Recognition of microbial and mammalian phospholipid antigens by NKT cells with diverse TCRs

Abstract

CD1d-restricted natural killer T (NKT) cells include two major subgroups. The most widely studied are Vα14Jα18(+) invariant NKT (iNKT) cells that recognize the prototypical α-galactosylceramide antigen, whereas the other major group uses diverse T-cell receptor (TCR) α-and β-chains, does not recognize α-galactosylceramide, and is referred to as diverse NKT (dNKT) cells. dNKT cells play important roles during infection and autoimmunity, but the antigens they recognize remain poorly understood. Here, we identified phosphatidylglycerol (PG), diphosphatidylglycerol (DPG, or cardiolipin), and phosphatidylinositol from Mycobacterium tuberculosis or Corynebacterium glutamicum as microbial antigens that stimulated various dNKT, but not iNKT, hybridomas. dNKT hybridomas showed distinct reactivities for diverse antigens. Stimulation of dNKT hybridomas by microbial PG was independent of Toll-like receptor-mediated signaling by antigen-presenting cells and required lipid uptake and/or processing. Furthermore, microbial PG bound to CD1d molecules and plate-bound PG/CD1d complexes stimulated dNKT hybridomas, indicating direct recognition by the dNKT cell TCR. Interestingly, despite structural differences in acyl chain composition between microbial and mammalian PG and DPG, lipids from both sources stimulated dNKT hybridomas, suggesting that presentation of microbial lipids and enhanced availability of stimulatory self-lipids may both contribute to dNKT cell activation during infection.

Fig. 1.

Mtb infected APCs and Mtb lipids stimulate dNKT hybridomas. (A) CD1d-transfected or untransfected RAW cells were infected with virulent Mtb H37Rv and cocultured with dNKT hybridomas. (B) Mtb polar or apolar lipids stimulate dNKT hybridomas in the presence of CD1d-transfected or untransfected RAW cells. IL-2 in culture supernatants was measured by ELISA. Data show mean ± SEM and are representative of two independent experiments.

Fig. 2.

Isolation of stimulatory Mtb lipids. (A) Using DEAE cellulose columns, Mtb polar lipids were separated into 10 semipreps (SP) that were tested for stimulation of 14S.10 cells. (B) Lipids from fraction 4.3 were further separated by preparative 1D and 2D TLC, resulting in the stimulatory 4.3.2 Mtb lipid. (C and D) MS analysis revealed lipid 4.3.2 (C) to be PG with various molecular species (D). Data are representative of two independent experiments.

Fig. 3.

Cg lipids stimulate dNKT hybridomas. (A) Polar or apolar Cg lipids were tested for reactivity of dNKT hybridomas as in Fig. 1B. (B) Cg polar lipids were separated by 2D TLC and analyzed for reactivity with 14S.10 cells. (C–E) MS analysis revealed (C) the stimulatory lipid p6 to be PG (C) and the active lipid p5 (D) to be DPG (or cardiolipin; CL) (E). (F) Stimulation of 14S.10 cells in the presence of RAW.CD1d or RAW.ut cells and purified Mtb or Cg PG. Data are representative of two independent experiments.

Fig. 4.

Range of reactivities to microbial lipids among dNKT hybridomas. dNKT hybridomas were stimulated with lipids isolated from Cg polar lipids (as in Fig. 3B) in the presence of RAW.CD1d cells. (A) 14S.6. (B) TBA7. (C) VII68. (D) 431.A11. Cg lipid p6 is PG and p5 is DPG (Fig. 3). Table 1 shows TCR-α and -β gene utilization and summarizes reactivities. Data are representative of two independent experiments.

Fig. 5.

Microbial lipids activate dNKT cells independent of APC TLR signaling and require uptake and/or processing. (A) 14S.10 dNKT hybridoma cells (Left) and 24.9 iNKT hybridoma cells (Center) or an iNKT cell line (Right) were stimulated with Mtb polar lipids, α-GalCer, or LPS, respectively, in the presence of WT or MyD88^−/− BMDCs. (B) RAW.CD1d cells were either pulse→fix or fix→pulse treated with lipids and cultured with 14S.10 dNKT or 24.9 iNKT hybridoma cells. (C) α-GalCer, Cg PG (contains C18:1/16:0), and synthetic C16:0/18:1 PG (representing mammalian PG) compete with biotin–PE binding to CD1d. (D) Stimulation of 14S.10 cells with Cg PG presented by plate-bound CD1d in the absence of APCs. Data are representative of two independent experiments.

Fig. 6.

Microbial and mammalian phospholipids stimulate dNKT cells. (A) Microbial Cg PG (containing C18:1/16:0) or mammalian PG (isolated from murine skin, containing C16:0/18:1) was added to RAW.CD1d or RAW.ut cocultures with 14S.10 hybridoma cells. (B) Synthetic versions representing the dominant microbial PG (C18:1/16:0) and mammalian PG (C16:0/18:1) species stimulated 14S.10 cells. (C) Cg and bovine DPG stimulated 14S.10 cells. Data are representative of two independent experiments.

Fig. 7.

Antigen specificities of Vα14⁺ iNKT, Vα10⁺ NKT, and dNKT cells. GSL-1 is found in Sphingomonas spp., Bb DAG in Borrelia burgdorferi, and Ms α-GlcA DAG in Mycobacterium smegmatis (3).