of May 14, 2018.This information is current as

T Cellsβα +Thymic NK1.1Transgene Promotes Positive Selection of

-Chainβ Thymocyte-Derived TCR +A NK1.1

and Charles A. Janeway, Jr.Christophe Viret, Olivier Lantz, Xin He, Albert Bendelac

http://www.jimmunol.org/content/165/6/3004doi: 10.4049/jimmunol.165.6.3004

2000; 165:3004-3014; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/165/6/3004.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2000 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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A NK1.11 Thymocyte-Derived TCR b-Chain TransgenePromotes Positive Selection of Thymic NK1.11 ab T Cells1

Christophe Viret,* Olivier Lantz, † Xin He,* Albert Bendelac,‡ and Charles A. Janeway, Jr.2,3*

As a consequence of the peptide specificity of intrathymic positive selection, mice transgenic for a rearranged TCRb-chain derivedfrom conventional ab T lymphocytes frequently carry mature T cells with significant skewing in the repertoire of the companiona-chain. To assess the generality of such an influence, we generated transgenic (Tg) mice expressing ab-chain derived fromnonclassical, NK1.11 ab T cells, the thymus-derived, CD1.1-specific DN32H6 T cell hybridoma. Results of the sequence analysisof genomic DNA from developing DN32H6b Tg thymocytes revealed that the frequency of the parentala-chain sequence, in thisinstance the Va14-Ja281 canonicala-chain, is specifically and in a CD1.1-dependent manner, increased in the postselectionthymocyte population. In accordance, we found phenotypic and functional evidence for an increased frequency of thymic, butinterestingly not peripheral, NK1.11 ab T cells in DN32H6 b Tg mice, possibly indicating a thymic determinant-dependentmaintenance. Thus, in vivo expression of the rearranged TCRb-chain from a thymus-derived NK1.11 Va141 T cell hybridomapromotes positive selection of thymic NK1.11 ab T cells. These observations indicate that the strong influence of productiveb-chain rearrangements on the TCR sequence and specificity of developing thymocytes, which operates through positive selectionon self-determinants, applies to both classical and nonclassicalab T cells and therefore represents a general phenomenon inintrathymic ab T lymphocyte development. The Journal of Immunology,2000, 165: 3004–3014.

T he intrathymic development of classicalab T cells relieson the interaction of the TCR with self-peptide:self-MHCcomplexes expressed on thymic stromal cells. A minimal

interaction is required to rescue immature thymocytes from pro-grammed cell death and allow them to complete their maturation(positive selection), whereas stronger interactions result in the in-duction of apoptosis to maintain tolerance to most self-determi-nants expressed on bone marrow-derived cells (negative selection)(1–4). A recent series of in vivo observations made using MHCclass II systems (reviewed in Refs. 5–8) extended pioneering ex-periments related to CD81 T cell development (9–12) and firmlyestablished that positive selection of immatureab thymocytes isself oriented. That is, positive selection relies on the recognition ofself-peptide:self-MHC complexes expressed on thymic cortical ep-ithelial cells.

One of the direct demonstrations of the crucial role of self-peptide recognition in positive selection of CD41 T cells tookadvantage of transgenic mice expressing the rearranged D10 TCRb-chain (13). It is emphasized that in TCRb-chain transgenic(Tg)4 mice, thymocytes undergo a normal developmental process;they do not display the early high expression of TCR seen in most

TCR ab Tg mice. The CD3 complex expression by CD42CD82

and CD41CD81 thymocytes is comparable to that of wild-typethymocytes, anda-chain selection occurs normally at the appro-priate stage (13–15). In the D10 TCRb-chain Tg mice, analysis ofthe D10 TCR VaJa rearrangement in mature T cells revealed thata limited set of junctional sequences is selected from a very diversepool of preselection sequences (13). This restriction was furtherenforced on the H-2 Ma2/2 background, which displays a highlylimited self-peptide complexity. In H-2 Ma2/2 mice, the MHCclass II expression level is intact, but the invariant chain-derived81–104 (CLIP) peptide is dominant (16–18). In a distinct TCRb-chain Tg system, both V and J gene segment usages of the com-paniona-chain were significantly biased in mature T cells (19).The impact of a TCRb-chain transgene on thea-chain repertoirewas also observed in mice expressing a single peptide:MHC classII complex unrelated to the parental TCR; both the Va usage andthe amino acid composition of the CDR3 loop were significantlyinfluenced (20). Finally, mice transgenic for theb-chain of theMCC88–103:I-Ek complex-specific 5C.C7ab TCR were ana-lyzed using MCC88–103:I-Ek tetramers, which stain 95% of5C.C7ab TCR Tg thymocytes (15). Virtually all tetramer-positivecells from thymus and lymph node expressed the parental Va seg-ment (Va11) and displayed the characteristic CDR3 loop lengthrestriction observed among other MCC/I-Ek reactive T cells.Amino acid sequences identical with the 5C.C7 TCRa-chain weredetected in both tetramerhigh and tetramerlow cells, but not amongVa111/tetramer-negative cells. Collectively, these studies suggestthat productive TCR b-chain rearrangements at theCD44lowCD251 stage of thymocyte development, and thereforethe so-calledb selection process (21–23), critically impact on theTCR a-chain repertoire of matureab T cells through intrathymicpositive selection. However, the fact that the vast majority of thesestudies used rearranged TCRb-chains derived from conventionalCD41 ab T cells raises the question of the generality of thisphenomenon.

