Oligoclonality and innate-like features in the TCR repertoire of type II NKT cells reactive to a beta-linked self-glycolipid

Abstract

TCR-mediated recognition of beta-linked self-glycolipids bound to CD1d is poorly understood. Here, we have characterized the TCR repertoire of a CD1d-restricted type II NKT cell subset reactive to sulfatide involved in the regulation of autoimmunity and antitumor immunity. The sulfatide/CD1d-tetramer(+) cells isolated from naïve mice show an oligoclonal TCR repertoire with predominant usage of the Valpha3/Valpha1-Jalpha7/Jalpha9 and Vbeta8.1/Vbeta3.1-Jbeta2.7 gene segments. The CDR3 regions of both the alpha- and beta-chains are encoded by either germline or nongermline gene segments of limited lengths containing several conserved residues. Presence of dominant clonotypes with limited TCR gene usage for both TCR alpha- and beta-chains in type II NKT cells reflects specific antigen recognition not found in the type I NKT cells but similar to the MHC-restricted T cells. Although potential CD1d-binding tyrosine residues in the CDR2beta region are conserved between most type I and type II NKT TCRs, CDR 1alpha and 3alpha regions differ significantly between the two subsets. Collectively, the TCR repertoire of sulfatide-reactive type II NKT cells exhibits features of both antigen-specific conventional T cells and innate-like cells, and these findings provide important clues to the recognition of beta-linked glycolipids by CD1d-restricted T cells in general.

Fig. 1.

Preferential use of TCR Vβ8.1/Vβ3.1 and Vα3/Vα1 gene segments by sulfatide-reactive type II NKT cells. (A) Flow cytometric profiles of liver MNCs from Jα18^−/− mice following staining with PE-labeled sulfatide/CD1d-tetramer (Sulf/CD1d) or unloaded tetramer (PBS/CD1d) and FITC-labeled anti-TCRβ, anti-NK1.1, or anti-CD69. (B Upper Left) Bar graphs depicting the percentage of staining of sulfatide/CD1d-tetramer⁺ cells with different anti-Vβ mAbs. Percentage was calculated in relation to total tetramer⁺ cells after subtracting background with unloaded tetramer. (B Upper Right) Tricolor flow cytometric analysis of liver MNCs following staining with PE-labeled unloaded (PBS) or sulfatide-loaded tetramers, PE-Cy5-labeled anti-TCRβ, and FITC-labeled anti-TCR Vα3. Numbers within boxes indicate the percentage of positive cells in total lymphocytes. (B Lower) Gel images showing RT-PCR products of indicated Vβ (Left) or Vα (Right) chains expressed by sorted tetramer⁺ cells or unsorted liver lymphocytes. A 50- or 100-bp DNA ladder was used. Data are representative of two to four individual experiments.

Fig. 2.

Spectratyping analysis of TCR Vβ-Jβ and Vα-Cα products reveals a few dominant clonotypes of limited CDR3 lengths in sulfatide-reactive type II NKT cells. RNA was extracted from sorted tetramer⁺ cells (Sulf/CD1d+), tetramer-negative cells (Sulf/CD1d-), and unsorted liver lymphocytes and splenocytes for Vα and Vβ, respectively. (Left) Vβ-Cβ PCR products subjected to Jβ run-off extension reactions, and significant spectratyping profiles of Vβ8.1-Jβ2.7 and Vβ3.1-Jβ2.7. (Right) Vα-Cα PCR products were subjected to Cα run-off extension reactions, and significant spectratyping profiles of Vα3-Cα and Vα1-Cα are shown. CDR3 lengths are depicted below. For comparison of profiles heights are shown in relation to a reference peak (10 aa for Vβ8.1, 8 aa for Vβ3.1, 12 aa for Vα3, and 11 aa for Vα1).

Fig. 3.

Analysis of CDR region amino acid sequences. (A) Conserved CDR3 amino acid residues (purple) and their frequency (%) among the TCR Vβ8.1, Vβ3.1, Vα3, or Vα1 chains. (B) Comparison of the CDR2β, CDR3α, and CDR1α regions between the type I and type II NKT cells. The type I and type II NKT TCR amino acid sequences are depicted in top and bottom rows, respectively. Conserved residues between type I and II are shown in green, whereas those among type II TCRs are depicted in purple. The underlined residues are crucial in binding to the αGalCer/CD1d complex (10).

Fig. 4.

Increased numbers of sulfatide-reactive type II NKT cells are present in sulfoglycolipid-deficient mice. (Left and Center) Flow cytometric profiles of liver MNCs from CST^−/− or CST^+/− mice following staining with sulfatide-loaded or unloaded (PBS) tetramers and anti-TCRβ. Numbers within boxes indicate the percentage of positive cells in total lymphocytes. Data are representative of three individual experiments. (Right) Bar graphs summarizing absolute numbers of sulfatide/CD1d-tetramer⁺ cells in CST^−/− vs. CST^+/− mice (n = 3 per group). *, P < 0.05.