Tissue-Specific Recognition of Mouse CD1 Molecules Se-Ho Park, Jessica H. Roark and Albert Bendelac This information is current as J Immunol 1998; 160:3128-3134; ; of September 25, 2021. http://www.jimmunol.org/content/160/7/3128

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Tissue-Specific Recognition of Mouse CD1 Molecules1

Se-Ho Park, Jessica H. Roark, and Albert Bendelac2

Although there is evidence that some members of the CD1 gene family may present particular types of foreign Ags, such as mycobacterial lipid Ags or synthetic hydrophobic peptides, to ␣␤ T cells, most CD1 isotypes share the unusual property of being recognized by a high frequency of naturally autoreactive ␣␤ T cells. In the case of mouse CD1.1 and its human counterpart CD1d, a significant fraction of the autoreactive T cells express semi-invariant TCRs. CD1.1-specific T cells have a restricted tissue distribution and very promptly secrete a large panel of potent cytokines, including IL-4 and IFN-␥, upon primary activation through their TCR, suggesting that they might regulate some immune responses in these tissues. We show here that their au- torecognition of mouse CD1.1 is highly dependent upon the cell type in which CD1.1 is expressed. For example, some of these T cells only respond to CD1.1 expressed by splenic dendritic cells, some respond preferentially to cortical , and others respond to splenic B cells. Tissue specificity of CD1.1 recognition is also observed with various cell lines transfected with CD1.1 cDNA. These results show that different CD1.1 self Ags are expressed in different tissues and can be specifically recognized by Downloaded from autoreactive T cells. They suggest that CD1.1 may be naturally associated with a variety of self ligands that overlap only partially in different cell types. The Journal of Immunology, 1998, 160: 3128–3134.

␤ he CD1 locus encodes 2m-associated proteins that are empty surface molecule (10, 12), it should be capable, in theory, of distant homologues of MHC molecules. Up to five dis- accommodating ligands such as small peptides or lipids. T tinct genes have been identified in humans, and interspe- Although recent evidence points to the potential role of CD1 in http://www.jimmunol.org/ cies sequence comparison, especially of the ␣1 and ␣2 domains, presenting foreign Ags to T cells, many CD1-restricted T cells has revealed the existence of two conserved subfamilies (1, 2). The appear to be autoreactive (5, 13, 14). Whether this unusual finding CD1d family is represented in humans, mice, rats, and rabbits, and reflects biases in culture conditions or some inherent property of the CD1a,b,c,e family is represented in humans and sheep, but not the CD1 system is unclear and the answer may vary according to in mice or rats. The observation that CD1 and MHC map to two particular CD1 isotypes. However, recent studies in the mouse paralogous regions of the genome suggests that they arose as a have uncovered an entire subset of CD1-autoreactive T cells (15), result of a chromosomal duplication that included the entire an- a large fraction of which express TCRs made of an invariant cestor locus (3, 4). In contrast to classical MHC molecules, how- V␣14-J␣281 ␣-chain and polyclonal ␤-chains belonging mainly to by guest on September 25, 2021 ever, CD1 proteins are nonpolymorphic and are conserved across the V␤8 family (16–18). A similar subset using V␣24-J␣Q and species. Therefore, they are not targets of either allogeneic re- V␤11 exists in humans (16, 19–22). Such cells do not seem to sponses or transplantation rejection, nor do CD1 molecules appear require exposure to pathogens or foreign Ags to expand, as they to act as restricting elements for responses to conventional Ags (5). are found in normal numbers in germfree animals and can be gen- There is evidence, however, that human CD1b can sample the erated in fetal thymic organ culture or in fetal liver cell suspension endosomal compartment using a targeting sequence encoded in its culture (14, 15, 23). Both the mouse and human subsets coexpress cytoplasmic tail (6, 7) and present unconventional Ags, such as receptors of the NK lineage and have a CD4-positive or CD4/CD8 mycobacterial cell wall mycolic acid and lipoarabinomannans (5, double-negative (CD8␣␣ in humans) phenotype (14). They are 8, 9), to ␣␤ T cells. Screening of a random peptide phage display absent in mice with targeted inactivation of CD1 (24–26) (S.-H. library indicated that mouse CD1.1, the homologue of human Park, J. H. Roark, and A. Bendelac, manuscript in preparation). CD1d, could bind synthetic peptides of 14 to 24 amino acids with Other CD1-autoreactive T cells that do not have restriction of TCR a defined hydrophobic motif and present them to ␣␤ T cells (10). ␣-or␤-chain usage and may not coexpress NK receptors have also The crystal structure of CD1.1 revealed an MHC-like fold with a been found among the residual CD4 T cells of MHC class II KO3 large hydrophobic Ag binding groove, which appeared to be empty mice (27). The frequency of CD1 autoreactivity in this population in the protein sample produced in insect cells (11). Structure anal- was also high, reaching 8%, compared with the 1% MHC class II ysis suggested that although CD1.1 may be stably expressed as an autoreactive cells in mainstream CD4 T cells. Overall, the size of the CD1.1-autoreactive population is quite significant, rang- ing from 0.5 to 2% of the lymphocyte population in the spleen to 20 to 40% in the liver or bone marrow (14). It is likely therefore Department of Molecular Biology, Princeton University, Princeton, NJ 08544 that CD1 recognition by naturally autoreactive cells, which are Received for publication September 17, 1997. Accepted for publication November numerous and secrete large amounts of influential cytokines, is 26, 1997. bound to regulate the outcome of some important immune The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance responses. with 18 U.S.C. Section 1734 solely to indicate this fact. In this report we set out to investigate the natural CD1 ligands 1 This work was supported by a grant from the National Institutes of Health (RO1- recognized by CD1-autoreactive mouse T cells. Combined results AI38339), a grant from the Mallinckrodt Foundation, a Juvenile Diabetes Foundation fellowship (to J.H.R.), and a Cancer Research Institute Investigator Award (to A.B.). 2 Address correspondence and reprint requests to Dr. Albert Bendelac, Department of 3 Abbreviations used in this paper: KO, knockout; NOD, nonobese diabetic; HSA, Molecular Biology, Princeton University, Washington Rd., Princeton, NJ 08544. E- heat-stable Ag; DC, dendritic cells; PE, phycoerythrin; high, high level; low, low mail address: [email protected] level.