In this study we have analyzed mice expressing a rearrangedTCR b-chain transgene derived from Va14-Ja281 expressing

*Section of Immunobiology and Howard Hughes Medical Institute, Yale UniversitySchool of Medicine, New Haven, CT 06520;†Institut National de la Sante´ et de laRecherche Medicale, Unite 25, Hopital Necker, Paris, France; and‡Department ofMolecular Biology, Princeton University, Princeton, NJ 08540

Received for publication April 5, 2000. Accepted for publication June 23, 2000.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported in part by the Howard Hughes Medical Institute and byGrant AI14579 (to C.A.J.).2 Address correspondence and reprint requests to Dr. Charles A. Janeway, Jr., YaleUniversity School of Medicine. Section of Immunobiology, LH 416, 310 CedarStreet, New Haven, CT 06520-8011. E-mail address: charles.janeway@yale.edu3 C.A.J. is an investigator with the Howard Hughes Medical Institute.4 Abbreviations used in this paper: Tg, transgenic;b2m, b2-microglobulin; HSA,heat-stable Ag; int, intermediate; CDR, complementarity-determining region.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00

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NK1.11 T cells, an atypical population of CD41 or CD42CD82

ab T cells that require for both their intrathymic development andtheir activation, interaction of their semicanonicalab TCR withthe TAP-independent,b2-microglobulin (b2m)-dependent, non-classical MHC class I molecule CD1.1 (reviewed in Refs. 24–26).We find that transgenic expression of a rearrangedb-chain isolatedfrom a thymus-derived Va14-Ja281-expressing NK1.11 T hybrid-oma (DN32H6) promotes positive selection of thymocytes carry-ing the canonical Va14-Ja281 a-chain of NK1.11 T cells. Ac-cordingly, a higher frequency of NK1.11 ab T cells was detectedin the thymus, but strikingly not in the periphery, of DN32H6 TCRb-chain Tg mice. The data identify the critical impact of produc-tive b-chain rearrangement on the subsequent specificity of devel-oping thymocytes as a general phenomenon inab T celldevelopment.

Materials and MethodsMice

Mice used were 6–8 wk old and were housed in the Yale ImmunobiologyMouse Unit (New Haven, CT). C57BL/6 (B6) andb2m-deficient mice(b2m

2/2) (27) were obtained from The Jackson Laboratory (Bar Harbor,ME). The CD1.12/2 mice (B6-129 mixed background) were a gift from Dr.L. Van Kaer (Howard Hughes Medical Institute, Nashville, TN) (28).DN32H6 TCR b-chain Tg mice were generated by microinjecting theb-chain transgene into (B63 SJL)F2 oocytes (Comparative MedicineTransgenic Facility, Yale University). Transgene integration was assessedby PCR using tail genomic DNA. Founder animals were backcrossed fourtimes to C57BL/6 mice in a specific pathogen-free environment. The studywas performed using one Tg line, because the three lines obtained werephenotypically similar. Screening of animals was performed by PCR afterextraction of tail genomic DNA using the following oligonucleotide prim-ers: Vb8 specific sense, 59-GCCTTGTATCGATTCCCCTTCTTGTCTGTA-39; and Jb1.4-specific antisense, 59-CCATGACCGAAAAATAATCTTTCGTTGGAC-39.

A new PCR assay was developed for the identification of TCRb TgCD1.1-deficient mice. The sequences of the oligonucleotide primers usedwere: sense, 59-AAGCGCAGAAGTCGGAGCCG-39, which is specific forthe 59 end of the CD1.1-coding sequence (29); and antisense, 59-CT-TCTCTAGGTCTGACTT-39, which is specific for the end of exon 3 of theCD1.1 gene where theneo-resistance gene was inserted (28). A 1.2-kbband was generated for the CD1.11/1 and CD1.11/2 samples, but not forthe CD1.12/2 samples. Conversely, theneo-resistance gene was detectedfor both CD1.12/2 and CD1.11/2 samples, but not for CD1.11/1 samples.

TCRb-chain construct

The DN32H6 TCRb-chain transgene was constructed using the rearrangedV(D)J sequence from the CD42CD82 DN32H6 T cell hybridoma (Vb8.2-Db1.1-Jb1.4) (30) and a modified version of the TCRb shuttle vector (31).The construct was tested for expression in vitro by cotransfection with ana-chain construct into theab-negative 58 T cell line (O. Lantz and A.Bendelac, unpublished observations). For microinjection, the transgenewas linearized (SalI/PvuI digest), isolated by electrophoresis on 1% aga-rose gel, electroeluted, purified using an ELUTIP minicolumn (Schleicher& Schuell, Keene, NH), and dialyzed.

