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TCR Chain Influences But Does Not Solely Control Autoreactivity of V14J281T Cells¹

Fox Chase Cancer Center, Philadelphia, PA 19111

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD1d-dependent accumulation of T cells bearing a canonical V14J281 -chain (V14⁺ T cells) is thought to model positive selection of lipid-specific T cells, based on their ability to recognize CD1d-presented self glycolipid(s). However, it has been difficult to demonstrate self ligand specificity in this system, as most V14⁺ T cells do not exhibit significant autoreactivity despite high reactivity to -galactosylceramide presented by CD1d (-GalCer/CD1d). To assess the role of TCR chain in determining the -GalCer/CD1d vs autoreactive specificity of V14⁺ T cells, we conducted TCR or TCR chain transduction experiments. In this study we demonstrate, by combining different TCR chains with the V14 -chain in retrovirally transduced T cell lines, that the V14 -chain plays a primary role, necessary but not sufficient for imparting -GalCer/CD1d recognition. -Chain usage alone is not the sole factor that controls the extent of autoreactivity in V14⁺ T cells, since transduction of TCR chains from a high CD1d autoreactive V14⁺ T cell line conferred the -GalCer/CD1d specificity without induction of autoreactivity. Thus, heterogeneity of V14⁺ T cell reactivity is due to both -chain diversity and control mechanism(s) beyond primary TCR structure.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
It appears that CD1d plays a critical role in the positive selection of CD1-restricted T cells, resulting in a repertoire of microbial and autologous lipid-specific cells (1, 2, 3). Distinctively, both mice and humans show a natural accumulation of T cells with a homologous canonical TCR chain together with a similar glycolipid specificity. They express V14 rearranged to J281 in mice (V14) and V24 rearranged to JQ in humans in association with a biased set of V chains (4, 5, 6, 7, 8, 9, 10) and exhibit specificity to glycosylceramide-containing -form anomeric sugars, such as -galactosylceramide presented by CD1d (-GalCer/CD1d)³ (11, 12). Accumulation of these T cells depends on the presence of CD1d⁺ hemopoietic cells, providing evidence for self ligand/CD1d-mediated positive selection (9, 13, 14). In this self Ag recognition, a lysosomal hybrolase -galactosidase A, an enzyme whose deficiency causes Fabry’s disease (15, 16), appears to be involved to generate the monosaccharide epitope in the APCs (17). Murine V14⁺ T cells have been shown to contribute to a variety of immune functions (1, 18). Considering the normal accumulation of such T cells in both mice and humans, it has been suggested that self glycolipid(s) with -galactosidase A-dependent epitope(s) elicits expansion of this self-reactive T cell pool, with fundamental importance in the immune system throughout all mammalian species.

However, demonstrating self glycolipid specificity for all V14⁺ T cells has been difficult. Some V14⁺ T cells exhibit specific reactivity to CD1d⁺ autologous/syngeneic cells, as CD1d autoreactive T cells, and several cellular phospholipids have been identified as potential candidates for the V14⁺ TCR self lipid ligand important in positive selection (8, 9, 19). However, many V14⁺ T cells do not, or only marginally, exhibit clear autoreactivity, despite their high reactivity to -GalCer/CD1d (3, 20). This discordant reactivity pattern among V14⁺ T cells has complicated identification of self ligand(s) and elucidation of the mechanism for generation of this T cell subset.

We undertook a study to clarify the roles of TCR and TCR chains in determining the -GalCer/CD1d specificity vs CD1d autoreactivity. First, we conducted a conventional approach, analyzing numerous V14⁺ T cell hybridomas generated from mice, as representative of the T cell repertoire positively selected on the basis of self lipid/CD1d affinity. Second, we used retroviral TCR transduction of TCR^-+ T cell hybridomas (derived from TCR knockout mice) to directly assess the role(s) of V14 -chain and -chain in the CD1d-restricted specificity. Our data demonstrate the key role of the canonical V14 -chain in the -GalCer/CD1d specificity, yet reveal a contribution of -chain in modulating this reactivity, consistent with the finding of nonrandom -chain usage in the V14 T cell pool in mice. Importantly, we found that transduction of TCR from a V14⁺ CD1d autoreactive T cell generated the -GalCer specificity, but without autoreactivity, suggesting the existence of a mechanism(s) to regulate self reactivity distinct from the TCR Ag binding site structure. Thus, -chain diversity is not a sole reason for the heterogeneous self reactivity among V14⁺ T cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

C57BL/6.ICR mice were maintained in our laboratory animal facility. Mice deficient for TCR chain due to gene targeting (C^-/-) (21) on a C57BL/6 background were purchased from The Jackson Laboratory (Bar Harbor, ME). Studies were performed according to institutional guidelines for animal use and care.