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 The Journal of Immunology 3129

from two previous studies had suggested that CD1 autorecognition mAbs 19G11 and 15F7 used in this study are rat IgG2bs that were purified by different T cells depended upon the type and the genetic back- over protein G-Sepharose and conjugated with biotin or were used pure in ␮ ground of the CD1-expressing cell (15, 27). However, it was not cell culture assays at a final concentration of 2 g/ml. clear whether differences in T cell recognition were related to the CD1.1 transfected lines expression of modified versions of the same protein, such as, for The human C1R B cell (34), the rat RBL basophil leukemia (35), and the example, CD1/glycolipid complexes, or to a range of other possi- B6 mouse C57SV fibroblast (36) lines were transfected with 10 to 30 ␮g ble factors. These included the expression of different membrane of linearized CD1.1 vector by the electroporation method using a Gene levels of CD1 by various cell types, as Abs were not available to Pulser (Bio-Rad, Hercules, CA), and stable transfectants were selected by monitor surface expression; CD1 gene polymorphism, as CD1 se- G418 treatment and cell sorting. By flow cytometry, transfected cells were quences were not available in the relevant mouse strains; and dif- found to express approximately 10 times more CD1.1 per cell than thymocytes. ferential expression and recognition of CD1 isotypes, as the mouse CD1 locus encodes two genes, CD1.1 and CD1.2, with different T cell hybridoma generation patterns of mRNA expression (28). Although CD1.1 and CD1.2 A new panel of T cell hybridomas was generated by fusion of cells stim- are 95% identical, their products can nevertheless be distinguished ulated for 4 days by anti-CD3 with BW5147␣␤Ϫ, as previously described by T cells (S.-H. Park and A. Bendelac, manuscript in preparation). (16). To enrich for CD1 specificity, the cell populations were obtained ␤b To address these confounding factors, we first generated CD1- from C57BL/6.I-A -deficient mice and depleted of HSA-positive and CD8-positive thymocytes with Ab and complement, and B220-positive and specific mAbs (52) to define the levels of CD1 surface expression CD8-positive splenocytes by panning on Ab-coated plates, as previously by different cell types using flow cytometry. We also took advan-

described (37). Hypoxanthine-aminopterin-thymidine-resistant hybrid- Downloaded from tage of the existence of a natural CD1.2 mutant mouse strain, omas were screened for IL-2 production upon stimulation with a 1/1 mix- C57BL/6, in which we identified a mutation that predicts the ab- ture of B6 spleen and thymus cells, and positive hybridomas were sub- sence of surface expression, thus considerably reducing the com- cloned. CD1 specificity was determined by blocking IL-2 production with an anti-CD1 mAb. Hybridomas expressing the canonical invariant V␣14- plexity of the system. Second, we derived a panel of CD1-autore- J␣281 TCR ␣-chain were identified by PCR with primers for V␣14 and active T hybridomas from splenic or thymic cells to study their J␣281 as previously described (16). CD1.1 recognition on various freshly purified CD1.1-presenting