Immunostaining and Abs

Thymus, spleen, and lymph nodes (axillary, lateral axillary, superficial in-guinal, and mesenteric) were removed, and single-cell suspensions wereprepared. Splenic RBC were lysed using Tris-buffered ammonium chlo-ride. Fluorescence-labeled mAbs were used for multicolor staining. Briefly,0.2 3 106 cells were incubated in microtiter U-bottom plates with a satu-rating concentration of labeled mAb in 20ml for 30 min on ice. Cells werewashed twice and analyzed immediately. For two-step staining, cells wereincubated first with purified mAbs in PBS with 2% FCS/0.1% NaN3, fol-lowed by a F(ab9)2 of goat anti-mouse Ig-FITC conjugate from Sigma (St.Louis, MO). The mAbs used were anti-Vb8.1-2-FITC (clone MR5-2), anti-Cb-PE (H57-597), anti-Va2,3.2,8,11-FITC (B20.1, RR3-16, B21.14,RR8-1) from PharMingen (San Diego, CA), anti-CD8a-PE/FITC (53-6.7)from Life Technologies (Grand Island, NY), and anti-CD4-quantum red(H129.19) from Sigma. The Y3JP (mouse IgG2a, anti-I-Ab), F23.2 (mouseIgG2, anti Vb8.2), 53-6.72 and 2.43 (both rat IgG2b, anti-CD8), PK136(mouse IgG2a, anti-NK1.1(NKR-P1C, Ly-55)), YCD3-1 (rat IgG2c, anti-CD3e), 20C9 (hamster IgG, anti-heat stable Ag (HSA)), and 14.8 (rat

IgG2b, anti-CD45RA/B220) mAbs were affinity purified from hybridomasupernatants using standard procedures.

Flow cytometry and cell sorting

For flow cytometry, a FACScan flow cytometer and CellQuest softwarefrom Becton Dickinson (Mountain View, CA) were used to collect andanalyze the data. Nonviable cells were excluded using forward and sidescatter electronic gating or propidium iodide. For cell sorting, freshly iso-lated thymocytes were triple stained for CD4, CD8, and Vb8. Vb8low andVb8int/high thymocytes were sorted after gating on CD41 cells using aFACStarPlus station (Becton Dickinson).

Cloning and sequencing of CDR3a regions

Genomic DNA from sorted cells was prepared immediately using a DNAextraction kit (Stratagene, La Jolla, CA) and was resuspended in distilledwater overnight. The CDR3 ofa-chains that use the Va14-Ja281 combi-nation was PCR-amplified (recombinant Taq DNA polymerase, Life Technol-ogies) by a two-step process. The first step used oligonucleotide primers spe-cific for the intronic sequences flanking the Va14 and Ja281 gene segments:Va14 Leader-V intron-specific sense, 59-CTTAGGATTTGGGGGAAAAAT-39; and post-Ja281 intron-specific antisense, 59-TCCGAGGTGCGGCCGCAAAGAAATACTTAAGAAAAATTCTTT-3 9. Reaction mixtures weredenatured by heating to 94°C for 5 min and then were subjected to 30 cyclesof amplification using a DNA engine thermocycler (MJ Research, Watertown,MA) under the following conditions: 94°C for 1 min, 55°C for 1.5 min, and72°C for 1.5 min. The final extension was performed at 72°C for 10 min. Thesecond amplification step used 1–5ml of the primary amplification andoligonucleotide primers specific for the coding sequences of the Va14 andJa281 gene segments: Va14-specific sense, 59-GATAAGAATTCTAAGCACAGCACGCTGCACA-39; and Ja281-specific antisense, 59-GAAACCGGATCCCCAGGTATGACAATCAGCTGAGTCC-39 (30 cycles as indicatedabove). Fresh PCR products were cloned into Bluescript II KS phagemid vec-tor (Stratagene) afterEcoRI-BamHI double digest (underlined sites) or werecloned directly into the PCR2.1 vector using the TA cloning kit (Invitrogen,San Diego, CA). Transformed DH5a cells (Life Technologies) were plated inthe presence of Bluo-gal (Life Technologies) and antibiotic, and recombinantindividual colonies were directly subjected to PCR using the M13F(-20) andM13R oligonucleotide primers (25 cycles as indicated above). PCR fragmentswere purified using silica gel membranes (QIAquick kit, Qiagen, Chatsworth,CA) and directly sequenced with the T7 oligonucleotide primer using the TaqDyeDeoxy Terminator Sequencing kit (Applied Biosystems, Foster City, CA)and an ABI 373A DNA sequencer. Sequence analysis of Va2-Ja4 rearrange-ments was performed using oligonucleotide primers published previously (13).

Functional assay

For IL-4 production assay, freshly isolated T cell suspensions were pre-pared and cultured in anti-CD3e (YCD3-1)-coated 24-well plates (BectonDickinson, Lincoln Park, NJ) overnight at 37°C in Click’s EHAA medium(Irvine Scientific, Santa Ana, CA) supplemented with 5% heat-inactivatedFCS (Intergen, Purchase, NY), 53 1025 M 2-ME (Bio-Rad, Richmond,CA), 2 mM L-glutamine, and 50mg/ml gentamicin (Life Technologies).IL-4 production was detected using the IL-4-dependent murine CT.4S cellsin a proliferation assay. The cells were incubated with supernatants induplicate wells for 72 h and 1mCi of [3H]thymidine/well was added to theculture for the last 24 h. The plates were then harvested, and counts perminute were determined using liquid scintillation counting. In some situ-ations cell suspensions were first enriched in NK1.11 T cells by negativeselection using magnetic beads (Bio Mag, Advanced Magnetic, Cam-bridge, MA) and mAbs. Thymocytes were negatively selected using anti-HSA and anti-CD8a mAbs, and ammonium chloride-treated splenocyteswere negatively selected using anti-B220, anti-MHC class II, andanti-CD8a mAbs.