Abs/reagents, flow cytometry sorting, and analysis

Reagents used for four-color flow cytometry analysis, cell sorting, and cytokine measurement were mostly prepared in our laboratory, as described elsewhere (22). Hybridoma Abs used in this study: CD4 (GK1.5), CD8 (53-6-7), heat-stable Ag (HSA)/CD24 (J11d, 30F1), GD1c (SM3G11), NK1.1 (PK136), CD3 (500A-A2), TCRC (H57-597), TCRC (H28-710), V8 (F23.1), IL-2 (JES6-1A12, JES6-5H4), and B220 (RA3-6B2). Fluorescein- or biotin-coupled reagents specific for various TCR V and V families were purchased from BD PharMingen (San Diego, CA). A FACStar^Plus flow cytometer (BD Biosciences, San Jose, CA) was used for cell purification and analysis.

Hybridomas

To establish NK1⁺ T cell hybridomas, thymocytes from six 3-mo-old C57BL/6.ICR mice were prepared, and HSA (30F1^-) cells were enriched by magnetic bead depletion (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany), then subsequently four-color stained with Abs to CD4, a CD8 plus HSA mixture, NK1.1, and TCRC for cell sorting. CD4⁺ or CD4^- cells in the CD8^-HSA^-NK1⁺C⁺ cell fraction were sorted as CD4⁺ or CD4^-NK1⁺ T cells. For NK1^- T cell purification, HSA^- thymocytes were MACS preenriched as for NK1⁺ T cell preparation, then stained with Abs to CD4, CD8, an HSA plus GD1c/3G11 mixture, and NK1.1. CD4⁺CD8^-HSA^-GD1c/3G11^-NK1^- cells were sorted. These CD4⁺NK1^- T cells were >95% C⁺. A total of 3–4 x 10⁵ of these purified cells were each separately stimulated with plate-coated anti-CD3 for 2 days together with lethally irradiated B6 spleen cells, then fused with the BW^-- cell line, as described previously (23). To establish TCR^-+ T cell hybridomas, spleen cells from two C^-/- mice were stained, and CD4⁺TCRC⁺CD8^-B220^- cells were purified by cell sorting then stimulated with anti-CD3 for fusion with the BW^-- cell line. Hybridomas were screened for positive surface CD3/C expression and anti-CD3-induced IL-2 secretion, and subsequently cloned by cell sorter. All hybridomas in this study were maintained in Opti-MEM (Life Technologies, Rockville, MD) plus 4% FCS plus 2-ME. Hybridomas selected for further study were periodically resorted on the basis of the surface CD3 expression to maintain original surface TCR/CD3 level using a flow cytometer.

Assays for CD1d-specific reactivity

Assays for -GalCer/CD1d reactivity and CD1d autoreactivity were described previously (20). In brief, CD1d-transfected (or nontransfected) A20 B lymphoma or RMA-S thymoma lines were used as stimulators. A total of 1 x 10⁵ stimulator cells, either -GalCer pulsed (50 ng/ml for 3 h, then extensively washed), control -GalCer pulsed, or nonpulsed, was incubated with 4 x 10⁴ responder hybridoma cells in a total of 150 µl of RPMI 1640 culture medium/well (U-bottom 96-well plate). In parallel, responder hybridomas were stimulated separately with either anti-CD3 (or anti-C)-coated plate (1 µg/ml), -GalCer (50 ng/ml), or -GalCer. -GalCer and -GalCer were kindly provided by Y. Koezuka (Kirin Brewery, Gunma, Japan). One-day culture supernatant was tested by ELISA to measure IL-2 level. rIL-2 standard was purchased from BD PharMingen. Autologous MLR activity was tested using lethally irradiated thymocytes from C57BL/6 or ₂-microglobulin-deficient C57BL/6 mice (purchased from The Jackson Laboratory) (2 x 10⁴ responder mixed with 4 x 10⁴ stimulator), as described previously (20, 23). Average of duplicate cultures is shown, in which SDs are <5% in all figures presented. Experiments were repeated more than three times; representative data are shown.