Cell preparations and FACS analysis/sorting http://www.jimmunol.org/ cell types as well as on a panel of CD1.1-transfected tumor lines. To our surprise, we found that a significant fraction of CD1-auto- Cells were stained for three-color FACS analysis with directly conjugated reactive hybridomas, whether using a semi-invariant V␣14-J␣281/ Abs obtained from PharMingen (San Diego, CA; RM4–5 anti-CD4, 53.6.7 ␤ anti-CD8, RA3–6B2 anti-B220, M1/69 anti-HSA, and 2.4G2 anti-Fc re- V 8 TCR or other diverse TCRs, displayed individual and unique ceptor) or made in the laboratory (33D1 anti-dendritic cell (DC)-specific patterns of CD1.1 reactivity. These results demonstrate that dif- surface marker). CD1.1 was surface stained with 19G11-biotin followed by ferent CD1 ligands are recognized by individual T cell clones and streptavidin-PE (Caltag, San Francisco, CA). FACS analyses were per- may suggest that CD1.1 is naturally associated with a set of self formed on a FACScan (Becton Dickinson, Mountain View, CA). FACS ligands that varies considerably from one tissue to the other. sorting was performed using a Vantage (Becton Dickinson, Lincoln Park, NJ) equipped with dual (argon and dye) lasers. by guest on September 25, 2021 Materials and Methods Cell subset enrichment Mice For DC enrichment, spleen cell suspensions (20 ϫ 106 cells/ml in culture C57BL/6.␤2MϪ/Ϫ (29) (backcrossed 10 times to B6), C57BL/6.MHC medium, as described below) were allowed to adhere on plastic culture Ϫ/Ϫ bϪ/Ϫ II (30, 31) (I-A␤ ; backcrossed eight times to B6), and C57BL/ dishes (Falcon 3025, Becton Dickinson Labware) for2hat37°C in a 5% Ϫ/Ϫ 6.TAP1 (32) (backcrossed four times to B6), were maintained under CO2 incubator. Nonadherent cells were then removed with several washes specific pathogen-free conditions in our barrier facility. Other mouse and vigorous pipetting, and adherent cells were reincubated overnight. strains were purchased from The Jackson Laboratory (Bar Harbor, ME), Cells that detached during the second culture period were recovered after except NOD, which was obtained from Taconic Farms (Germantown, NY). centrifugation over a 50% Percoll gradient and contained Ͼ95% DC iden- CD1-congenic mice, referred to as B10.NOD-CD1 and NOD.B6-CD1 tified by morphology, expression of 33D1, and lack of staining with 2.4G2 (38). Splenic B cells, T cells, and CD4ϩCD8ϩ, CD4ϩCD8Ϫ, here, were gifts from Drs. Linda Wicker and Larry Peterson (Merck Re- ϩ Ϫ search Laboratories, Rahway, NJ) and correspond to B10.NOD-H2g7Idd10 and CD8 CD4 subsets were obtained by cell sorting after staining spleen and NOD.B6-Idd3Idd10, respectively (as CD1 maps to the Idd10 cells with anti-B220-FITC and CD5-PE or staining thymocytes with anti- high low locus) (33). CD4-FITC and CD8-PE. HSA and HSA single-positive thymocytes were sorted after staining thymocyte suspensions with anti-CD4-PE, CD8- CD1.1 and CD1.2 cloning and sequencing APC, and HSA-FITC. B cell-depleted spleen cells were obtained after panning (1 spleen equivalent in 10 ml of PBS-0.1% BSA) for 30 min at 4°C CD1.1 and CD1.2 cDNAs were PCR amplified using the following set of over plates (Falcon 1013) coated with goat anti-␮ Ab (Southern Biotech- Ј specific primers: CD1.1–5 , AAGCGCAGAAGTCGGAGCCG; CD1.1– nology Associates, Birmingham, AL) and were Ͻ5% B220 positive. 3Ј, GCAGGTACGCACATTTGCAGTTGTG; CD1.2–5Ј, AGCAGAGTA AAGTGCAGAAGTCGG; and CD1.2–3Ј, CCTCACTCCGGCACATA Ag stimulation of T cell hybridomas CATG. Amplified products were subcloned into the PCR3 expression vector (Invitrogen, San Diego, CA), and sequences from multiple sub- Unirradiated whole thymocytes or splenocytes, purified cellular fractions clones were determined using the Sequenase 2.0 (Amersham, Arlington (5 ϫ 105 cells, except DC used at 3 ϫ 105 cells, per well), or CD1.1- Heights, IL). Sequencing was also performed on total PCR products using transfected cell lines (5 ϫ 104 cells) were incubated in flat-bottom micro- the dsDNA cycling sequencing system (Life Technologies, Gaithersburg, wells in the presence of 5 ϫ 104 hybridoma T cells in a final volume of 0.2 MD) and on genomic DNA. ml of a 1/1 mixture of Click’s medium and RPMI (Biofluids, Rockville, MD) enriched with 10% heat-inactivated FCS, glutamine, antibiotics, and Anti-CD1 mAbs 5 ϫ 10Ϫ5 M 2-ME. The IL-2 content of supernatants collected after an incubation period of 20 h was measured using the CTLL bioassay as pre- A large panel of anti-CD1 mAbs was generated and is described in detail viously described (39). elsewhere (52). Briefly, the mAb-secreting hybridomas were generated by fusion with the Sp2/0 myeloma of spleen cells from rats repeatedly im- munized with various CD1.1-transfected cell lines or with mouse thymo- Results cytes. CD1 specificity was established by the appropriate pattern of stain- CD1.1 expression by C57BL/6 thymocytes and splenocytes ing of transfected cell lines, by the lack of staining of cells from CD1 KO mice, and by immunoprecipitation. All mAbs, except 15F7, cross-reacted In earlier studies, we found that three hybridomas derived from with CD1.2, as judged by staining of RBL cells transfected with a B6 normal thymic NK1 T cells reacted to B6 thymocytes but only CD1.2 cDNA in which the frameshift mutation had been corrected. The moderately to splenocytes (15, 39). Independently, Cardell et al. 3130 TISSUE-SPECIFIC RECOGNITION OF CD1