ResultsTransgenic mice expressing a NK1.11 T cell-derived TCRb-chain transgene

The majority of NK1.11 ab T cells are thymus-derived T lym-phocytes with unusual features. They are CD41CD82 orCD42CD82, express an intermediate TCR level, display an acti-vated/memory cell surface phenotype (CD1221, CD44high,CD62Llow), and coexpress receptors of the NK cell lineage, suchas NK1.1 and Ly49. Another major characteristic of NK1.11 Tcells is the highly restricted diversity of theirab TCR; theb-chainuses essentially the Vb8, Vb7, and Vb2 segments along with quite

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variable VDJ junctions, whereas thea-chain is invariant and con-sists of the Va14-Ja281 rearrangement. The V-J junction has aninvariant size and, in most cases, contains the valine (V) 92-gly-cine (G) 93-aspartate (D) 94-arginine (R) sequence (VGDR) (re-viewed in Refs. 24–26). The TCR repertoire analysis of NK1.11

T cells was facilitated by the generation of T cell hybridomas fromsorted CD41 or CD42CD82 mature CD44high thymocytes (30).One such hybridoma (DN32H6) was used to prepare a TCRb-chain construct (seeMaterials and Methods). Mice with germ-line transmission of the DN32H6 TCRb-chain transgene (Vb8.2-Jb1.4) were derived and backcrossed to C57BL/6 mice. Expres-sion of the transgenicb-chain was evaluated using three-colorstaining and flow cytometric analysis of cell suspensions fromlymphoid organs. Compared with that in nontransgenic littermates,expression of the DN32H6 TCRb-chain transgene resulted in anincrease in Vb81 cells in the periphery and thymus, but a nearlynormal TCR expression level (Fig. 1) as is usually the case forsingle TCRb-chain Tg mice. About 95–97% of peripheralab1 Tcells were Vb8.21, indicating that efficient allelic exclusion occursat the TCRb-chain locus (not shown). The thymic cellularity wasrepeatedly comparable to that of transgene-negative littermates,and peripheral DN32H6 TCRb Tg T cells displayed a virtuallynormal CD4/CD8 coreceptor distribution (Fig. 1,bottom). Thus,intrathymicab T cell development appears to operate normally inmice expressing the DN32H6 TCRb-chain transgene.

High frequency of the canonical Va14-Ja281 a-chain jointamong mature thymocytes from NK1.11 T cell-derived TCRb-chain Tg mice

The selection of the DN32H6 TCRa-chain (the canonical Va14-Ja281 chain of NK1.11 ab T cells) was analyzed by sorting outthymocytes that were CD41Vb8low or CD41Vb8int/high and se-quencing the junctional region of the Va14-Ja281 rearranged se-quences (seeMaterials and Methods). In the preselection (TCRlow)thymocyte population, the Va14-Ja281 junction was diverse;combining results from two DN32H6b Tg mice, a total of 31sequences contained threea-chain canonical junctions (VGDR;9.6%) encoded by three distinct nucleotide sequences (Fig. 2), 18out-of-frame sequences (58%), and 10 noncanonical, highly di-verse junctions (32.2%) without obvious bias in either length oramino acid composition. In sharp contrast with the preselectionpopulation, the postselection thymocyte population showed a highfrequency of the canonical junction as well as a severely reducedfrequency of out-of-frame sequences; up to 80% (82.8%) of thejoints were canonical (15/171 14/185 29/35 for two mice ana-lyzed) and were encoded in four distinct nucleotide sequences.Such a frequency is higher than that we observed in a non-Tgcontrol mouse when we used stringent sorting conditions; the fre-quency of the canonical junction was about 65% amongCD41Vb8.21 TCRint thymocytes from a transgene-negative lit-termate (Fig. 3). Despite the fact that the glycine residue of thecanonical junction can be generated from the GGC germline codon(see Fig. 2,top), it frequently results from N additions, because theGGC codon is observed for only a low number of canonical se-quences, about 15% of all canonical junctions identified (Figs. 2and 3). In DN32H6 TCRb-chain Tg mice, the frequency of out-of-frame Va14-Ja281 rearrangement among postselection thymo-cytes was clearly reduced; 17% (6 of 35) as opposed to 58% (18of 31) in the preselection population. These observations indicatethat in the DN32H6 TCRb Tg thymus, the enrichment in thecanonical VGDR joint, which occurs at the immature to maturethymocyte transition, is associated with a reduction of the fre-quency of out-of-frame sequences.