RT-PCR and sequencing of TCR

The presence of V14-J281 message was tested by RT-PCR, simultaneously comparing with VBWB message from the BW fusion partner serving as an mRNA control, as previously described (23). Primer sets used: V14-J281 (5'-TCCTGGTTGACCAAAAAGAC, 3'-CAGGTATGACAATCAGCTGAGTCC), and VBWB-C (5'-CATTCGCTCAAATGTGAACAG, 3'-GAAGCTTGTCTGGTTGCTCCAG). For TCR DNA sequencing, cDNA from hybridomas was amplified using the following V primers in combination with a C2 primer: V8.2, (5'-GCATGGGCTGAGGCTGATCCA); V14, (5'-ACGACCAATTCATCCTAAGCAC); V6 (5'-CTCTCACTGTGACATCTGCC); V11, (5'-GAACGATTCTCAGCTCAGAT); and C2, (3'-CCAAGCACACGAGGGTAGCCTT). Amplified DNA was then cloned using TA vector (Invitrogen, Carlsbad, CA), and plasmid clones containing appropriate size insert were sequenced on an ABI-377 instrument (Applied Biosystems, Foster City, CA), as described elsewhere.

Retroviral TCR transduction

V14-GFP. mRNA was prepared from the CD1d autoreactive hybridoma N38-2C12 and reverse transcribed into cDNA. The TCR coding sequence was amplified by PCR using PFU DNA polymerase (Stratagene, La Jolla, CA) with a V14-C primer pair engineered to contain EcoRI and XhoI restriction sites (5'-GGAATTCATGAAAAAGCGCCTGAGTGCC, 3'-CCGCTCGAGTCAACTG GACCACAGCCTC). The 837-nt amplified fragment was cloned into TA vector following the manufacturer’s protocol and verified by sequence analysis. The ExoRI/XhoI fragment released by double restriction digest was gel purified, then ligated with EcoRI/XhoI-cut pBMN-internal ribosomal entry site (IRES)-enhanced green fluorescent protein (EGFP) retroviral vector (24). Plasmid clones containing appropriate size insert were purified and introduced into the Phoenix ecotropic packaging line (provided by G. Nolan, Stanford University, Stanford, CA) by lipofection (LipofectAMINE; Life Technologies), following the manufacturer’s protocol. Viral supernatant (serum-free OptiMEM, 6-h culture the day following transfection) was harvested, 1 µl/ml lipofectAMINE was added, and C⁺ hybridoma cells were resuspended in this supernatant mixture. OptiMEM containing 4% FCS was added after 3–6 h. The infection was repeated on the next day. Green fluorescent protein (GFP)⁺ cells were sorted by flow cytometry 2–3 days after the last infection. Cells with high levels of TCR/CD3 were subsequently sorted by using allophycocyanin-coupled anti-CD3, together with GFP fluorescence.

V8-YFP and V6-GFP. For V8 -gene transduction, cDNA was made from V8.2⁺ hybridomas then amplified with PFU using a V8-C primer pair (5'-GGATCCATGTCTAACACTGCCTTCCCTGACCC, 3'-GCGGCCGCCTGTTTCAGAGTCAAGGTGTCAACG). The 1.1-kb fragment was TA cloned and sequenced for verification as above. The ExoRI/XhoI fragment was ligated with the pBMN-IRES-yellow fluorescent protein (YFP) retroviral vector (in which EGFP was replaced by enhanced YFP) (25). Transduction was as described above. Flow cytometry was set to specifically purify GFP⁺YFP⁺ double-expressing cells (when GFP⁺ cells were used as recipients) by compensating with control cell lines transfected with either V14.GFP or V8.YFP alone (into CD3^- variant T hybridoma cell line, A50G1.CD3^-; M. Gui and K. Hayakawa, unpublished observations). Subsequently, high surface V8⁺ cells among GFP⁺ cells were sorted by staining with allophycocyanin anti-V8. For V6 -gene transduction, cDNA from H41-2D9 was amplified with V6 primer (5'-GGATCCATGAACAAGTGGGTTTTCTGCTGG) paired with the antisense C primer used for V8 -gene cloning, generating a 1.1-kb fragment that was cloned and ligated with pBMN-IRES-EGFP retroviral vector. GFP⁺ and surface V6⁺ cells were sorted by staining with biotin anti-V6 plus Texas Red avidin.