FIGURE 1. Map of the CD1 locus with the position of the frameshift mutation in CD1.2. In the B6 strain, a single nucleotide (G) deletion at position 690 from the translation start site causes a frameshift near the 5Ј end of the ␣3 exon of CD1.2. The sequences of B6 and 129 are aligned for comparison. E, EcoRI site. derived seven CD1-specific hybridomas from the residual splenic CD4 cells of MHC II KO mice, and these reacted well to spleen cells, although no analysis of their reactivity to thymocytes was presented (27). Interestingly, two out of seven hybridomas did not ␮ FIGURE 2. CD1.1 expression by B6 thymocytes and splenocytes. Ar- Downloaded from recognize spleen cells from MT KO (B cell-less) mice, and five rows point to the average level of expression of CD1.1 by indicated cell of seven failed to respond to spleen cells from at least one genet- subsets (identified by multiparameter analysis). The level of CD1 expres- ically different strain of mouse. Although these observations could sion by splenic DC was determined on purified DC recovered after over- be explained by differential expression of CD1.1 vs CD1.2 in dif- night culture (see Materials and Methods). ferent cell types and/or by some degree of polymorphism, a more intriguing possibility raised by Cardell et al. was that such auto- reactive T cells recognized modified CD1 molecules, for example thymocytes (producing 7–36 times more IL-2 than with spleno- http://www.jimmunol.org/ associated with as yet undefined, tissue-specific coligands. cytes), one (1C8.DC1) responded mainly to splenocytes (produc- We set out to distinguish between these possibilities and deter- ing 16 times more IL-2 than with splenocytes), and seven re- mine whether CD1-autoreactive ␣␤ TCRs may indeed recognize sponded to both thymocytes and splenocytes (IL-2 values within a modified CD1 molecules. First, we sequenced CD1.1 and CD1.2 in threefold range). Thus, the results obtained with the extended T some relevant mouse strains and discovered that the B6 strain har- hybridoma panel demonstrated that spleen cells could be as good bors a frameshift mutation at the beginning of the fourth exon or even better CD1.1-presenting cells than thymocytes for some encoding the ␣3 domain of CD1.2, which is predicted to abolish CD1.1-autoreactive T cells and confirmed that for other CD1.1- surface expression (Fig. 1). autoreactive T cells, thymocytes were the best stimulators, imply- by guest on September 25, 2021 Next, we produced a panel of anti-CD1 mAbs. By flow cytom- ing the existence of qualitative differences in CD1.1 presentation etry, we then quantitatively analyzed the level of CD1.1 expressed by different cell types. We confirmed that CD1.2 recognition is not by B6 thymic and splenic cells. Surprisingly, we found that a ma- involved in the B6 system by showing that autoreactive T cells jority of spleen cells expressed a level of CD1.1 that was similar were inhibited to respond to B6 thymocytes or splenocytes by to or higher than that observed on thymocytes (Fig. 2). More de- blocking with 15F7 (Table I), a CD1.1-specific mAb (see tailed studies defined a hierarchy of CD1.1 expression with DC Ͼ Footnote 4). B cells ϭ double-positive thymocytes Ͼ single-positive thymo- Since the panel of T hybridomas included cells derived from cytes Ͼ mature peripheral T cells (see Footnote 4). thymus cells and others derived from spleen cells, it was possible These results suggested that the poor recognition of spleen to examine whether the tissue specificity of recognition correlated CD1.1 by our first series of NK1 T cell-derived hybridomas was with the tissue of origin. Although a large proportion (58%) of not due to a lower level of surface expression on spleen, and there- hybrids reacted to both splenic and thymic cells, it is striking to fore, that these T cells must discriminate qualitatively between note that all four thymocyte-specific clones (DN32.D3, DN32.F3, CD1.1 expressed by B6 thymocytes and splenocytes. TA.H1, and 431.A11) were derived from the thymus, and that 1C8.DC1, the single spleen cell-specific clone, was derived from Tissue-specific recognition of CD1.1 the spleen. In other words, no spleen-specific hybrid was found To examine this possibility, we derived a new series of hybridomas among thymus-derived CD1.1-autoreactive cells, and conversely, by fusing both thymic and splenic CD8-negative T cells obtained no thymus-specific hybrid was found in the spleen cell-derived from B6.MHC IIϪ/Ϫ mice with BW5147␣␤Ϫ. Hypoxanthine-am- population. Although the dataset is relatively limited, it is strength- inopterin-thymidine-resistant hybrids were screened for IL-2 se- ened by the fact that the CD1.1-autoreactive hybrids used in this cretion upon stimulation with a 1/1 mixture of thymocytes and study were selected with an unbiased screening assay testing for splenocytes, and CD1-specific clones were identified by blocking responsiveness to either thymic or splenic cells. The results sug- their response with anti-CD1 mAbs. Nine of these new clones and gest, therefore, that CD1.1-autoreactive cells that reside in a par- three previously described ones (DN32.D3, DN32.F3, and ticular tissue may have accumulated or expanded in response to 431.A11, derived from B6 thymic NK1 T cells) (15, 16) were used local CD1.1 presentation. for further studies. Although some of the T cell hybridomas used the same invariant V␣14-J␣281 TCR ␣-chain (Table I, left col- Fine tissue specificity of CD1.1-responding hybridomas umn), they differed in their TCR ␤-chain usage (15, 16) (data not To investigate the basis of this tissue specificity, we purified some shown). of the major cell types in thymic and splenic populations and com- When stimulated with thymocytes or splenocytes, the hybrid- pared their ability to stimulate eight of the hybridomas. To our omas displayed three main patterns (Table I). Four of 12 surprise, the level of specificity increased further, in that six pat- (DN32.D3, DN32.F3, TA.H1, and 431.A11) responded mainly to terns now emerged (Table II). One hybridoma, 1C8.DC1, which The Journal of Immunology 3131