The impact of the DN32H6 TCRb-chain transgene on thefrequency of the canonical Va14-Ja281 a-chain is specific

To assess the specificity of the effect we observed, we used de-veloping DN32H6 TCRb Tg thymocytes to analyze the frequencyof a TCR a-chain junctional domain that was unrelated to theNK1.11 T cell canonicala-chain. To do so, we examined thefrequency of the CDR3 segment of thea-chain (Va2-Ja4) of theD10.G4.1 (D10) TCR- which also uses the Vb8.2 segment andrecognizes a conalbumin peptide in the context of I-Ak (32, 33).The CDR3 of Va2-Ja4 rearrangeda-chains was previously shownto be highly diverse among preselection thymocytes in D10 TCRb Tg mice (13). In contrast with the analysis of the NK1.11 T cellcanonical CDR3a (Fig. 2), the parental D10 TCR CDR3a was notdetected among Va2-Ja4-rearranged sequences derived from thepostselection DN32H6 TCRb Tg thymocyte population (Fig. 4).The CDR3 sequence of Va2-Ja4-rearranged chains is very diversein both pre- and postselection thymocytes, virtually as diverse asamong TCRlow D10 TCR b Tg thymocytes analyzed previously(13). In addition, the frequency of both in-frame and out-of-framesequences was virtually unchanged in the postselection population(about 1/3 and 2/3, respectively, in both thymocyte populations). Thisresult indicates that expression of the DN32H6 TCRb-chain does notpromote the selection of a NK1.11 T cell-unrelated Vb8.21 TCRa-chain (the CDR3a of the D10 TCR), indicating that its effect on theselection of the NK1.11 T cell invarianta-chain is specific.

FIGURE 1. Intrathymic ab T cell development occurs normally inDN32H6 TCRb Tg mice. Flow cytometric analysis of thymic (top) andsplenic (bottom) single-cell suspensions from a 6- to 8-wk-old DN32H6TCR b-chain Tg mouse and a transgene-negative littermate. Cell suspen-sions were triple stained with anti-CD4, anti-CD8, and anti-Vb8.1-2 Abs.Vb8.1-2 expression is shown as a histogram (x-axis, log fluorescence in-tensity;y-axis, cell number), the splenocyte CD4/CD8 coreceptor distribu-tion is represented as a dot plot without (left) and with (right) electronicgating on Vb8.1-21 peripheral T cells. Lymph node cells analysis revealeda similar phenotype (not shown). Quadrant statistics are indicated. In thisparticular analysis, the thymic cellularity was 137.23 106 for the DN32H6b Tg mouse and 129.93 106 for the control littermate.

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The presence of the canonical Va14-Ja281 a-chain junction inpostselection DN32H6b Tg thymocytes is entirely dependent onthe expression of the positively selecting ligand CD1.1

Because positive selection of Va14-Ja281-expressing NK1.11 abT cells is directed by recognition of the nonclassical MHC class Imolecule CD1.1 whose expression on CD41CD81 cortical thy-

mocytes is TAP independent andb2m dependent (34–37), we an-alyzed the Va14-Ja281 a-chain junction of thymocytes fromDN32H6 TCR b Tg mice lacking a functionalb2m gene (27).Sequencing results from two mice (Fig. 5) indicate that in theabsence ofb2m, there is no enrichment in the canonical VGDRjoint in postselection thymocytes carrying the Va14-Ja281

FIGURE 2. Enrichment in the ca-nonical VGDR junction and reducedfrequency of out-of-frame sequencesupon positive selection of DN32H6TCR b Tg thymocytes carrying theVa14-Ja281 rearrangement. Nucleo-tide sequences from genomic DNA andamino acid translations of the Va14-Ja281 CDR3a region from preselection(TCRlow) and postselection (TCRint/

high) CD41 DN32H6 TCRb Tg thymo-cytes are represented. Distinct nucleo-tide sequences encoding identicalamino acid sequences are clustered to-gether. The germline sequences encod-ing the Va14 and Ja281 segments areshown on thetop of the figure. Nucle-otides resulting from trimming and/oradditions are underlined. The sequencefrequencies are indicated on therightand are summarized in bold charactersfor each mouse. The number of differentsequences among the out-of-frame se-quences is indicated. Data derived fromtwo 6- to 8-wk-old DN32H6 TCRb-chain Tg mice are shown.

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a-chain rearrangement (0/31), whereas there were 2/37 (;5.5%)of these sequences in preselection thymocytes. To obtain moredirect evidence of the role of CD1.1, we subsequently repeated thisanalysis using DN32H6 TCRb Tg mice on a CD1.1 deficientbackground when CD1.12/2 mice became available (28). TheVb8low thymocyte population analysis (Fig. 6) revealed a se-quence profile basically indistinguishable from those obtainedfrom immature DN32H6 TCRb Tg and DN32H6 TCRb Tgb2m

2/2 thymocytes (see Figs. 2 and 5): a low frequency of thecanonical junction (3/295 10.3%) and a majority of out-of-framesequences (18/295 62%). The postselection thymocyte popula-tion analysis confirmed that in the absence CD1.1 molecules, noNK1.11 T cell a-chain canonical junction can be detected in thy-mocytes that are beyond the stage of positive selection (0/28). Thisindicates that the high frequency of the canonical VGDR junctiondetected among postselection DN32H6 TCRb Tg thymocytes

(Fig. 2) is entirelyb2m and CD1.1 dependent and therefore effec-tively results from intrathymic positive selection on CD1.1.