Immunochemical analysis

Two-dimensional electrophoresis analysis of surface TCR complex was performed as described elsewhere (26). In brief, hybridoma cells were surface biotinylated, lysed with 1% digitonin (CalBiochem-Novabiochem, La Jolla, CA), and immunoprecipitated with anti-C-coupled protein A-Sepharose B beads (Sigma-Aldrich, St. Louis, MO). Immunoprecipitates were applied to two-dimensional electrophoresis (nonequilibrium pH-gradient gel electrophoresis (NEPHGE/13% SDS-PAGE) gels under reducing conditions, blotted onto polyvinylidene fluoride membrane, incubated with HRP-avidin, and revealed by ECL. Western blotting of hybridomas for TCR detection (27) was conducted following a standard procedure with 1% Nonidet P-40 lysate. A total of 10⁶ cell equivalent lysates was run on 13% SDS-PAGE with 2-ME, blotted with anti-C (2 µg/ml), then incubated with HRP-coupled protein A (Kirkegaard & Perry Laboratories, Gaithersburg, MD) and revealed by ECL.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
V14⁺ T cells with -GalCer/CD1d reactivity in mice express considerably diverse -chains

The majority of CD1d-dependent V14⁺ T cells in mice are confined to a small CD4⁺ or CD4^-8^-NK1⁺ TCR population in the thymus and peripheral sites that exhibits a surface phenotype shared with NK cells, hence the term NK T subset (1). In addition, an autoreactive NK1^- T cell subset is detectable in the CD4⁺8^- T cell fraction in thymus (22, 23). We made hybridomas from these NK1⁺ and NK1^- populations in thymus and found that 77 of 92 NK1⁺ and three of 17 NK1^--derived hybridomas were V14J281⁺ (as V14⁺), based on analysis of RNA. The majority of V14⁺ hybridomas, but not V14^- cells, showed a strong -GalCer/CD1d-specific response regardless of NK1⁺ or NK1^- origin (Table I). In addition to previously reported V8 (8.1, 8.2, 8.3), V7, V2, and V10 usage (1, 20), we found V9⁺ and V14⁺ V14 lines with -GalCer/CD1d reactivity, as shown in Fig. 1, suggesting that -GalCer/CD1d-specific V14⁺ T cells selected in mice express considerable -chain diversity. As Fig. 1 also presents, most of these V14⁺ T cells showed only marginal (or no) reactivity to the CD1d transfectant, unless it was prepulsed with -GalCer. Some lines showed high spontaneous secretion of IL-2 and/or IL-3 (data not shown).

View this table: [in this window] [in a new window]   Table I. -GalCer/CD1d reactivity in association with V14J281 usage1

View larger version (32K): [in this window] [in a new window]   FIGURE 1. Diverse V gene family usage in the -GalCer response by V14+ T cell hybridomas. Depicted V14+ hybridomas in the figure were from the collection of V14+J281+ T cell hybridomas in Table I. ELISA of IL-2 levels in culture supernatant after 1-day stimulation. Average of duplicate cultures (<5% SD) is shown. CD1.A20: CD1d-transfected B lymphoma A20; GalCerCD1.A20: -GalCer-pulsed CD1d A20 transfectant. Relative V gene family representation among the NK1+ T cell-derived hybridomas in Table I was V8 > V7 > V2 > V9. Of three V14+ hybridomas from NK1- cells in Table I, two were V14+ and one was V8.2+, V9+, or V14+ V14 hybridomas did not express other Vs, as determined by surface staining and message analysis (23 ).

Detection of -chains nonpermissive for -GalCer/CD1d specificity together with V14 -chain

To assess whether the close linkage between V14⁺ T cells and -GalCer/CD1d reactivity is mediated solely by the V14 -chain, we conducted retroviral V14 -chain transduction. For this purpose, TCR^-+ hybridomas were made from the spleen of TCR knockout mice (C^-/-) to serve as V14-GFP gene transfer recipients. In spleen of C^-/- mice, a few CD4⁺C⁺ T cells are produced independent of the class I/CD1 environment (21, 28); thus, their -chains should not be preselected on the basis of CD1-restricted specificity. These splenic C^-+ cells express pre-TCR^b and are functionally competent, being able to secrete diverse cytokines upon TCR stimulation (29).