Table I. Tissue specificity of CD1.1 recognition by T cell hybridomasa

IL-2 (U/ml)

Thymic Stimulators Splenic Stimulators

B6 B6 TH/SPL stim V␣14 No Ab 15F7 B6.TAPϪ B6.TAPϪ No Ab 15F7 By.TAPϪ B6.␤2Ϫ ratio

Thymus-derived hybrids DN32.D3 ϩ 36 Ͻ133 Ͻ1 Ͻ1 Ͻ1 Ͻ1 Ͻ1 >36 DN32.F3 ϩ 19 Ͻ120 Ͻ12Ͻ12Ͻ1 9.50 TB.B2 ϩ 4 Ͻ13Ͻ19Ͻ113Ͻ1 0.44 TA.H1 ϩ 7 Ͻ111 Ͻ1 Ͻ1 Ͻ12Ͻ1 >7.00 TB.H1 ϩ 11 Ͻ18Ͻ122Ͻ125Ͻ1 0.50 431.A11 Ϫ 19 Ͻ1154 22 2 3 9.50 TB.A7 Ϫ 46 2 37 2 84 Ͻ134Ͻ1 0.55 TB.D7 Ϫ 22 Ͻ118 Ͻ119Ͻ127Ͻ1 1.16 TC.B11 Ϫ 13 Ͻ123 Ͻ141Ͻ130Ͻ1 0.32 Spleen-derived hybrids 1C8.DC1 Ϫ 14 Ͻ1 11 2 216 3 198 2 0.06 SB.C12 Ϫ 6 Ͻ16Ͻ15Ͻ14Ͻ1 1.20 Downloaded from SA.A8 Ϫ 3 Ͻ16Ͻ16Ͻ118Ͻ1 0.50