Thymic, but not peripheral, increase in NK1.11 ab T cells inDN32H6 TCRb Tg mice

Besides restrictions in the Va-Ja junction (38, 39), several studieson TCR b-chain Tg mice reported skewing toward Ag-specificmature T cells (40–42). In addition, mice carrying ab-chain trans-gene derived from autoreactive T cells showed accelerated onset ofdisease in the case of both diabetes (43) and collagen-inducedarthritis (44). In the case of the DN32H6 TCRb Tg mice, we triedto determine whether the sequence analysis of the Va14-Ja281junction correlates with a detectable phenotype. We first looked atthe frequency of NK1.11 T cells in thymus and spleen. A simpleNK1.1 staining repeatedly revealed a slight increase in thymic

FIGURE 3. Sequence analysis ofthe junction of the Va14-Ja281 rear-rangement among TCRlow and TCRint

thymocytes from a non-Tg mouse. Nu-cleotide sequences from genomic DNAand amino acid translations of theVa14-Ja281 CDR3a region fromCD41Vb8.2low and CD41Vb8.2int

thymocytes from a transgene-negativelittermate are shown. The F23.2 mAbwas used to analyze specificallyVb8.21 thymocytes. Sequences arerepresented as described in Fig. 2.

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NK1.11 cells in DN32H6 TCRb Tg mice compared with trans-gene-negative littermates. Such an increase was not detectedamong splenocytes (Fig. 7A,left panels). To obtain better resolu-tion, we repeated the NK1.1 staining after enrichment of the cellsuspensions in NK1.11 T cells; thymocytes were depleted ofCD81 and immature (HSA1) cells, and splenocytes were depletedof CD81 T cells and B cells. The enriched populations confirmedan increased frequency of NK1.11 cells in the thymic, but not thesplenic, compartment (Fig. 7A,right panels). A NK1.1/Cb doublestaining indicated more precisely that DN32H6 TCRb Tg micehave an increased frequency of NK1.11 ab T cells in the thymus(Fig. 7B). A similar phenotypic analysis of both unmanipulatedand NK T cell-enriched cell suspensions from bone marrow ofDN32H6 TCRb Tg mice and control littermates revealed no sig-nificant differences in the frequency of NK1.11 ab T cells (datanot shown). Because the thymic cellularity is virtually unmodified

in DN32H6 TCRb Tg mice (see Figs. 1 and 7), this increasedfrequency corresponds to a significant increase in the absolutenumber of thymic NK1.11 ab T cells. In addition, the fraction ofVa(2-3.2-8-11)1 cells was reduced among Vb8.2int thymocytes inthe presence of the DN32H6 TCRb-chain transgene (data notshown). We then searched for a functional reflection of this ob-servation. It is well documented that NK1.11 ab T cells are ableto produce various types of cytokines. Anti-CD3 stimulation causesrapid and massive secretion of IL-4, but also significant amounts ofIFN-g and TNF-b (45–48). This potential is further extended by theability to elicit IFN-g secretion upon stimulation through NK1.1 (49)or the IL-12R (50, 51). To determine whether DN32H6 TCRb Tglymphocytes have a modified capacity to produce IL-4, thymocytesand splenocytes from transgenic and control mice were stimulatedovernight using plate-bound anti-CD3e (YCD3-1) mAb, and IL-4 se-cretion was measured by the IL-4-specific CT.4S cell-based bioassay.

FIGURE 4. The DN32H6 TCRb-chain does notpromote positive selection of thymocytes carryingthe NK1.11 T cell-unrelated D10 TCR CDR3a.The sequence of the Va2-Ja4 rearrangement that isused by thea-chain of the Vb8.21 D10 TCR wasanalyzed in pre- and postselection DN32H6 TCRbTg thymocyte populations. The sequencing resultsare organized as described in Fig. 2. The parentalD10 TCR CDR3a junction is indicated (top). Notethat in both immature and mature thymocyte pop-ulations, the frequency of in-frame and out-of-frame sequences remains stable, and that in-framesequences are equally diverse. Analysis of a secondDN32H6 TCRb Tg mouse produced similar results(not shown).

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FIGURE 5. The high frequency of the canonicalVGDRG joint among postselection DN32H6 TCRbTg thymocytes is entirelyb2m dependent. DN32H6TCR b Tg b2m-deficient mice were generated bybreeding and were analyzed at 6–8 wk of age. Se-quencing data derived from two mice are repre-sented as described in Fig. 2.

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Under these conditions, no obvious difference was observed amongsplenocytes, whereas transgenic thymocytes revealed a slight increasein IL-4 production (Fig. 8A). Stimulation of NK1.11 T cell-enrichedthymic and splenic populations confirmed this result (Fig. 8B). Theseobservations are consistent with the sequence analysis of genomicDNA (Fig. 2) and indicate that in the presence of the NK1.11 Tcell-derived DN32H6 TCRb transgene, the number of NK1.11 ab Tcells is augmented in the thymus, and that these additional cells are

functional, at least in terms of their ability to release IL-4 upon anti-CD3 stimulation. The data also suggest that an increased frequency ofNK1.11 ab T cells does not occur or, alternatively, is not sustainedin the periphery of DN32H6 TCRb-chain Tg mice.

