
==== Front
J Exp MedJ. Exp. MedThe Journal of Experimental Medicine0022-10071540-9538The Rockefeller University Press 9874567ArticlesDistinct Subsets of CD1d-restricted T Cells Recognize Self-antigens Loaded in Different Cellular Compartments  Chiu Ya-Hui *Jayawardena Jayanthi *Weiss Angela *Lee Daniel *Park Se-Ho *Dautry-Varsat Alice ‡Bendelac Albert *From the * Department of Molecular Biology, Princeton, New Jersey 08544; and the ‡ Institut Pasteur, Unite de Biologie des Interactions Cellulaires, 75724 Paris Cedex 15, FranceAddress correspondence to Albert Bendelac, Department of Molecular Biology, Princeton, NJ 08544. Phone: 609-258-5454; Fax: 609-258-2205; E-mail: abendelac@molbio.princeton.edu


4 1 1999 189 1 103 110 15 9 1998 28 10 1998 1999This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).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.

CD1self-antigenendosomeinterleukin 4interferon γ
==== Body
Classical MHC class I and class II molecules in vertebrates capture pathogen-derived peptides in the endoplasmic reticulum or in the endosomal compartment, and present them on the cell surface for recognition by CD8+ or CD4+ α/β T lymphocytes, respectively (1). In contrast, CD1 molecules, a family of MHC-like, non– MHC-encoded molecules, seem to present a novel antigenic universe, made of lipids rather than peptides, to T cells (2). Human CD1b and CD1c molecules can present various lipid and glycolipid components of mycobacterial cell walls (3–5). CD1d, a conserved isotype that is expressed by all mammals studied to date, and the only one that is expressed by mice and rats, may also bind various glycolipids, including glycosyl phosphatidyl inositols (6) and glycosylated ceramides (7).

Although little is known about the frequency and phenotype of T cells that use CD1 as a presenting molecule, or about the antigen presentation pathways associated with CD1, several observations suggest that they differ from those defined in the classical MHC system. In humans, a few CD1b- and CD1c-restricted T cell lines have been reported. Most of them have an unusual CD4/CD8 double negative phenotype (3–5), while some are CD8+ (8). Their recognition of mycobacterial lipid antigens depends on a tyrosine-based motif encoded in the cytoplasmic tail of CD1b itself that targets CD1b to the endosome, a different mechanism of endosomal trafficking than that of the MHC class II pathway (9, 10). CD1d, the only CD1 isotype in mice, interacts with a prominent subset of CD1d-restricted T cells that has been identified in vivo on the basis of its unique phenotype and functional properties. This subset comprises the NK1.1 receptor–expressing α/β T cells (NK T cells) that preferentially use an invariant Vα14-Jα281 TCR α chain paired with a Vβ8 TCR β chain in mice (11, 12) or the homologous Vα24-JαQ/Vβ11 in humans (11, 13, 14). This cell subset, which includes both double negative and CD4 T cells, accounts for 15% of mature thymocytes, 5% of spleen T cells, and 30% of liver T cells. The unusual functional properties of these NK T cells (15), which include their ability to explosively release key cytokines such as IL-4 and IFN-γ upon TCR engagement, are thought to be the basis for their role in various intracellular infections (16, 17), in tumor rejection (18), and in autoimmune diseases (19, 20). A large fraction of these cells can be shown to be autoreactive to CD1d-expressing cells (12). Thus, a recent report showing that alpha galactosyl ceramide, a component of marine sponges, can specifically stimulate most Vα14-Jα281/Vβ8 T cells in a CD1d-restricted fashion (7) suggests that these cells might survey a single, yet unidentified family of self-glycolipids with homology to alpha galactosyl ceramide. There have been reports of other CD1d-autoreactive T cell hybridomas that do not use Vα14 TCRs (11, 12, 21), but the phenotype and the functional properties of their precursor cell type have not been characterized in vivo. Therefore, we asked whether these hybridomas belonged to the NK1.1+ subset or were perhaps a window into a new T cell subset.

Here, we report the existence of a novel, prominent subset of CD1d-restricted T cells that do not express the NK1.1 receptor or the Vα14 TCR. Like Vα14+ NK1.1+ T cells, these T cells exhibit a high frequency of autoreactivity to CD1d, use a restricted 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, Vα14+ NK1.1+ and Vα14− NK1.1− T cells differ markedly in their requirements for self-antigen presentation. Vα14+ NK1.1+ cells require endosomal targeting of CD1d through a tail-encoded tyrosine-based motif for antigen recognition, whereas Vα14− NK1.1− T cells do not. Altogether, these experiments suggest that there are two phenotypically different subsets of CD1d-restricted T cells that survey antigens loaded in distinct cellular compartments. These results have significant implications for the antigen presenting functions of CD1 molecules.