Table II lists the deduced TCR CDR3 regions of C^-+ hybridomas (H41) used for -chain transduction. Two H41 lines expressed V14, similar to -GalCer/CD1-reactive V14/V14 lines from B6 mice (N57), but their CDR3/J regions showed clonal differences. Other C⁺ hybridomas chosen were V11⁺ and V6⁺. As shown in Fig. 2a, these C^-+ lines produced significant IL-2 upon anti-CD3 stimulation, but none showed CD1-specific -GalCer or autoreactivity before V14 transduction. After V14GFP transduction (Fig. 2b), both V14⁺ lines (2C9 and 3C5) acquired specific -GalCer/CD1 reactivity. Thus, V14 -chain conferred -GalCer/CD1 reactivity in combination with V14⁺ -chains, without obvious restriction of CDR3/J. However, both V11⁺ (3A6) and V6⁺ (2D9) lines failed to show such reactivity under identical conditions, despite acquisition of high GFP fluorescence and -chain protein production, at levels comparable with the V14⁺-transduced lines (Fig. 3).

View this table: [in this window] [in a new window]   Table II. The deduced TCR CDR3 region of hybridomas1

View larger version (20K): [in this window] [in a new window]   FIGURE 2. Selective acquisition of GalCer/CD1 responsiveness by V14-GFP-transduced -expressing hybridomas. IL-2 ELISA data are shown. a, TCR-+ hybridomas (H41 series in Table II) before transduction. b, After V14 transduction. All lines responded to plate-coated anti-C. The 3A6.V14 and 2D9.V14 lines did not secrete IL-3, IFN-, IL-4, or IL-5, as determined by ELISA, upon -GalCer CD1.A20 stimulation.

View larger version (49K): [in this window] [in a new window]   FIGURE 3. -Chain production by all V14 gene-transduced lines. Upper part of figure, Western blot ECL analysis. SDS-PAGE of 106 cell lysates/lane, blotted with anti-C, then detected by HRP-protein A. N57-2C12 is a wild-type V14+ hybridoma. The 2D9 at far right represents a nontransduced -+ hybridoma, H41-2D9. V14-GFP levels compared by flow cytometry analysis are shown on the bottom. Fluorescence (FL) intensity unit, (2), was autofluorescence level.

View larger version (66K): [in this window] [in a new window]   FIGURE 4. Two-dimensional electrophoresis analysis (NEPHGE/SDS) of surface TCR. Anti-C-immunoprecipitated surface-biotinylated material (from 1–2 x 106 cell lysates) was applied to a NEPHGE/13% SDS gel under reducing condition, then detected by HRP-avidin. TCR and TCR chain areas are marked. Surface -chain levels at the time of surface biotinylation, as assessed by flow cytometry anti-C-staining analysis, were: N57-2C12 (40 FL units), 2C9.V14 (20 U), 3C5.V14 (40 U), 3A6.V14 (50 U), 2D9.V14 (60 U), and 2D9 (50 U). Right columns show a summary of surface TCRV usage and -GalCer/CD1d responsiveness.

Transduction of autoreactive V14⁺ TCR generates -GalCer/CD1d reactivity without autoreactivity

We took advantage of this -GalCer/CD1d nonreactive 2D9.V14 line as a recipient for secondary transduction with different -chains to address the significance of -chain usage in CD1d-restricted specificities. In the studies described above, with either V14⁺ native or V14-transduced T cells, identification of cells with high CD1d autoreactivity was rare. That is, only two of 70 NK T cell-derived V14⁺ hybridomas were found to show clearly high CD1d autoreactivity in our analysis. One such autoreactive hybridoma was N38-2C12 (2C12), as shown in Fig. 5a and compared with N38-3C3 (3C3), a line with prototypic -GalCer/CD1d reactivity with little autoreactivity. A third line, the V14^- (V3⁺) hybridoma N37-2A9, lacked any CD1-restricted reactivity (2A9). These were all of NK T cell origin and expressed V8.2⁺ TCR and CD3 at comparable levels on the surface (data not shown). The 2C12 was also reactive with CD1d⁺ syngeneic (and allogeneic) thymocytes and spleen cells, but not with cells from class I-deficient mice (₂-microglobulin^-) (Fig. 5a); thus, it recognizes a physiologic CD1d/cellular target expressed on normal cells, as do other typical autoreactive V14⁺ T cells (8, 9). These V8.2⁺ hybridomas shown in Fig. 5a expressed different CDR3/J junctional sequences (Table II). The 2D9.V14 GFP line was transduced with these different V8.2 -chain genes using a retroviral IRES-YFP vector.