DiscussionTCR b-chain Tg mice have normal T cell development; their thy-mocytes display a normal TCR expression level, and selection of

FIGURE 6. The high frequency of the canoni-cal VGDRG joint among postselection DN32H6TCRb Tg thymocytes is entirely dependent on theexpression of CD1.1. CD1.12/2 mice were used toderive DN32H6 TCRb Tg CD1.1-deficient mice.Genomic DNA-derived sequences from two miceare represented as described in Fig. 2.

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the TCR a-chain occurs at the appropriate stage. In such mice,analysis of genomic DNA derived from developing thymocytesallows the examination of both the immature and maturea-chainrepertoires while avoiding issues such as the effects of environ-mental Ags on the composition of the mature TCR repertoire. In-deed, the sequence analysis of the companiona-chain from TCRb-chain Tg thymocytes has been successfully used to demonstratethe central role of self-peptide:self-MHC complex recognition inintrathymic positive selection ofab T cells (13, 42). These andsubsequent studies revealed that the expression of rearranged TCRb-chain has a crucial influence on the specificity of the matureTCR repertoire of CD41 ab T cells. Here, we report that the

transgenic expression of the rearranged TCRb-chain derived fromVa14-Ja281-expressing NK1.11 ab T cells promotes intrathymicpositive selection of T cells carrying the NK1.11 T cell canonicala-chain (Va14-Ja281); in the transgenic animals, an increased fre-quency of the canonical VGDR junction is observed among Va14-Ja281-rearranged sequences, and an augmented fraction ofNK1.11 ab T cells is detected in the thymus. Thus, the criticalinfluence of theb-chain transgene on the specificity of matureabT lymphocytes, best analyzed to date for classical CD41 ab Tcells, does apply to a major subset of nonconventional T lympho-cytes, the Va14-Ja281-expressing NK1.11 ab T cells. This im-plies that in normal circumstances, productive rearrangements atthe TCRb-chain locus have a major influence on the structure andthe specificity of the Ag receptor of developing classical and non-classicalab T cells. This influence operates through the process ofintrathymic positive selection on self-determinants, in this partic-ular case, CD1.1 molecules, most likely complexed to glycolipidicself-Ags (52).

In the course of the analysis of Va14-Ja281-rearranged se-quences, we observed that among the preselection repertoire, aVADR junction was detected twice (Fig. 2). Because a VVDR

FIGURE 7. Increased frequency of thymic, but not splenic, NK1.11 abT cells in DN32H6 TCRb-chain Tg mice.A, Expression of the NK1.1receptor by total and NK1.11 T cell-enriched thymocyte and splenocytesuspensions from 8-wk-old DN32H6 TCRb Tg and littermate controlmice. NK1.1 is detected using the PK136 mAb.x-axis, log fluorescenceintensity; y-axis, cell number. Histogram statistics are indicated. For en-richment, thymocytes were negatively selected using magnetic beads andanti-HSA (20C9) and anti-CD8a (53-6.72) mAbs, and ammonium chlo-ride-treated splenocytes were negatively selected using anti-B220 (14.8),anti-I-Ab (Y3JP), and anti-CD8a (53-6.72) mAbs.B, NK1.1/ab TCR dis-tribution of total and NK1.11 T cell-enriched thymic cell suspensions fromDN32H6 TCRb Tg and transgene-negative littermates.ab T cells weredetected using the anti-Cb H57 mAb. The frequency of NK1.11 ab T cellsis indicated after electronic gating on NK1.11/ab TCRint thymocytes. Inthis experiment the thymic cellularity was: DN32H6 TCRb Tg, 1653106; and control littermate, 171.23 106. Results are representative of threeexperiments.

FIGURE 8. Increased IL-4 production by freshly isolated DN32H6TCR b Tg thymocytes exposed to anti-CD3e mAb. A, Total thymocytesand splenocytes from DN32H6 TCRb Tg mice (b Tg) and transgene-negative littermate (B6) were exposed overnight to polystyrene-immobi-lized anti-CD3e mAb (YCD3- 1). Detection of IL-4 production was as-sessed by incubating supernatants with the IL-4-dependent murine CT.4Scells.B, Anti-CD3e stimulation of NK1.11 T cell-enriched thymocyte andsplenocyte suspensions from DN32H6 TCRb Tg and wild-type litter-mates. Cell suspensions were enriched in NK1.11 T cells as described inFig. 7. IL-4 production was detected as described inA.

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junction has been reported for hybridomas derived from matureCD44high NK1.11 thymocytes (30) and given that V and A areboth small and hydrophobic amino acids, the usage of the VADRjunction by NK1.11 ab T cells is theoretically possible. In linewith this possibility, the VADR junction is indeed observed amongCD41 Vb8.21 TCRint thymocytes from a nontransgenic controlmouse (Fig. 3). However, the confirmation of the usage of aVADR junction by the Va14-Ja281 a-chain of NK1.11 T cellTCR would require a functional analysis, for instance usingtransfectants.