Materials and Methods
Mice.
C57BL/6 and C57BL/6 IAβb−/− (MHC II−/−) mice were obtained from Taconic Farms. Vα14-Jα281 transgenic mice in a C57BL/6 or C57BL/6 Cα knockout background were described previously (22). All mice were raised in a specific pathogen–free barrier environment at Princeton University, according to institutional animal care and use guidelines.

T Cell Subset Staining and Purification.
Pooled splenocytes obtained from 10 C57BL/6.MHC II−/− mice were enriched in CD4 cells by incubation with 53.6.7, an anti-CD8 mAb followed by panning onto goat anti–rat/mouse Ig (Southern Biotechnologies)-coated plates. After staining with anti-CD4–FITC and anti-NK1.1–PE (PharMingen), NK1.1-positive and -negative CD4+ cells were FACS® sorted with a >95% purity.

In Vitro T Cell Stimulation Assay.
T cells were cultured for 18 h in the presence of CD1d-expressing cells (5 × 104 responders and 5 × 104 transfectant or 5 × 105 thymocyte or splenocyte stimulators, unless otherwise stated) in 100 μl of a 1:1 mixture of Click's medium and RPMI (Biofluids) enriched with 10% heat-inactivated FCS, glutamine, antibiotics, and 5 × 10−5 2-ME. IL-2 or IL-4 released in the supernatant was measured using the CTLL or CT4S bioassays, respectively, as described (23).

T Cell Hybridoma Generation.
Purified T cell subsets were cultured for 5 d with anti-CD3 and IL-2 and fused with BW5147αβ− as described (11). Vα14-Jα281–positive hybridomas were identified by PCR (11). CD1d-autoreactive hybridomas specifically secreted IL-2 (as well as IL-4) upon culture with CD1d-transfected RBL or C57SV cells but not with untransfected cells, or with a mixture of thymocytes and splenocytes from 129 but not 129.CD1−/− mice.

Competitive Reverse Transcription PCR Quantification of Cytokine mRNA.
Messenger RNA was purified from 105 FACS®-sorted cells using the RNeasy Mini Kit (QIAGEN), reverse transcribed, and PCR amplified using IL-4, IFN-γ, and hypoxanthine phosphoribosyl transferase (HPRT)1-specific primers in the presence of titrated amounts of competitor plasmids as described (24).

TCR Gene Sequencing.
Reverse transcription (RT)-PCR, primers, and methods were as described previously (11).

CD1d Tail–mutant Constructs and Transfectants.
Complementary DNA for CD1-TD was generated by PCR using full-length CD1d cDNA (25) as template. First, primers S3 (5′-CCCTGGGAATGCTTCGG-3′) and tR2 (5′-GGCAGGTGTAAGGAAGAGTCATCTCCTTCTCCAGATATAGTA-3′) were used to amplify the 600-bp fragment A, and primers tNotI (5′-AAAAAGCGGCCGCGCAGGTACGCACATTTGCAGTT-3′) and tF2 (5′-TACTATATCTGGAGAAGGAGATGACTCTTCCTTACACCTGCC-3′) were used to amplify the 240-bp fragment B. The sequences of the tR2 and tF2 primers are complementary to each other. Fragments A and B were then used together as templates to amplify the chimeric PCR fragment C using the S3 and tNotI primers. To generate the CD1-TD plasmid Tdel2, fragment C was digested by BstEII and NotI and then subcloned into pCD113 (25) to replace its wild-type counterpart CD1-WT. The cDNA for Y332F was generated by PCR using pCD113 as template. Primers used were t-F332 (5′-GGAGAAGGAGAAGCGCTTTTCAAGACATCCGG-3′) and CD1-R (5′-AAACTCGAGGCAGGTACGCACATTTGCAGT-3′). A single mutation introduced in the t-F332 primer is underlined. The amplifed 270-bp fragment was digested with Eco47III and XhoI and then subcloned into pCD113 to replace its wild-type counterpart. The sequence mutations were confirmed by sequencing. Plasmids (pCD113, Tdel2, and Y332F) were linearized by PvuI and XmnI before transfection. Transfection and selection of stable transfectants were as described (25).

Confocal Microscope Analysis.
Labelings were performed essentially as described (26). Cells washed twice in cold PBS were fixed in 4% paraformaldehyde and 4% sucrose in PBS for 20 min at room temperature. Subsequent steps were performed at room temperature. After quenching for 20 min in 50 mM NH4Cl in PBS, the cells were washed once in PBS and permeabilized for 5 min in 0.05% saponin in the buffer used for washing. Cells were then incubated with anti–LAMP-1 (PharMingen) in the permeabilizing buffer for 45 min. After two washes in this permeabilizing buffer, the presence of anti–LAMP-1 antibodies was revealed by incubating the cells for 45 min in permeabilizing buffer containing Rhodamine-labeled rabbit secondary antibodies (1:50; DAKO). After two washes in permeabilizing buffer, the cells were incubated for 45 min with FITC-labeled rat anti-CD1d mAb 19G11 (27). After three washes in permeabilizing buffer and one wash in PBS, the cells were mounted in 100 μg/ml 1,4 diacylbicyclo (2.2.2) octane (Dabco; Sigma Chemical Co.), 100 μg/ ml moviol (Calbiochem Corp.), 25% (vol/vol) glycerol, 100 mM Tris-HCl, pH 8.5. The samples were examined under an LSM 510 confocal microscope attached to an axiovert microscope equipped with an argon and a helium-neon laser (Carl Zeiss, Inc.). The Rhodamine and FITC emissions were recorded sequentially. Optical sections were recorded with a 63× lens and a pinhole aperture such that the thickness of the sections was ∼0.7 μm. No immunofluorescence staining was observed when second antibodies were used without the first antibody or with an irrelevant first antibody.