View larger version (38K): [in this window] [in a new window]   FIGURE 5. Acquisition of -GalCer/CD1d responsiveness, but not CD1d autoreactivity, by V8.2 -chain-transduced 2D9.V14 cell lines. a, Reactivity differences among three V8.2 hybridomas used as -chain-YFP sources. b, Reactivity of the 2D9.V14 line before (shaded bar) and after -chain transduction with N38-3C3 (filled bar)-, N37-2A7 (hatched bar)-, and N38-2C12 (open bar)-derived -chains.

View larger version (16K): [in this window] [in a new window]   FIGURE 6. Induction of surface V8 expression after transduction of three different V8-YFP constructs into the 2D9.V14 GFP line, with or without down-regulation of V6. All -chain-transduced cell lines maintained the high level V14-GFP fluorescence of the 2D9.V14 host cell line (data not shown). Surface -chain expression was examined by staining with allophycocyanin anti-V8 and biotin anti-V6/Texas Red avidin. All -chain-transduced cells showed similar unstained allophycocyanin/Texas Red background levels to 2D9.V14, as shown in the left panel.

Fig. 7 presents further analysis with one such 2C12 V8-YFP-transduced subclone, 2D9. 2C12[2F7] (2F7), as compared with the original autoreactive hybridoma N38-2C12. N38-2C12 was the cell line used to clone V14 -chain gene for transduction experiments as well as the -chain. This 2F7 V14/V8.2 double-transduced subclone expressed high V8 levels with negligible surface V6, comparable with N38-2C12 (Fig. 7a, two left panels). As a retroviral transduction control, the V6 gene derived from 2D9 was transduced into N38-2C12, generating a line expressing both V8 and V6 (Fig. 7a, right). The 2F7 expressed a normal ratio of TCR to TCR chain on the surface, similar to native N38-2C12 (Fig. 7b). However, there was a clear difference in response. Different from N38-2C12, the 2F7 cell line could be stimulated in the presence or absence of presenting cells, typical of -GalCer/CD1d responders, but showed no autoreactivity (Fig. 7c). In particular, this positive outcome after transduction, i.e., a gain of capacity by 2F7 to respond to -GalCer stimulation alone, was distinctive, because the original CD1d autoreactive line was refractory to such treatment. The lack of CD1d autoreactivity by 2F7 was not a consequence of retroviral transduction or a problem due to the presence of mixed TCRs, because the V6 -chain-transduced N38-2C12 hybridoma retained its original CD1d autoreactivity (Fig. 7c). These data reveal that T cells with identical TCR can exhibit different reactivity patterns: either -GalCer/CD1d without autoreactivity or CD1d autoreactivity.

View larger version (48K): [in this window] [in a new window]   FIGURE 7. Conversion from CD1d autoreactive to -GalCer/CD1d reactive by reconstituting the 2C12 V14/V8.2 TCR in the H41-2D9 cell line. The 2D9.2C12[2F7] was a subclone of the 2D9.V14.2C12V8 line sorted for high TCR expression. N38-2C12.V6 was V6-GFP-transduced N38-2C12. a, Flow cytometry analysis of surface TCR expression. Cell lines were stained with allophycocyanin anti-V8 and biotin anti-V6/Texas Red avidin. Background staining showed average FL intensity of 1 U for biotin-TR and 0.6 U for allophycocyanin with all cell lines. b, NEPHGE/SDS analysis of anti-C coimmunoprecipitation. TCR and TCR chains are marked. c, In vitro stimulation assay. The 2F7 also failed to respond to RMA-S CD1d transfectant, C57BL/6, or BALB/c thymocytes or spleen cells, whereas reactivity to these CD1d+ cells was intact in N38-2C12.V6.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

-GalCer/CD1d recognition

The importance of a canonical V14 -chain in positive selection of the -GalCer/CD1d-reactive T cell pool has been demonstrated previously by analysis of J281 knockout mice (11). NK T cell development was defective in such mice, resulting in a defective -GalCer/CD1d response. Our findings in this work are in agreement with the previous notion of the critical importance of V14, demonstrating a close linkage between -GalCer/CD1d specificity and V14 (J281) usage, and the capacity of the V14 -chain in conferring -GalCer/CD1d specificity. In terms of the role of TCR chain, participation of the -chain in -GalCer/CD1d recognition has been suggested previously from studies using CD1d mutants affecting -GalCer presentation to TCR (30) and by CD1d-GalCer tetramer-binding analysis of V14 T cells in V14 TCR-transgenic mice (31). In this study, we report a V14⁺ TCR line with a V6 -chain that is unresponsive to -GalCer/CD1d, providing clear evidence that -chain can modulate the V14 -GalCer/CD1d specificity. Thus, the V14 -chain is necessary, but not sufficient, for imparting -GalCer/CD1d recognition.

