The majority of CD1d-sulfatide-specific T cells in human blood use a semiinvariant Vδ1 TCR

Abstract

αβ T-cell lines specific for sulfatide, an abundant myelin glycosphingolipid presented by various CD1 molecules, have been previously derived from PBMCs of patients with demyelinating diseases such as multiple sclerosis (MS) but also from healthy subjects. Using an unbiased tetramer-based MACS enrichment method to enrich for rare antigen-specific cells, we confirmed the presence of CD1d-sulfatide-specific T cells in all healthy individuals examined. Surprisingly, the great majority of fresh sulfatide-specific T cells belonged to the γδ lineage. Furthermore, these cells used the Vδ1 TCR variable segment, which is uncommon in the blood but predominates in tissues such as the gut and specifically accumulates in MS lesions. Recombinant Vδ1 TCRs from different individuals were shown to bind recombinant CD1d-sulfatide complexes in a sulfatide-specific manner. These results provide the first direct demonstration of MHC-like-restricted, antigen-specific recognition by γδ TCRs. Together with previous reports, they support the notion that human Vδ1 T cells are enriched in CD1-specific T cells and suggest that the Vδ1 T-cell population that accumulates in MS lesions might be enriched in CD1-sulfatide-specific cells.

Fig. 1. CD1d-sulfatide tetramer staining and MACS enrichment of healthy PBMC

(A) Loading of human CD1d molecules with purified bovine sulfatide demonstrated by native IEF. Data are representative of two independent experiments. (B) CD3/tetramer staining before and after tetramer MACS enrichment in PBMCs from 3 healthy subjects. Starting population included ~1×10⁸ PBMCs, with ~1×10⁵ cells typically recovered after MACS enrichment. Gated CD3⁺ tetramer⁺ cells were further characterized for TCRαβ, TCRγδ and Vδ1 expression. Numbers indicate percentages in the gated population. Data are representative of 1 to 5 experiments per individual. (C) Frequency of CD1d-sulfatide tetramer⁺ CD3⁺ PBMCs in 8 healthy individuals; nd, not determined; for individual #4, presence of Vδ1 T cells was inferred from the amplification of a Vδ1-Jδ1sequence by RT-PCR after CD1d-sulfatide tetramer MACS enrichment followed by FACS sorting of 200 tetramer⁺ cells. (D) Frequency of CD1d-sulfatide tetramer⁺ cells among gated CD3⁺ Vδ1⁺ cells PBMCs. Values indicate average percentage ± SEM of 6 individuals examined.

Fig. 2. TCR γ and δ sequences of CD1d-sulfatide-specific Vδ1⁺ T cells

Nucleotide and amino acid sequences of TCR γ and δ CDR3 regions in 4 different individuals. The top 2 sequences were obtained from clones derived from individuals #1 and #2, DP10.7 and AB18.1 respectively, and the bottom 2 sequences were obtained from freshly sorted CD1d-sulfatide tetramer⁺ cells of individuals #3 and #4.

Fig. 3. Antigen specificity of CD1d-sulfatide tetramer⁺ V

δ1⁺ T cell clones.<br>(A) Clone DP10.7 stained with tetramers loaded with bovine sulfatide (B sulfatide), synthetic sulfatide acC26, αGalCer or unloaded as indicated (top left). Tetramer staining was inhibited by anti-TCR γδ (top right). Decay of tetramer staining (MFI) at indicated time and temperature is shown in the bottom plots. Data are representative of two independent experiments. (B) Similar analysis as in (A) for clone AB18.1. Data are representative of two independent experiments. (C) Native gel electrophoresis of the DP10.7 TCR, AB18.1 TCR, and CD1d demonstrates a specific interaction between the TCRs and sulfatide-loaded CD1d. Equimolar amounts of the TCR (produced in insect cells) and CD1d proteins were mixed and incubated at room temperature for 15 min, before running on a native gel along with control proteins as indicated. The binding of sulfatide-loaded, but not unloaded, CD1d by DP10.7 was observed with different preparations of TCR produced in bacteria or in insect cells.