Results
Two Subsets of CD1d-restricted T Cells.
To determine whether there are CD1d-restricted CD4 cell subsets other than the NK T cell subset, we reexamined the residual CD4 cell population found in C57BL/6 mice bearing a targeted mutation of MHC class II (IAβb−/−, referred to as MHC II−/−). Fig. 1 shows that the residual 2 or 3% CD4+ cells in MHC II−/− mice could be divided into CD4+ NK1.1+ (one third) and CD4+NK1.1− (two thirds) cells, as reported previously (21, 28–30). To examine these cells at the single cell level, we generated separate panels of T cell hybridomas from these sorted cell subsets. The hybrids were screened for autoreactivity to CD1d-transfected cells or to cells that naturally express CD1d, such as thymocytes and splenocytes, and for expression of the canonical Vα14-Jα281 TCR α chain. Significant numbers of CD1d-autoreactive cells were found in both NK1.1+ (27%) and NK1.1− (32%)–derived subsets (Fig. 1). Most of the hybrids derived from NK1.1+ cells used the Vα14-Jα281 TCR α chain (81%), as expected, whereas most of those derived from NK1.1− cells (86%) did not. Though the NK1.1− CD1d-autoreactive subset does not use the canonical Vα14-Jα281 TCR, it is nevertheless not a highly heterogeneous set but expresses a rather restricted set of TCR genes. For example, out of 13 such Vα14-negative CD1d-autoreactive hybridomas collected in several independent fusion experiments and used in the experiments depicted below in Fig. 4, 5 used Vα8, and 3 of them, derived from 2 different mice, had the Vα8 gene rearranged to the same Jα19 segment. Studies in progress on larger panels of hybridomas confirm that CD4+NK1.1− cells have a biased use of Vα gene families (data not shown). In addition, a majority of the Vα14-negative CD1d-autoreactive hybrids (9 out of 13), like the Vα14-positive hybrids (11), used Vβ8.2 (see legend to Fig. 4) with diverse junctional regions (not shown). Thus, the results demonstrate the existence of a second novel, phenotypically distinct set of CD1d-restricted T cells that are NK1.1−, use limited sets of TCRs, are autoreactive to CD1d, and whose frequency is comparable to that of Vα14+ NK1.1+ cells. 

Evidence for an Endosomal Pathway of Antigen Presentation by CD1d to Vα14+ T Cell Hybridomas.
The use of a limited set of TCR families by both sets of CD1d-autoreactive cells suggests that they might recognize a limited number of CD1d-associated self-antigens. To examine the origin of self-molecules potentially presented by CD1d, we took advantage of the existence of a targeting motif in the cytoplasmic tail of CD1d that gives it access to the endosomal compartment (9, 31). We constructed a tail-deleted variant (CD1-TD) lacking the SAYQDIR COOH-terminal end of the cytoplasmic tail which contains the endosomal targeting motif (underlined), and generated stable transfectants expressing CD1-TD or the wild-type CD1 (CD1-WT) using two different cell lines, mouse C57SV fibroblasts (32) and rat RBL basophils (33). CD1-TD was well expressed on the plasma membrane (Fig. 2 a) in a highly glycosylated form similar to that of CD1-WT (not shown). Fig. 2 b shows that CD1-WT exhibited a prominent intracytoplasmic distribution with a diffuse vesicular pattern that colocalized extensively with LAMP-1, indicating that a significant fraction of the CD1 molecules pass through a late endosome/lysosome location. A similar distribution was found in BCL-1, a B cell line that naturally expresses CD1d (not shown). In contrast, most LAMP-1–positive vesicules were devoid of CD1d in CD1-TD–transfected cells, despite the matched expression levels of surface CD1d. These results, shown in Fig. 2 for C57SV, a mouse fibroblast cell line, are identical to those recently reported for a mouse B cell line, A20, transfected with CD1-WT or CD1-TD (31), and are similar to those established previously in the human CD1b system (9). 