a T hybridomas are designated ϩ or Ϫ according to their utilization of V␣14-J␣281. The calculated ratio of IL-2 released in the presence of thymocytes vs splenocytes is shown in the rightmost column and is representative of 3 to 5 experiments (bold values highlight the tissue-specific hybridomas). 15F7 is a CD1.1-specific rat IgG2b mAb used at 2 ␮g/ml. It specifically blocked CD1 recognition when compared with control rat IgG2b mAbs, and, conversely, did not impair activation of control non-CD1-specific T hybridomas (data not shown). http://www.jimmunol.org/ predominantly responded to spleen cells, reacted only to purified cortical thymocytes, but poorly to B cells (Table II); however, DC, secreting up to 1500 U of IL-2, and was blind to other cell unlike 431.A11, its recognition of CD1.1 was independent of the types, including splenic B cells or T cells, or to cortical or med- mouse genetic background (data not shown and see Fig. 3), im- ullary thymocytes. DN32.D3 responded to cortical thymocytes, plying that TA.H1 recognizes a sixth distinct CD1.1 ligand. Test- and although no reactivity was initially detected against whole ing of these hybridomas against a panel of mouse, rat, and human spleen cells, a minimal response was obtained after removal of B cell lines transfected with CD1.1 confirmed the high level of tissue cells, and exposure to purified splenic DC revealed a good re- specificity, in that TA.H1 reacted to none, 431.A11 only responded sponse, equivalent to that against cortical thymocytes. 431.A11 to mouse fibroblast line C57SV, DN32.D3 responded to C57SV by guest on September 25, 2021 responded to cortical thymocytes, but also exhibited some variable and also to the rat basophil leukemia cell RBL, and 1C8.DC1, degree of reactivity to SP thymocytes. Purified immature TB.A7, TB.D7, and TC.B11 responded to the human C1R B cell (HSAhigh) SP thymocytes, which constitute 20 to 80% of SP cells line in addition to C57SV and RBL (Table IV). In summary, we and have not yet modulated CD1.1 expression (see Footnote 4), obtained evidence for up to six distinct CD1.1 Ags by simply appeared to stimulate 431.A11 as efficiently as the DP cells, testing eight CD1.1-specific T cell hybridomas against a limited whereas they failed to stimulate DN32.D3 (Table III). Again, de- sample of CD1.1-presenting cell types. spite the lack of a strong response to whole spleen cells, reactivity of 431.A11 to DC could be revealed after exposure to purified DC Most hybridomas responded to thymocytes or splenocytes from (Table II). A fourth pattern of specificity was revealed for TB.H1 a variety of mouse strains (not shown). However, one of them, and TC.B11, which responded strongly to B cells and DC and 431.A11, only recognized thymocytes from the genetically related poorly to cortical thymocytes (Table II). TB.A7 and TB.D7 also strains C57BL/6, C57BL/10, C58, C57BR, and C57L, but not had a unique pattern of specificity, including DC, B cells, and from BALB/c, SJL, C3H, AKR, DBA/2, 129, New Zealand White, cortical thymocytes. TA.H1, like 431.A11, responded to DC and New Zealand Black, or NOD (Fig. 3). Again, this difference was not related to polymorphism of the CD1 genes themselves. We took advantage of the existence of reciprocal B6/B10 and NOD Table II. Fine cell type specificity of CD1.1 recognition by autoreactive congenics for the Idd10 locus, which encompasses CD1 (33). Fig- T cell hybridomasa ure 3 shows that 431.A11 recognized thymocytes with the B10, but not the NOD, genetic background regardless of the origin of CD1. IL-2 (U/ml)

