Tracking the Response of Natural Killer T Cells to a Glycolipid Antigen Using Cd1d Tetramers

Abstract

A major group of natural killer (NK) T cells express an invariant Vα14+ T cell receptor (TCR) specific for the lipoglycan α-galactosylceramide (α-GalCer), which is presented by CD1d. These cells may have an important immune regulatory function, but an understanding of their biology has been hampered by the lack of suitable reagents for tracking them in vivo. Here we show that tetramers of mouse CD1d loaded with α-GalCer are a sensitive and highly specific reagent for identifying Vα14+ NK T cells. Using these tetramers, we find that α-GalCer–specific T lymphocytes are more widely distributed than was previously appreciated, with populations of largely NK1.1− but tetramer-binding T cells present in the lymph nodes and the intestine. Injection of α-GalCer leads to the production of both interferon γ and interleukin 4 by nearly all NK T cells in the liver and the majority of the spleen within 2 h. These cells mostly disappear by 5 h, and they do not reappear after 1 wk. Curiously, tetramer-positive thymocytes do not rapidly synthesize cytokines, nor do they undergo decreases in cell number after lipid antigen stimulation, although they express equivalent TCR levels. In summary, the data presented here demonstrate that α-GalCer–specific NK T cells undergo a unique and highly compartmentalized response to antigenic stimulation.

Cd1d–Glycolipid Tetramers

“…As described previously 5 6 7 and emphasized by the current detailed analysis of CD1d–α-GalCer tetramer–positive cells 10 11, CD1d-dependent NK T cells in all tissues analyzed (irrespective of NK1.1 expression) consist of a majority (60–80%) of CD4 + cells, with the remaining cells being CD4 − CD8 − . Essentially no CD8 + tetramer-positive cells can be detected.…”

“…The third (and potentially most novel) T cell subset defined by Matsuda et al 10 and Benlagha et al 11 are NK1.1 − but tetramer positive (Table ). Despite the absence of NK1.1 expression, these cells are CD1d dependent and express semiinvariant TCRs, as would be expected from their ability to bind CD1d–α-GalCer tetramers.…”

“…Despite recent reports of fluorescent tetramers incorporating MHC class Ib molecules, the successful production of CD1d–glycolipid tetramers by Matsuda et al 10 and Benlagha et al 11 was somewhat unexpected. In the first place, all previous MHC heavy chains had been refolded with well characterized short peptides that could be produced in large quantity and high purity, and in many cases the crystal structure of the relevant MHC–peptide complex was known.…”

“…This confusion has now been clearly resolved by the studies of Matsuda et al 10 and Benlagha et al 11. Both groups provide definitive evidence for the presence of three distinct T cell subsets defined by simultaneous staining with NK1.1 and CD1d–α-GalCer tetramers (Table ).…”

“…To circumvent these problems, Matsuda et al 10 in this issue (as well as Benlagha et al 11 in the June 5 issue) have succeeded in producing tetrameric CD1d–glycolipid complexes that bind selectively to NK T cells expressing the semiinvariant Vα14/Vβ8.2 or Vα24/Vβ11 TCR.…”