To determine whether the CD1d molecules trafficking through the endosome pick up a distinct set of self-antigens, we compared the ability of CD1-WT and CD1-TD transfectants to stimulate CD1d-autoreactive T cells. To achieve a dose titration of the CD1d molecules, we used multiple rounds of FACS® sorting to select several sublines expressing different surface levels of CD1-WT or CD1-TD. Fig. 3 shows that DN32.D3, a canonical Vα14-Jα281/Vβ8 hybridoma, responded 10-fold less well to CD1-TD than to CD1-WT, whether the CD1d molecule was expressed by rat RBL basophils or by mouse C57SV fibroblasts. In contrast, 1C8.DC1, a Vα14-negative hybridoma, showed identical responses to CD1-WT and CD1-TD over a wide range of surface concentrations, indicating that the CD1d-associated antigens recognized by 1C8.DC1 were essentially unaffected by the drastic changes in intracellular trafficking associated with the tail truncation, and therefore that they are most likely loaded in the secretory pathway. Again, identical results were obtained for both C57SV and RBL transfectants, as shown in Fig. 3, left and right, respectively, and confirmed in independent transfection experiments. An additional mutant of CD1d, CD1-Phe, which contains a Tyr to Phe mutation in the cytoplasmic tail motif, reproduced the CD1-TD phenotype (not shown, and see Table I below). 

These results suggested the existence of two separate pathways of antigen presentation by CD1d, one dependent on and one independent of endosomal trafficking, and therefore the existence of two separate pools of self-antigens loaded in distinct intracellular compartments. To test whether the differences in recognition exhibited by DN32.D3 and 1C8.DC1 were characteristic of their representative subsets, we tested an extended panel of CD1d-autoreactive T cell hybridomas generated from normal or MHC II−/− splenocytes or thymocytes over the course of 3 yr in seven fusion experiments. Fig. 4 is a compilation of several experiments comparing their recognition of C57SV and RBL transfectants expressing matched levels of CD1-TD and CD1-WT. We found that none of the 13 Vα14-negative hybridomas discriminated between CD1-WT and CD1-TD, whereas 10 out of 14 (71%) Vα14-positive hybridomas clearly did, exhibiting a 3–15-fold impaired recognition of CD1-TD. These patterns of recognition of CD1-WT and CD1-TD suggest that there is a systematic difference between the two T cell subsets in their antigen presentation requirements.

Fresh NK1.1+ Vα14+ T Cells Require Endosomal Trafficking of CD1d for Efficient Recognition.
A few (4 out of 14) Vα14-positive hybridomas were able to recognize CD1-TD as well as CD1-WT, possibly because of residual access of CD1-TD to the endosomal compartment, or because they crossreacted with other antigens. To assay a larger population of Vα14-positive T cells, and to rule out potential biases associated with studies of in vitro–derived hybridomas, we studied cells from a transgenic mouse where the invariant Vα14-Jα281 TCR α chain is expressed by all T cells, in association with endogenous, polyclonal TCR β chains (22). These T cells constitute a fresh polyclonal population of Vα14-Jα281–positive CD1d-autoreactive T cells. Table I shows that Vα14-Jα281 transgenic thymocytes responded strongly to CD1-WT– and poorly to CD1-TD–transfected cells. The response of 5 × 104 transgenic cells to RBL.CD1-TD was much lower than the response of 104 cells to RBL.CD1-WT, suggesting that >80% of fresh Vα14-positive cells specifically see endosomally loaded antigens. The control Vα14-negative T cell hybridoma 1C8.DC1 used in this experiment responded equally to both. We next compared fresh NK1.1+ and NK1.1− CD4+ cells purified from the spleens of MHC II−/− mice. Although these are not pure populations of Vα14-positive and -negative cells, they are significantly biased, containing 81 vs. 14% Vα14-positive cells, respectively (see Fig. 1). Here again, the NK1.1+ (Vα14-rich) subset responded much more strongly to CD1-WT than to CD1-Phe (or CD1-TD, not shown), whereas the NK1.1− (Vα14-poor) subset responded well to both (Table I).

In a recent study, a single hybridoma out of two Vα14+ hybrids tested was found to react less well to CD1-TD than to CD1-WT (31). The reaction patterns of our 27 hybridomas as well as those of fresh Vα14 transgenic and fresh NK1.1+ and NK1.1− cells conclusively demonstrate that the ability to discriminate CD1-TD from CD1-WT is the common, characteristic pattern of the Vα14 T cell subset.

Both NK1.1+ and NK1.1− T Cells Contribute to the Early Burst of IL-4 Induced by Anti-CD3 Injection In Vivo.
A hallmark of NK T cells is their extraordinary ability to synthesize and secrete large amounts of cytokines, especially IL-4, at peak levels very quickly after anti-CD3 injection in vivo, a unique property that is likely to influence the outcome of the responses in which they are involved (22, 34). To determine whether CD4+NK1.1− cells can contribute to this early cytokine burst, we purified CD4+NK1.1− and CD4+ NK1.1+ splenocytes 1.5 h after intravenous injection of 1 μg of 2C11 anti-CD3 antibody to MHC II−/− mice, and measured their IL-4 and IFN-γ mRNA using a competitive RT-PCR procedure (24). Fig. 5 shows that both subsets contributed significantly to the early cytokine burst, although NK1.1+ T cells tended to produce two to three times more IL-4 and IFN-γ mRNA than NK1.1− T cells within this short time-frame. In conventional in vitro mitogen stimulation assays, both subsets released the same amount of both IL-4 and IFN-γ proteins after a period of 48 h (not shown). Thus, we conclude that both subsets can produce IL-4 and IFN-γ upon primary stimulation, although the kinetics of in vivo production may be faster for NK T cells. 

