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

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.

NKT cells derive from double-positive thymocytes that are positively selected by CD1d.

CD1d-reactive NKT cells are a separate T cell sublineage. Instructive models propose that NKT cells branch off the mainstream developmental pathway because of their T cell antigen receptor specificity, whereas stochastic models would propose that they develop from precursor cells committed to this sublineage before variable-gene rearrangement. We show here that immature double-positive (DP) thymocytes form the canonical rearranged Valpha gene of NKT cells at nearly equivalent frequencies in the presence or absence of CD1d expression. After interacting with CD1d in the thymus, these cells give rise to expanded populations of NKT cells-including both CD4+ and double-negative lymphocytes in the thymus and periphery-that express this alpha chain. These results confirm the existence of a DP intermediate for CD1d-reactive NKT cells. They also show that the early developmental stages of these T cells are not governed by a distinct mechanism, which is consistent with the TCR-instructive model of differentiation.

Presentation of self and microbial lipids by CD1 molecules.

CD1 molecules present both self lipids and microbial lipids. Recent studies have elucidated novel antigenic structures that can be presented by CD1 for T cell stimulation, as well as new pathways for lipid-antigen presentation. Additionally, the development of lipid-CD1 tetramers now permits the tracking of CD1-reactive T cells during immune responses. Despite this, the roles of CD1-reactive T cells in both host defense and immune regulation remain to be unequivocally defined.

Tracking the response of natural killer T cells to a glycolipid antigen using CD1d tetramers.

A major group of natural killer (NK) T cells express an invariant Valpha14(+) T cell receptor (TCR) specific for the lipoglycan alpha-galactosylceramide (alpha-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 alpha-GalCer are a sensitive and highly specific reagent for identifying Valpha14(+) NK T cells. Using these tetramers, we find that alpha-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 alpha-GalCer leads to the production of both interferon gamma 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 alpha-GalCer-specific NK T cells undergo a unique and highly compartmentalized response to antigenic stimulation.

Systemic activation and antigen-driven oligoclonal expansion of T cells in a mouse model of colitis.

Transfer of CD4+CD45RBhigh T cells into immunodeficient mice results in both the expansion of the transferred T cells and colitis. Here we show that colitis pathogenesis requires expression of MHC class II molecules by the immune-deficient host. Analysis of the TCRbeta repertoire of the cells found in the large intestine of diseased mice revealed a population with restricted TCR diversity. Furthermore, nucleotide sequence analysis demonstrated the selection for particular CDR3beta amino acid sequence motifs. Collectively, these data indicate that the expansion of T cells in the intestine and colitis pathogenesis are likely to require the activation of Ag-specific T cells, as opposed to nonspecific or superantigen-mediated events. There is relatively little overlap, however, when the TCR repertoires of different individuals are compared, suggesting that a number of Ags can contribute to T cell expansion and the generation of a T cell population in the intestine. Surprisingly, many of the expanded clones found in the large intestine also were found in the spleen and elsewhere, although inflammation is localized to the colon. Additionally, donor-derived T cells appear to be activated in both the intestine and the spleen at early time points after cell transfer. Together, these results strongly suggest that disease induction in this model involves either the early and systemic activation of antigen-specific T cells or the rapid dispersal of T cells activated at a particular site.