Spleen Thymus B cell Whole DP SP Whole depleted T B DC Table III. Stage-specific recognition of thymic CD1.1 by T cell hybridomas 1C8.DC1 1 0.5 3 48 159 1 1 1500 DN32.D3 54 45 Ͻ0.5 Ͻ0.5 2 Ͻ0.5 1 42 431.A11 36 24 2 3 3 Ͻ0.5 2 21 IL-2 (U/ml) TB.H1 6 2 Ͻ0.5 6 51 Ͻ0.5 18 75 TC.B11 6 2 Ͻ0.5 30 78 Ͻ0.5 72 120 Thymocytes TB.A7 18 9 Ͻ0.5 12 93 Ͻ0.5 18 90 Ͻ Ͻ CD4 CD4 CD8 CD8 TB.D7 15 6 0.5 6 9 0.5 9 54 Whole DP HSAhigh HSAlow HSAhigh HSAlow TA.H1 12 9 Ͻ0.5 0.5 12 Ͻ0.5 0.5 57 Ͻ Ͻ a DN32.D3 20 20 0.6 0.2 0.4 0.2 DP, double-positive; SP, single-positive; DC, dendritic cells. Results are repre- 431.A11 13 9 8 0.7 5 Ͻ0.2 sentative of 2 to 5 experiments. 3132 TISSUE-SPECIFIC RECOGNITION OF CD1

does not transport peptides with the size or sequence required to bind CD1.1 efficiently. Conversely, the fact that TAP-deficient cells, which express much less classical MHC class I molecules but similar amounts of CD1.1 on their surface, did not induce a higher level of activation of the CD1.1 autoreactive hybrids is noteworthy. It suggests that the CD1.1-autoreactive hybridomas, including those derived from NK1 T cells that express Ly49 re- ceptors, are not susceptible to MHC class I-mediated inhibition of activation. This finding correlates with our observation that Ly49 receptor expression is lost upon fusion of NK1 T cells with BW5147 ␣␤Ϫ (data not shown).

Discussion Among the most intriguing aspects of the mouse CD1 and human CD1d systems are the high frequency of autoreactive T cells that respond to CD1, the expression of semi-invariant TCRs and NK receptors by many of these cells, and their unique cytokine secre- Downloaded from tion properties. These properties are linked developmentally, since transgenic expression of the V␣14 TCR ␣-chain by mainstream developing thymocytes is sufficient to impart the NK1 phenotype and the IL-4 secretion potential (42). The expression of NK re- FIGURE 3. The genetic background alters CD1 recognition by the T ceptors appears to be a late, separate event in the thymic matura- cell hybridoma 431.A11. IL-2 release by the T cell hybridomas 431.A11 tion process that requires expression of the common cytokine re- and DN32.D3 cultured with thymocytes of individual mice from various ceptor ␥-chain (43). In mice deficient for the common ␥-chain, a http://www.jimmunol.org/ strains is plotted as the percentage of that obtained with B6 thymocytes. typical IL-4 producer V␣14/V␤8 population is found in the thy- B10.NOD-CD1 and NOD.B6-CD1 mice correspond to B10.NOD- H2g7Idd10, and NOD.B6-Idd3Idd10, respectively (as Idd10 encompasses mus, but is arrested at a pre-NK1 stage where it does not express CD1) (33). Results with the CD1 congenics were confirmed in a separate NK receptors such as NK1.1 and Ly49. It is not found in the experiment. periphery, possibly because the lack of inhibitory NK receptors, which may be necessary to keep the TCR autoreactivity in check, prevents escape from negative selection. The control of NK1 T cell TAP-independent recognition of CD1.1 activation may therefore be a complex phenomenon that results