Discussion
We have identified the in vivo counterpart of the non– Vα14-expressing CD1d-autoreactive α/β T cell hybridomas reported previously by several laboratories, and showed that they belong to a novel subset of T cells that shares some characteristics with the Vα14+NK1.1+ T cells but differs in many others. Like Vα14-positive NK T cells, these T cells exhibit a high frequency of autoreactivity to CD1d, use restricted families of TCRs, and contribute to the early burst of IL-4 and IFN-γ induced by intravenous injection of anti-CD3. However, they do not express the NK1.1 receptor. In addition, they recognize different subsets of CD1d molecules. We showed using a large panel of hybridomas as well as polyclonal fresh populations of Vα14-positive and -negative T cells that the Vα14+ NK1.1+ T cells require endosomal targeting of CD1d through a tail-encoded tyrosine-based motif for recognition of CD1d, whereas the newly discovered Vα14− NK1.1− T cells do not. This dichotomy between NK1.1-positive and -negative T cells is independent of the expression of NK1.1, because NK T cell–derived hybridomas fail to express NK1.1 (35; and data not shown). Altogether, these experiments clearly establish that there are two phenotypically different subsets of CD1d-restricted T cells that survey antigens loaded in distinct cellular compartments.

The results imply that CD1d may load self-antigens in the two cellular compartments that are sampled separately by the classical MHC class I and class II molecules. To sample the endosome, it uses an endosomal targeting motif without which it can efficiently only sample the secretory pathway. In support of the existence of a secretory pathway of antigen loading is the recent report that a soluble, secretory form of CD1d could be loaded with endogenous cellular glycosyl phosphatidyl inositols (6). The existence of a second, endosomal pathway has also been previously suggested by the finding that presentation of alpha galactosyl ceramide, a mimic of the self-antigen recognized by Vα14 cells, is chloroquine dependent (7). As suggested (9), CD1d may differ from MHC class II in that it might reach the cell surface first and only secondarily be internalized, using its tail-encoded tyrosine-based motif to access the endosome, load new antigens, and recycle to the cell surface. Indeed, experiments in progress in our laboratory indicate that the rate of internalization of CD1-TD is significantly reduced compared with that of CD1-WT (data not shown)

Therefore, like MHC class I and class II–restricted CD8 and CD4 T cells, CD1d-restricted Vα14-negative and Vα14-positive cells may survey different pathways of antigen presentation. This dichotomy is reinforced by the distinct phenotypes of the two subsets, in particular with respect to the expression of the NK1.1 receptor. The emerging picture is that CD1d-restricted T cells constitute at least two subsets of α/β T lymphocytes, comparable in numbers to the NK cells (on the order of 1–20% of the lymphocyte compartment in various tissues), that are particularly enriched in some tissues, such as the liver, spleen, and bone marrow, and that use a limited number of TCRs to focus on a limited number of distinct self- and foreign antigens. Although the self-antigens recognized by the two subsets of CD1d-restricted T cells described here remain to be characterized, the evidence that the T cells can recognize both mouse and rat CD1-WT– and CD1-TD–transfected cells indicates that the nature and the cellular distribution of these self-antigens are conserved. An intriguing possibility, suggested by current models of CD1/antigen/ TCR interactions (7, 36), is that the CD1d-restricted T cells survey glycosylation changes in some conserved families of glycolipids, and thus act as sentinels in various conditions of stress, infection, or tumor growth where glycosylation processes may be affected. The finding that the phenotypic properties of these cells are distributed according to the antigens they recognize raises interesting developmental and functional issues.

We thank C. Carnaud and P. Matzinger for reviewing the manuscript, A. Beavis for cell sorting, and D. Hasara and L. Antonucci for managing the mouse colonies.

This work was supported by grants from American Cancer Society IM 788, a Cancer Research Institute Investigator Award (A. Bendelac), a grant from the Mallinckrodt Foundation, and a postdoctoral fellowship from the Cancer Research Institute (Y.-H. Chiu). The confocal microscope was purchased thanks to a donation from Marcel and Liliane Pollac.

Abbreviations used in this paper
HPRThypoxanthine ribosyl transferase

mfimean fluorescent intensity

RTreverse transcriptase

Figure 1 Two subsets of CD1d-autoreactive T cells. Residual CD4+ cells in the spleen of MHC II−/− mice were enriched by panning against CD8 and Ig, sorted into NK1.1-positive and -negative subsets, activated for 5 d with anti-CD3 and IL-2 in vitro, and fused with BW5147αβ− to obtain T hybridomas. Individual hybridomas were screened for CD1d-autoreactivity and for Vα14-Jα281 rearrangement.

