MAIT) cells, although CD26− Vδ2+ cells exhibit cytotoxic, effector-like profiles. At birth, the Vδ2+Vγ9+ population is dominated by CD26hiCD94lo cells; during adolescence and adulthood, Vδ2+ cells acquire CD94/NKG2A expression and the relative frequency of the CD26hiCD94lo subset declines. Critically, exposure of the CD26hiCD94lo subset to phosphoantigen in the context of interleukin-23 (IL-23) and CD26 engagement drives the acquisition of a cytotoxic program and concurrent loss of the MAIT cell-like phenotype. The ability to modulate the cytotoxic potential of CD26hiCD94lo Vδ2+ cells, combined with their adenosine-binding capacity, may make them ideal targets for immunotherapeutic expansion and adoptive transfer.

Vδ2

Mucosal associated invariant T (MAIT) cells have properties of the innate and acquired immune systems. While the response to vigorous exercise has been established for most leukocytes, MAIT cells have not been investigated. Therefore, the purpose was to determine if MAIT cell lymphocytosis occurs with acute maximal aerobic exercise and if this response is influenced by exercise duration, cardiovascular fitness, or body composition. Twenty healthy young males with moderate fitness levels performed an extended graded exercise test until volitional fatigue. Peripheral blood mononuclear cells were isolated from venous blood obtained prior and immediately after exercise and were labeled to identify specific T cell populations using flow cytometry. The percentage of MAIT cells relative to total T cells significantly increased from 3.0 to 3.8% and absolute MAIT cell counts increased by 2.2-fold following maximal exercise. MAIT cell subpopulation proportions were unchanged with exercise. Within cytotoxic T lymphocytes (CTL), MAIT cells consisted of 8% of these cells and this remained constant after exercise. MAIT cell counts and changes with exercise were not affected by body composition, VO2peak, or exercise duration. Maximal exercise doubled MAIT cell numbers and showed preferential mobilization within total T cells but the response was not influenced by fitness levels, exercise duration, or body composition. These results suggest that acute exercise could be used to offset MAIT cell deficiencies observed with certain pathologies. MAIT cells also make up a substantial proportion of CTLs, which may have implications for cytotoxicity assays using these cells.