However, -chain usage is much less restricted than previously thought. We show in this work V chains from six gene families that combine with V14 in -GalCer/CD1d recognition. We also demonstrate that various TCR chains, originally unrelated to the CD1-restricted specificity, can combine with V14 -chain in generating -GalCer/CD1d responsiveness. Therefore, it appears that the canonical -chain plays the major role, with -chain facilitating stabilization of this interaction with an antigenic epitope presented by CD1d. Consistent with this view, it was previously reported that NK T cell subset generation was normal in V8.2 TCR-transgenic mouse lines expressing V8 -chains derived from TCRs with CD1d irrelevant specificities (11, 27). Although self ligand(s) for V14 T cells remains elusive, our binding studies using -GalCer/CD1d reasonably explain why the V14⁺ T cell pool is comprised of cells with a biased, yet relatively diverse, -chain repertoire.

Control of autoreactivity by V14⁺ T cells

V14⁺ T cells gradually accumulate in mice as an NK T subset with an activated phenotype, suggesting continuing selection by Ags in the microenvironment during V14⁺ T cell differentiation. This V14⁺NK T cell subset shows a more biased V gene family representation and CDR3 constraints (1, 3) than anticipated from our in vitro -GalCer/CD1d stimulation analyses. This may reflect further selection based on affinity differences or selective cross-reactivity to allow differentiation and survival, dependent on -chain usage. Affinity difference among V14⁺ T cells to the physiologic self ligand to account for CDR3 constraints has been suggested previously: only autoreactive hybridomas, but not -GalCer/CD1d-reactive hybridomas, were activated by plate-coated purified CD1d, which was promoted by the addition of cellular lipid extract (19, 32). In this stimulation system, Ag presentation was not in a cellular form; thus, signaling modification by non-TCR receptor/ligand interactions was not possible, suggesting a role for TCR chain in determining affinity to the physiologic self ligand.

If affinity is the sole reason determining the extent of CD1d autoreactivity, transduction of -chains from autoreactive vs nonautoreactive T cells should reveal such differences. Surprisingly, we show in this work that precisely reconstituting the high autoreactive 2C12 TCR failed to induce autoreactivity, instead generating -GalCer/CD1d reactivity similar to the majority of V14⁺ T cell lines. This was not due to the loss of TCR-mediated IL-2 signaling, but rather showed a gain of ability to be directly stimulated by -GalCer. Thus, the extent of autoreactivity among V14⁺ T cells or cell lines is not determined solely by differences in the TCR or -chain usage, but also by distinctions in the host cell expressing the TCR. Attenuation of autoreactivity may occur by either gain of inhibitory receptors, loss of coreceptors, or alteration of intracellular signaling pathways in the T cell host, as extensively documented with NK cells and cytotoxic T cells (33). Differences in glycosylation (34) or in lipid raft dynamics (35) in the T cell host are other possibilities. In terms of receptor expression, the autoreactive 2C12 and nonautoreactive 2F7 showed a similar surface phenotype: CD4^-, CD8^-, CD90^high, CD44^high, Ly-6C⁺, CD28⁺, NK1^-, DX5^-, Ly-49A^-, Ly-49C^-, CD16/32^-. Compared with 2C12, 2F7 showed higher CD1d, lower CD45RB, and lower Ly-6A/E. Although our preliminary analysis did not reveal any clear association between such surface receptor levels and autoreactivity, it is clearly a subject for further scrutiny. In addition to such receptor-mediated downstream signaling, possible differences in self Ag presentation between V14⁺ T cells is another consideration. Nonetheless, our results reveal a complexity of autoreactive expression beyond TCR usage, and suggest that affinity to CD1d/self ligand of V14⁺ TCRs or ligand diversity need not be as heterogeneous as originally thought.