Figure 4 Most Vα14-Jα281–positive T cell hybridomas do not efficiently recognize CD1-TD. Compilation of multiple experiments performed with 14 Vα14-Jα281–positive and 13 Vα14-Jα281–negative hybridomas stimulated with RBL (filled circles) or C57SV (open circles) transfectants. Results are expressed as the ratio of IL-2 production elicited by cells expressing CD1-TD over that elicited by cells expressing similar levels of CD1-WT. Hybridomas stimulated with untransfected C57SV or RBL cells did not secrete detectable IL-2. The hybridomas were obtained from a total of seven independent fusions of B6 CD44hiHSAlo thymocytes (431.A11, DN32.D3, DN32.F3); B6.MHC II−/− HSAloCD4+ thymocytes (TB.A7, TC.B11); B6.MHC II−/− NK1.1+CD4+ thymocytes (P4, P6, P9, P12, P17, P41, P72); B6.MHC II−/− NK1.1+ (S2P4, S2P16, S2P23, S2P26, S2P29) and NK1.1− (S2M6, S2M7, S2M9, S2M10, S2M16) CD4+ splenocytes; B6.MHC II−/− CD8− splenocytes (1C8.DC1); and B6.Vα14-Jα281 Tg Cα−/− splenocytes (Vα14.C7, Vα14.A2, Vα14.D21). Vα-Jα gene usage is indicated. All hybridomas used Vβ8.2 TCR β chains with the exception of P17 (Vβ14), 1C8.DC1 (ND), S2M7 (Vβ11), S2P16 (Vβ4), and P72 (ND).

Figure 2 Cellular localization of CD1-WT and CD1-TD. (a) Mouse C57SV fibroblasts were stably transfected with CD1-WT or CD1-TD cDNA and selected by flow cytometry after staining with anti-CD1d 19G11-PE to express matched surface levels of CD1. (b) Representative medial optical cuts of CD1-WT (left) or CD1-TD (right) expressing C57SV cells double stained with anti-CD1d (FITC, top) and anti– LAMP-1 (Rhodamine, bottom) demonstrate that most LAMP-1–positive intracytoplasmic vesicules contain CD1-WT but not CD1-TD.

Figure 3 CD1-WT and CD1-TD are differently recognized by some T cell hybridomas. DN32.D3, a Vα14-Jα281–positive hybridoma, and 1C8.DC1, a Vα14-negative hybridoma, were stimulated with CD1-WT (filled squares) or CD1-TD (open squares)–transfected RBL (left) or C57SV (right) cells, and the IL-2 release was measured using the CTLL bioassay. Stable transfectants were FACS® selected for expression of a range of surface concentrations of CD1d. Surface levels of CD1d were estimated at saturation of staining antibody on a fluorescent bead–calibrated FACScan® (Becton Dickinson) and were proportional to the mean fluorescent intensity (mfi). Thus, the top left panel shows that RBL.CD1-TD expressing 239 mfi of CD1d was less stimulatory for DN32.D3 than RBL.CD1-WT expressing six times less CD1d (mfi = 43), whereas both forms of CD1d stimulated 1C8.DC1 equally efficiently (bottom left). Similar results with C57SV transfectants are shown on the right. Hybridomas stimulated with untransfected C57SV or RBL cells did not secrete detectable IL-2 (<0.2 U/ml).

Table I Fresh CD1-WT–reactive Vα14-Jα281 T Cells Respond Poorly To CD1-TD 

		No. of cells		RBL.CD1-WT		RBL.CD1-TD	
		
×104
		
IL-4 (U/ml)
	
Vα14 Tg thymocytes		5		975		 32	
 (100% Vα14)		1		306		 22	
1C8.DC1		5		240		320	
 (Vα14-neg)		1		103		 87	
				RBL.CD1-WT		RBL.CD1-Phe	
CD4 NK1.1+
		1		370		 49	
 (81% Vα14)							
Vα14 Tg thymocytes		1		130		  5	
 (100% Vα14)							
CD4 NK1.1−
		1		695		400	
 (14% Vα14)							
1C8.DC1		1		120		       85	
 (Vα14-neg)							
Thymocytes from Vα14-Jα281 TCR α transgenic, Cα−/− B6 mice or sorted CD4+NK1.1+ or CD4+NK1.1− MHC II−/− splenocytes (same as in Fig. 1, containing an estimated frequency of 81 and 14% Vα14-positive TCRs, respectively) were cultured for 5 d with 2.25 μg/ml Con A and IL-4 (10 ng/ml), washed, and restimulated for 20 h at indicated cell numbers with 104 RBL.CD1-WT, RBL.CD1-TD, or RBL.CD1-Phe cells expressing similar levels of surface CD1d, in the presence of 20 U/ml IL-2. IL-4 release was measured using the CT4.S bioassay. 1C8.DC1 is a control Vα14-negative CD1d-autoreactive hybridoma. Untransfected RBL cells elicited <10 U/ml IL-4 in all cases. Results are representative of seven experiments with Vα14-Jα281 TCR α transgenic cells of either thymic or splenic origin, and of two experiments with sorted CD4+NK1.1+ or CD4+NK1.1− MHC II−/− splenocytes. Tg, transgenic.  

