The mouse CD1d-restricted repertoire is dominated by a few autoreactive T cell receptor families

Abstract

To define the phenotype and T cell receptor (TCR) repertoire of CD1d-dependent T cells, we compared the populations of T cells that persisted in major histocompatibility complex (MHC)-deficient mice, which lack mainstream T cells, with those from MHC/CD1d doubly deficient mice, which lack both mainstream and CD1d-dependent T cells. Surprisingly, up to 80% of the CD1d-dependent T cells were stained by tetramers of CD1d/alpha-galactosylceramide, which specifically identify the previously described CD1d autoreactive Valpha14-Jalpha18/Vbeta8 natural killer (NK) T cells. Furthermore, zooming in on the CD1d-dependent non-Valpha14 T cells, we found that, like Valpha14 NK T cells, they mainly expressed recurrent, CD1d autoreactive TCR families and had a natural memory phenotype. Thus, CD1d-restricted T cells differ profoundly from MHC-peptide-specific T cells by their predominant use of autoreactive and semiinvariant, rather than naive and diverse, TCRs. They more closely resemble other lineages of innate lymphocytes such as B-1 B cells, gammadelta T cells, and NK cells, which express invariant or semiinvariant autoreactive receptors. Finally, we demonstrate that the MHC-restricted TCR repertoire is essentially non-cross-reactive to CD1d. Altogether, these findings imply that lipid recognition by CD1d-restricted T cells may have largely evolved as an innate rather than an adaptive arm of the mouse immune system.

Figure 1

Residual αβ T cell subsets in MHC-versus MHC/CD1d–deficient mice. (A) NK1.1⁺ and NK1.1⁻ subsets. FACS^® dot plots represent the TCR-β¹ gated splenocytes from MHC class II– and MHC class I–deficient mice stained with antibodies against NK1.1, CD4, and/or CD8. In the bar graphs, bars represent the average ratio of the numbers of each cell type in CD1d^−/− mice versus CD1d^+/− littermates (5–14 littermate pairs per data point). MHC class I^−/− mice were either K^bD^b−/− or TAP^−/−; results with K^bD^b−/− mice (shown in the FACS^® dot plots) and TAP^−/− mice (summarized in the bar graphs) were similar. Error bars are not shown for graphic clarity. ND, not detected (frequency <0.05%). *Statistically significant (P < 0.05). (B) CD1d/αGalCer⁺ and CD1d/αGalCer⁻ subsets. The FACS^® dot plots show the CD4 and CD1d/αGalCer tetramer profiles of TCR-β¹ gated cells from MHC class II^−/−CD1d^+/− and MHC class II^−/−CD1d^−/− littermates, as indicated. The bar graphs represent the average ratio of the numbers of CD1d/αGalCer⁻ cells in CD1d^−/− mice versus CD1d^+/− littermates (four to eight littermate pairs per data point).

Figure 2

CD1d-dependent Vα3.2/Vβ8 T cells. Liver lymphocytes from a pair of MHC class II–deficient and MHC class II/CD1d doubly deficient littermates were four-color stained with CD1d/αGalCer tetramers and anti-CD4, Vα3.2, and Vβ8. Right panels are gated from CD4⁺CD1d/αGalCer⁻ cells as indicated.

Figure 3

Memory phenotype of CD1d-restricted T cells. Panels represent the FACS^® dot plots of CD4 ⁺TCR-β¹ gated spleen cells of littermates of the indicated genotypes.

Figure 4

CD1d-deficient mice do not efficiently generate killer cells against CD1d. C57BL/6.CD1d^−/− female mice were immunized with C57BL/6 male spleen cells and killing responses against LPS blasts from C57BL/6 males (H-Y + CD1), C57BL/6.CD1d^−/− males (H-Y), C57BL/6 females (CD1), and C57BL/6.CD1d^−/− females (none) were measured in vitro. Each graph represents separate experiments with three mice each.