The results described above strongly suggested that most CD1.1 from the balance between positive signals such as those mediated by guest on September 25, 2021 molecules expressed at the cell membrane are recognized in asso- by TCR autorecognition of CD1 and possibly also by NK receptors ciation with a set of coligands. The results shown in Table I dem- such as NK1.1 (44) and by negative signals such as those trans- onstrate, however, that all 12 CD1-specific hybridomas, whether duced by members of the Ly49 family upon binding to MHC class using the invariant V␣14 TCR ␣-chain or other TCRs, recognized I molecules. Thresholds and balance between these signaling path- CD1.1 expressed by fresh thymocytes or splenocytes equivalently ways may be set in the thymic environment or, as suggested for in the presence or the absence of TAP. This correlates with the fact true NK cells, may be flexible and calibrated in different tissue that surface expression of CD1.1 by TAP-deficient cells is equiv- environments (45). alent to that of TAP-sufficient cells (12, 27, 40, 41). By compar- By showing that CD1.1 is constitutively expressed by peripheral ␤ ison, recognition of 2m-deficient cells, which express low to un- APCs, notably splenic B cells and DC, and that the level of ex- detectable CD1.1, was completely abolished. The results confirm pression is stimulatory to a large fraction of CD1-specific T cell and extend previous observations (12, 27, 40, 41), clearly indicat- hybridomas, the present results suggest that a significant popula- ing that the peptide pool contributed by TAP does not influence T tion of T cells residing in peripheral tissues may be constitutively cell recognition of CD1.1, either because CD1.1 is protected from triggered through their CD1-autoreactive TCRs. This is supported binding peptides in the endoplasmic reticulum or because TAP by the fact that all resident CD1.1-autoreactive cells were found to respond prominently to their own tissue of origin, whereas in some cases they did not respond to CD1.1-expressing cells of other tis- Table IV. Cell-type specificity of recognition of CD1.1-transfected sues. Thus, resident CD1.1-autoreactive cells may have expanded linesa or accumulated in response to previous local stimuli. Autoreactive responses may be down-regulated through the MHC class I-spe- IL-2 (U/ml) cific, inhibitory NK receptors expressed by many of these CD1- RBL- C57SV- C1R- autoreactive cells, since engagement of MHC-specific inhibitory CD1.1 CD1.1 CD1.1 receptors can inhibit TCR-mediated activation (46–48). In addi- 1C8.DC1 7 32 7 tion, we have found that none of the T hybridomas used in this DN32.D3 75 30 1 study expressed such NK receptors as NK1.1, Ly49A, C, or I (not 431.A11 Ͻ114 2shown) even though many originated from the fusion of T cells TB.H1 9 ND ND that did express these receptors. A similar observation was recently TC.B11 18 44 18 TB.A7 57 40 28 reported by Shimamura et al. (18). Together, these results suggest TB.D7 10 39 12 that the latent autoreactivity of NK1 T cells may be revealed in TA.H1 Ͻ1 Ͻ1 Ͻ1 hybridomas, not because of inherent differences in activation a Similar results were obtained in 2 to 5 experiments. Untransfected cell lines did thresholds, but because they have lost the suppressor arm of the not stimulate T cell hybridomas (data not shown). ND, not determined. activation control. The Journal of Immunology 3133

Key to understanding the biology of CD1 is the question of the for CD1.1 binding, whereas DC that express higher levels of nature of the CD1 ligands recognized by T cells. There is evidence CD1.1 also seem to express a fuller set of CD1 ligands. that CD1.1 may be expressed on the membrane as a stable empty It remains unclear whether tissue-specific CD1 ligands elicit dis- molecule and also that it may bind hydrophobic peptides with a tinct functional subsets of autoreactive T cells, or whether their putative anchoring motif (10–12). In addition, sequence analysis diversity has little functional significance but is merely a reflection has revealed two remarkable features that may govern crucial as- of the fact that the self Ags associated with CD1 only partially pects of intracellular trafficking and Ag presentation. First, the in- overlap in different tissues. In any case, the results presented in this tracytoplasmic tail of CD1.1 displays an endosomal targeting sig- paper suggest that CD1.1 is naturally associated with self ligands nal similar to that of human CD1b (6), and second, the CD1.1 and therefore imply that it might also present foreign ligands. Fu- leader peptide itself (14) displays the canonical CD1.1 binding ture studies aiming at identifying these putative self and foreign motif elucidated by Castano et al. (10). These observations have ligands associated with CD1.1 should provide major insights into led us to propose a model where the head (leader peptide) and the the biology of the CD1 system and the pathway of immune reg- tail of CD1.1 play a role that is similar in essence to that of the C- ulation by CD1-autoreactive T cells. and the N-termini of the invariant chain involved in the MHC class II pathway, protecting the groove from Ag loading in the endo- Acknowledgments plasmic reticulum and driving CD1.1 to sample Ags in the endo- We thank J.-P. Kinet, Barbara Knowles, Larry Peterson, Hidde Ploegh, and somal compartment (14). The model is compatible with the idea Linda Wicker for the gifts of reagents; Andy Beavis for advise on flow that CD1.1 may be expressed and recognized in association with a cytometry and cell sorting; and Darren Hasara for managing the mouse Downloaded from variety of self as well as foreign hydrophobic ligands of peptidic or colonies. lipidic nature. The results presented in this paper support the existence of such References ligands. Indeed, cell type specificity of antigenic recognition by T 1. Martin, L. H., F. Calabi, and C. Milstein. 1986. Isolation of CD1 genes: a family cells usually reflects the recognition of tissue-specific peptides of major histocompatibility complex-related differentiation antigens. Proc. Natl. Acad. Sci. USA 83:9154.

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