Figure 5 Both CD1d-restricted T cell subsets contribute to the early burst of IL-4 and IFN-γ induced by anti-CD3. RT-PCR quantitation of cDNA (lower bands), obtained from 105 FACS®-purified CD4+NK1.1+ and CD4+NK1.1− T cells from MHC II−/− mice injected 1.5 h earlier, was performed against threefold dilutions of competitor IL-4, IFN-γ, and HPRT plasmids (upper bands), as described in Materials and Methods. Results of two independent experiments, each with duplicate samples of 105 sorted cells, showed that both subsets contributed to the early cytokine release, although on a per cell basis the production of IL-4 and IFN-γ by CD4+NK1.1+ T cells was on average three- and twofold, respectively, above that of CD4+NK1.1− T cells.
==== Refs
References
1 Germain RN   Immunology. The ins and outs of antigen processing and presentation Nature 1986 322 687 689 3489186 
2 Porcelli SA  Brenner MB   Antigen presentation: mixing oil and water Curr Biol 1997 7 R508 R511 9259536 
3 Beckman EM  Porcelli SA  Morita CT  Behar SM  Furlong ST  Brenner MB   Recognition of a lipid antigen by CD1-restricted αβ+ T cells Nature 1994 372 691 694 7527500 
4 Sieling PA  Chatterjee D  Porcelli SA  Prigozy TI  Mazzaccaro RJ  Soriano T  Bloom BR  Brenner MB  Kronenberg M  Brennan PJ    CD1-restricted T cell recognition of microbial lipoglycan antigens Science 1995 269 227 230 7542404 
5 Beckman EM  Melian A  Behar SM  Sieling PA  Chatterjee D  Furlong ST  Matsumoto R  Rosat JP  Modlin RL  Porcelli SA   CD1c restricts responses of mycobacteria-specific T cells. Evidence for antigen presentation by a second member of the human CD1 family J Immunol 1996 157 2795 2803 8816382 
6 Joyce S  Woods AS  Yewdell JW  Bennink JR  Silva ADD  Boesteanu A  Balk SP  Cotter RJ  Brutkiewicz RR   Natural ligand of mouse CD1d1: cellular glycosylphosphatidylinositol Science 1998 279 1541 1544 9488653 
7 Kawano T  Cui J  Koezuka Y  Toura I  Kaneko Y  Motoki K  Ueno H  Nakagawa R  Sato H  Kondo E    CD1d-restricted and TCR-mediated activation of Vα14 NKT cells by glycosylceramides Science 1997 278 1626 1629 9374463 
8 Stenger S  Mazzaccaro RJ  Uyemura K  Cho S  Barnes PF  Rosat JP  Sette A  Brenner MB  Porcelli SA  Bloom BR  Modlin RL   Differential effects of cytolytic T cell subsets on intracellular infection Science 1997 276 1684 1687 9180075 
9 Sugita M  Jackman RM  van Donselaar E  Behar SM  Rogers RA  Peters PJ  Brenner MB  Porcelli SA   Cytoplasmic tail-dependent localization of CD1b antigen-presenting molecules to MIICs Science 1996 273 349 352 8662520 
10 Jackman RM  Stenger S  Lee A  Moody DB  Rogers RA  Niazi KR  Sugita M  Modlin RL  Peters PJ  Porcelli SA   The tyrosine-containing cytoplasmic tail of CD1b is essential for its efficient presentation of bacterial lipid antigens Immunity 1998 8 341 351 9529151 
11 Lantz O  Bendelac A   An invariant T cell receptor α chain is used by a unique subset of MHC class I–specific CD4+  and CD4− 8− T cells in mice and humans J Exp Med 1994 180 1097 1106 7520467 
12 Bendelac A  Lantz O  Quimby ME  Yewdell JW  Bennink JR  Brutkiewicz RR   CD1 recognition by mouse NK1+ T lymphocytes Science 1995 268 863 865 7538697 
13 Porcelli S  Yockey CE  Brenner MB  Balk SP   Analysis of T cell antigen receptor (TCR) expression by human peripheral blood CD4− 8− α/β T cells demonstrates preferential use of several Vβ genes and an invariant TCR α chain J Exp Med 1993 178 1 16 8391057 
14 Dellabona P  Padovan E  Casorati G  Brockhaus M  Lanzavecchia A   An invariant Vα24-JαQ/Vβ11 T cell receptor is expressed in all individuals by clonally expanded CD4− 8− T cells J Exp Med 1994 180 1171 1176 8064234 
15 Bendelac A  Rivera MN  Park S-H  Roark JH   Mouse CD1-specific NK1 T cells. Development, specificity, and function Annu Rev Immunol 1997 15 535 562 9143699 