However, -chain usage may still be significant in determining cell fate. Interestingly, we found that transduction of the 2C12 -chain into the 2D9.V14⁺ T cell line resulted in a distinctively unstable level of surface TCR expression, which might result from continual TCR internalization due to high affinity specifically associated with the 2C12 -chain (compared with other -chains). A few subclones initially showing autoreactivity became unresponsive to stimulation through the TCR within 1 wk, as determined by down-regulation of IL-2 and IL-3, and lack of IFN-, IL-4, IL-5, or IL-10 secretion, despite maintaining a high surface TCR density and the ability to secrete IL-2 upon PMA plus calcium ionophore treatment (data not shown). The fact that most autoreactive V14⁺ TCRs recognize immature thymocytes expressing high CD1d (9) suggests that V14⁺ TCRs may have affinity to their own cellular products, including CD1d itself at some level, as a unique feature of this specificity. In normal V14⁺ T cell maturation, the CD1d level decreases and several NK receptors are induced (1, 9). These normal maturation-associated events may prevent endogenous self activation and induction of anergy, while maintaining reactivity to various exogenously presented self Ags. Fusion with the CD1d⁺ thymoma hybridization partner or TCR transduction into the non-NK T cell hybridoma, as done in this study, may have facilitated endogenous self Ag presentation and abrogation of normal physiologic regulatory mechanisms. N38-2C12 or other autoreactive V14 T cell lines may be infrequent exceptions as hybridomas, retaining both high-affinity TCR on the surface and CD1d autoreactivity. Taken together, our results suggest an intricate balance between appropriate TCR affinity to self ligand and control of autoreactivity in NK T cell development.

-chain restriction before positive selection

In early T cell development, precursors for V14⁺ T cells appear strongly dependent on the pre-TCR stage (36) and have stringent -chain structural requirements to allow formation of the TCR complex and expression on the cell surface, apparently more than for other -chains (27). Our V14 transduction data with one V11 -chain, showing difficulty in efficient surface expression, are consistent with this type of stringent early -chain selection. V11⁺ T cell lines repeatedly showed failure to express surface V14 by using two other independently isolated T cell lines from TCR-deficient mice (our unpublished observation). Therefore, V14 may already constrain -chain diversity to some extent, before positive selection initiates.

Concluding remarks

Our results establish a strong case for the primary, but not exclusive, role that V14 -chain plays in both early development and subsequent positive selection of CD1d-restricted glycolipid-specific T cells, which mediate critical functions in the immune system. -chain plays a modulating role, influencing cell fate, differentiation, and fine tuning of autoreactivity. In addition, it appears that autoreactivity in these cells has a requirement beyond TCR structure. A normal expansion of T cells with predominant canonical usage of single Ag receptors has also been found among T cells in specific locations (37). These observations suggest that positive selection through affinity to self ligand of particular Ag receptors encoded by distinctive V regions can lead to a predominance of such cells, resulting in eventual expansion of a natural autoreactive cell pool, without pathology. Investigation of the mechanisms that control TCR self reactivity remains a fertile area of research.

	Acknowledgments

 
We thank Drs. M. Kronenberg and N. Burdin for providing CD1d transfectants; Dr. Y. Koezuka for -GalCer and -GalCer; Dr. G. Nolan for Phoenix ecotropic packaging line and pBMN-IRES-EGFP retroviral vector; J. Dashoff, S. A. Shinton, and A. Cywinski for technical help; Dr. X.-X. Zeng for establishment and initial characterization of hybridomas; Dr. P. Nakajima for cell fusion; and Drs. D. Kappes, K. Campbell, and D. Wiest for critical reading of this manuscript.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant R01AI41726 (to K.H.).

² Address correspondence and reprint requests to Dr. Kyoko Hayakawa, Fox Chase Cancer Center, Reimann Building, 7701 Burholme Avenue, Philadelphia, PA 19111. E-mail address: K_Hayakawa{at}fccc.edu

³ Abbreviations used in this paper: -GalCer/CD1d, -galactosylceramide presented by CD1d; GFP, green fluorescent protein; EGFP, enhanced GFP; FL, fluorescein; HSA, heat-stable Ag; IRES, internal ribosomal entry site; NEPHGE, nonequilibrium pH-gradient gel electrophoresis; YFP, yellow fluorescent protein.

Received for publication July 12, 2001. Accepted for publication September 28, 2001.

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