The CD1 family of major histocompatibility complex (MHC)-like molecules specializes in presenting lipid and glycolipid antigens to α/β T lymphocytes, but little is known about the size of the CD1-restricted T cell population or the frequency of T lymphocytes specific for a given glycolipid antigen. Here, we report the generation and use of mouse CD1d1–glycolipid tetramers to visualize CD1d-restricted T cells. In contrast with previous BIAcore-based estimates of very short half-lives for CD1d–glycolipid complexes, we found that the dissociation rate of several different CD1d–glycolipid complexes was very slow. Fluorescent tetramers of mouse CD1d1 complexed with α-galactosylceramide (αGalCer), the antigen recognized by mouse Vα14-Jα281/Vβ8 and human Vα24-JαQ/Vβ11 natural killer T (NKT) cell T cell receptors (TCRs), allowed us for the first time to accurately describe, based on TCR specificity, the entire population of NKT cells in vivo and to identify a previously unrecognized population of NK1.1-negative “NKT” cells, which expressed a different pattern of integrins. In contrast, natural killer (NK) cells failed to bind the tetramers either empty or loaded with αGalCer, suggesting the absence of a CD1d-specific, antigen-nonspecific NK receptor. Mouse CD1d1–αGalCer tetramers also stained human NKT cells, indicating that they will be useful for probing a range of mouse and human conditions such as insulin-dependent diabetes mellitus, tumor rejection, and infectious diseases where NKT cells play an important role.

Although recent studies have indicated that the major histocompatibility complex–like, β2-microglobulin–associated CD1 molecules might function to present a novel chemical class of antigens, lipids and glycolipids, to α/β T cells, little is known about the T cell subsets that interact with CD1. A subset of CD1d-autoreactive, natural killer (NK)1.1 receptor–expressing α/β T cells has recently been identified. These cells, which include both CD4−CD8− and CD4+ T cells, preferentially use an invariant Vα14-Jα281 T cell receptor (TCR) α chain paired with a Vβ8 TCR β chain in mice, or the homologous Vα24-JαQ/Vβ11 in humans. This cell subset can explosively release key cytokines such as interleukin (IL)-4 and interferon (IFN)-γ upon TCR engagement and may regulate a variety of infectious and autoimmune conditions. Here, we report the existence of a second subset of CD1d-restricted CD4+ T cells that do not express the NK1.1 receptor or the Vα14 TCR. Like the Vα14+ NK1.1+ T cells, these T cells exhibit a high frequency of autoreactivity to CD1d, use a restricted albeit distinct set of TCR gene families, and contribute to the early burst of IL-4 and IFN-γ induced by intravenous injection of anti-CD3. However, the Vα14+ NK1.1+ and Vα14− NK1.1− T cells differ markedly in their requirements for self-antigen presentation. Antigen presentation to the Vα14+ NK1.1+ cells requires endosomal targeting of CD1d through a tail-encoded tyrosine-based motif, whereas antigen presentation to the Vα14− NK1.1− cells does not. These experiments suggest the existence of two phenotypically different subsets of CD1d-restricted T cells that survey self-antigens loaded in distinct cellular compartments.

The essential hematopoietic transcription factor PU.1 is expressed in multipotent thymic precursors but downregulated during T lineage commitment. The significance of PU.1 downregulation was tested using retroviral vectors to force hematopoietic precursors to maintain PU.1 expression during differentiation in fetal thymic organ culture. PU.1 reduced thymocyte expansion and blocked development at the pro-T cell stage. PU.1-expressing cells could be rescued by switching to conditions permissive for macrophage development; thus, the inhibition depends on both lineage and developmental stage. An intact DNA binding domain was required for these effects. PU.1 expression can downregulate pre-Talpha, Rag-1, and Rag-2 in a dose-dependent manner, and higher PU.1 levels induce Mac-1 and Id-2. Thus, downregulation of PU.1 is specifically required for progression in the T cell lineage.

PU.1 is essential for early stages of mouse T cell development but antagonizes it if expressed constitutively. Two separable mechanisms are involved: attenuation and diversion. Dysregulated PU.1 expression inhibits pro-T cell survival, proliferation, and passage through β-selection by blocking essential T cell transcription factors, signaling molecules, and Rag gene expression, which expression of a rearranged T cell antigen receptor transgene cannot rescue. However, Bcl2 transgenic cells are protected from this attenuation and may even undergo β-selection, as shown by PU.1 transduction of defined subsets of Bcl2 transgenic fetal thymocytes with differentiation in OP9-DL1 and OP9 control cultures. The outcome of PU.1 expression in these cells depends on Notch/Delta signaling. PU.1 can efficiently divert thymocytes toward a myeloid-like state with multigene regulatory changes, but Notch/Delta signaling vetoes diversion. Gene expression analysis distinguishes sets of critical T lineage regulatory genes with different combinatorial responses to PU.1 and Notch/Delta signals, suggesting particular importance for inhibition of E proteins, Myb, and/or Gfi1 (growth factor independence 1) in diversion. However, Notch signaling only protects against diversion of cells that have undergone T lineage specification after Thy-1 and CD25 up-regulation. The results imply that in T cell precursors, Notch/Delta signaling normally acts to modulate and channel PU.1 transcriptional activities during the stages from T lineage specification until commitment.