16 Denkers EY  Scharton-Kersten T  Barbieri S  Caspar P  Sher A   A role for CD4+ NK1.1+  T lymphocytes as major histocompatibility complex class II–independent helper cells in the generation of CD8+ effector function against intracellular infection J Exp Med 1996 184 131 139 8691126 
17 Flesch IE  Wandersee A  Kaufmann SH   IL-4 secretion by CD4+ NK1+ T cells induces monocyte chemoattractant protein-1 in early listeriosis J Immunol 1997 159 7 10 9200431 
18 Cui J  Shin T  Kawano T  Sato H  Kondo E  Toura I  Kaneko Y  Koseki H  Kanno M  Taniguchi M   Requirement for Vα14 NKT cells in IL-12-mediated rejection of tumors Science 1997 278 1623 1626 9374462 
19 Gombert JM  Herbelin A  Tancrede-Bohin E  Dy M  Carnaud C  Bach JF   Early quantitative and functional deficiency of NK1+-like thymocytes in the NOD mouse Eur J Immunol 1996 26 2989 2998 8977295 
20 Wilson SB  Kemt SC  Patton KT  Orban T  Jackson RA  Exley M  Porcelli S  Schatz DA  Atkinson MA  Balk SP    Extreme Th1 bias of invariant Vα24JαQ T cells in type I diabetes Nature 1998 391 177 181 9428763 
21 Cardell S  Tangri S  Chan S  Kronenberg M  Benoist C  Mathis D   CD1-restricted CD4+ T cells in MHC class II–deficient mice J Exp Med 1995 182 993 1004 7561702 
22 Bendelac A  Hunziker RD  Lantz O   Increased interleukin 4 and immunoglobulin E production in transgenic mice overexpressing NK1 T cells J Exp Med 1996 184 1285 1293 8879200 
23 Bendelac A  Schwartz RH   CD4+ and CD8+ T cells acquire specific lymphokine secretion potentials during thymic maturation Nature 1991 353 68 71 1831881 
24 Reiner SL  Zheng S  Corry DB  Locksley RM   Constructing polycompetitor cDNAs for quantitative PCR J Immunol Methods 1993 165 37 46 8409467 
25 Park S-H  Roark JH  Bendelac A   Tissue-specific recognition of mouse CD1 molecules J Immunol 1998 160 3128 3134 9531267 
26 Ojcius DM  Niedergang F  Subtil A  Hellio R  Dautry-Varsat A   Immunology and the confocal microscope Res Immunol 1996 147 175 188 8817746 
27 Roark JH  Park S-H  Jayawardena J  Kavita U  Shannon M  Bendelac A   CD1.1 expression by mouse antigen presenting cells and marginal zone B cells J Immunol 1998 160 3121 3127 9531266 
28 Cosgrove D  Gray D  Dierich A  Kaufman J  Lemeur M  Benoist C  Mathis D   Mice lacking MHC class II molecules Cell 1991 66 1051 1066 1909605 
29 Grusby MJ  Johnson RS  Papaioannou VE  Glimcher LH   Depletion of CD4+ T cells in major histocompatibility complex class II-deficient mice Science 1991 253 1417 1420 1910207 
30 Bendelac A  Killeen N  Littman D  Schwartz RH   A subset of CD4+ thymocytes selected by MHC class I molecules Science 1994 263 1774 1778 7907820 
31 Brossay L  Tangri S  Bix M  Cardell S  Locksley R  Kronenberg M   Mouse CD1-autoreactive T cells have diverse patterns of reactivity to CD1+ targets J Immunol 1998 160 3681 3688 9558068 
32 Knowles BB  Koncar M  Pfizenmaier K  Solter D  Aden DP  Trinchieri G   Genetic control of the cytotoxic T cell response to SV40 tumor-associated specific antigen J Immunol 1979 122 1798 1806 87443 
33 Kulczycki A Jr  Isersky C  Metzger H   The interaction of IgE with rat basophilic leukemia cells. I. Evidence for specific binding of IgE J Exp Med 1974 139 600 616 4812630 
34 Yoshimoto T  Paul WE   CD4+  NK1.1+ T cells promptly produced IL-4 in response to in vivo challenge with anti-CD3 J Exp Med 1994 179 1285 1295 7908323 
35 Shimamura M  Ohteki T  Beutner U  MacDonald HR   Lack of directed Vα14-Jα281 rearrangements in NK1+ T cells Eur J Immunol 1997 27 1576 1579 9209513 
36 Moody DB  Reinhold BB  Guy MR  Beckman EM  Frederique DE  Furlong ST  Ye S  Reinhold VN  Sieling PA  Modlin RL    Structural requirements for glycolipid antigen recognition by CD1b-restricted T cells Science 1997 278 283 286 9323206

