Natural killer T cells reactive to a single glycolipid exhibit a highly diverse T cell receptor beta repertoire and small clone size

Abstract

CD1d-restricted natural killer (NK) T cells reactive with the glycolipid alpha-galactosylceramide (alpha-GalCer) are a distinct lymphocyte sublineage. They express an invariant Valpha14-Jalpha18 T cell receptor (TcR), but the role of the beta chain has been controversial. Here, we have used CD1d tetramers to identify and isolate NK T cells based on their antigen specificity. In mice lacking germline Vbeta8, most of the alpha-GalCer-reactive T cells express either Vbeta2 or Vbeta7, strong Vbeta selection being revealed by the lack of an increase in other Vbeta regions. By contrast to the selection for complementarity determining region (CDR) 3beta sequences in some anti-peptide responses, alpha-GalCer-reactive T cells have polyclonal CDR3beta sequences. There is little CDR3beta sequence redundancy between organs or individual mice, and, surprisingly, there also is no evidence for organ-specific CDR3beta sequence motifs. These data argue against a T cell receptor-mediated self-reactivity for tissue-specific CD1d-bound ligands. Each NKT clone is represented by only 5-10 cells. This clone size is similar to naive conventional T cells, and much lower than that reported for memory T cells, although NK T cells have an activated/memory phenotype.

Figure 1

Normal α-GalCer responsiveness in mice lacking Vβ8. (A) Intracellular cytokine staining with α-IL-4 and α-IFN-γ mAbs of lymphocytes isolated from the liver of a C57BR/J mouse 2 h after i.v. injection of α-GalCer. (B) Comparison of α-GalCer/CD1d tetramer staining of lymphocytes from the liver of C57BR/J mice at 2 h and 18 h after i.v. injection of α-GalCer. These data are representative of three animals tested in each condition.

Figure 2

Analysis of TcR diversity of α-GalCer reactive T cells. α-GalCer/CD1d tetramer⁺ cells were sorted by flow cytometry from the liver, thymus, and spleen of C57BL/6 mice. Lymphocytes from the organs of two mice were pooled. (A) The CDR3β profiles are depicted for selected Vβ-Cβ PCR amplifications with the indicated Vβ primers. The intensity of fluorescence is represented in arbitrary units as a function of CDR3 length, in amino acids. (B) The stringency of the sort for tetramer⁺ cells was confirmed by analysis of the Vα chain usage. Vα-Cα PCR amplifications were performed with primers for Vα14 and primers for Vα2, a negative control for Vα regions typically not expressed by α-GalCer reactive T cells. Two independent sorting experiments were performed, and each gave similar results.

Figure 3

Diverse Vβ-Jβ rearrangements of α-GalCer/CD1d-reactive T cells. Fragment length analysis profiles of Vβ7-Jβ products are depicted for tetramer-binding cells from the liver. The intensity of fluorescence is represented in arbitrary units as a function of CDR3 length in amino acids. The results from this analysis are representative of the polyclonality of all Vβ-Jβ rearrangements in tetramer-reactive cells observed in the liver, spleen, and thymus for Vβ8.1, Vβ8.2, Vβ7, and Vβ2. Two independent experiments gave similar results.

Figure 4

α-GalCer/CD1d tetramer⁺ populations are detected in HY Vβ8.2 TcR transgenic mice. The FACS dot plots display the α-GalCer/CD1d tetramer vs. TcRβ profiles of cell suspensions obtained from the liver of C57BL/6 and Vβ8.2 (anti-H-Y) transgenic mice. The expression of the Vβ8.2 transgene by tetramer⁺ T cells in the liver of transgenic mice was ensured by the absence of staining with another endogenous Vβ (anti-Vβ7) Abs (data not shown). The percentage of tetramer⁺ cells from one representative experiment is shown; three mice from each strain were analyzed.

Figure 5

NK T cell repertoires of distinct animals are mostly nonoverlapping. α-GalCer/CD1d tetramer⁺ cells were sorted by flow cytometry from the liver, thymus, and spleen of two individual C57BL/6 mice. The percentages, less than 1% in every case, of recurrent sequences of the Vβ7-Jβ1.2 CDR3 region of NK T cells between the different organs of two individual mice are presented.