With respect to NK1.11 ab T cell development, both a com-mitted precursor model and a mainstream precursor model havebeen considered (reviewed in Ref. 26). The first model postulatesthe existence of a precursor committed to become an NK1.11 abT cell and implies that the canonical Va14-Ja281 rearrangement isgenetically programmed. Important developmental differences ap-pear to exist between NK1.11 ab T cells andab T cells. Forinstance, the expression of pTa has been shown to be absolutelyrequired for NK1.11 ab T cell development (53). In addition, theSrc protein kinase Fyn is mandatory for the CD1-dependent de-velopment of NK1.11 ab T cells, but not for the development ofconventional T cells (54, 55). However, the occurrence of randomrearrangements at the seconda locus of NK1.11 ab T cells (30,56), the diversity of nucleotide sequences encoding the canonicala-chain amino acid sequence (30) and the lack of evidence fordirected Va14-Ja281 rearrangements among thymic NK1.11 abT cells (57) argue against such a committed precursor model. Thissuggests, rather, that the Va14-Ja281 rearrangement is stochasticin nature and that positive selection on CD1.1 rescues the raremainstreamab thymocytes carrying the canonicala-chain and apermissive Vb segment (mainstream precursor model) (26). Therepertoire analysis we performed here using genomic DNA fromimmature DN32H6 TCRb Tg and wild-type thymocytes indicatesthat despite the germline encoded sequence of the canonical joint,about 50–65% of the Va14-Ja281 rearrangements are not pro-ductive, and about 25–40% produce noncanonical joints. Suchproportions are drastically reduced in the postselection thymocytepopulation, which, in the presence of CD1.1, is dominated by thecanonical VGDR joint selected at the amino acid level. Our ob-servations are best explained by a strong ligand-mediated selectionand directly support the mainstream precursor model of NK1.11

ab T cell development. Sequences derived from immature thymo-cytes also suggest that under the experimental conditions usedhere, the probability of observing the canonical VGDR junctionamong the rearranged Va14-Ja281 a-chains ranges from 1/15 to1/19 in most mice analyzed (five of seven; see Figs. 2, 3, 5, and 6).

Using immunostaining and functional assay, we observed anincreased frequency of NK1.11 ab T cells in the thymus, but,strikingly, not in the spleen and bone marrow of DN32H6 TCRb-chain Tg mice. This increase of about 2-fold appears moderateand could be regarded as the reflection of a rather inefficient pos-itive selection of Va14-Ja281/DN32H6 TCRb Tg thymocytes.However, in C-a-deficient mice expressing a canonical Va14-Ja281 chain transgene, that is, in mice in which any permissiveb-chain rearrangement allows the assembly of a Va14-Ja281-ex-pressing NK1.11 T cell TCR, the increase in NK1.11 ab T cellsobserved was about 7-fold (58). These increases are thus charac-terized by a similar order of magnitude. This may suggest thatlimiting factors control positive selection of Va14-Ja281-express-ing NK1.11 ab T cells. Possibly, the abundance of appropriateCD1.1-presented glycolipidic self-Ags may have a limiting effect.The reason for the thymus-restricted increase in NK1.11 ab Tcells in DN32H6 TCRb-chain Tg mice remains unclear. One pos-sibility is that a mechanism exists that tightly regulates the number

of NK1.11 ab T cells in vivo. Thus, although expression of theDN32H6 TCRb transgene seems to promote positive selection ofNK1.11 ab T cells, their number could be rapidly reduced tonormal before or concomitantly with their departure from the thy-mus. Such a homeostatic hypothesis is supported by the observa-tion that in MHC class II-deficient (I-Abb2/2) mice that lack mostconventional CD41 ab T cells, the absolute number of CD41

NK1.11 T cells is virtually unchanged (59). However, this possi-bility appears in conflict with the increased number of NK1.11 abT cells in the periphery of Va14-Ja281 Tg mice (58). An alter-native possibility can be considered in light of the documentedtissue-specific recognition of CD1.1. It is known that CD1.1-spe-cific T hybridomas respond differently to CD1 presentation by thy-mic and splenic CD1.11 cells and that further differences are seenwhen analyzing distinct CD1.11 cell types derived from thespleen. Indeed, a careful functional analysis of 12 hybridomas re-vealed that no spleen-specific hybridoma was found among thy-mus-derived hybridomas, and that no thymus-specific hybridomawas identified among spleen-derived hybridomas (29). This obser-vation supports a model in which tissue-resident CD1.1-specificNK1.11 ab T cells recognize CD1.1 locally, that is CD1.1 asso-ciated with tissue-specific coligands that may only partially over-lap. According to this model, DN32H6 TCRb Tg NK1.11 T cellscould recognize a thymus-specific ligand in the context of CD1.1.This would lead to their maintenance in the thymic microenviron-ment, but not in the periphery. Such a scenario appears consistentwith the idea that the thymus, but not the spleen, is highly enrichedin CD1.1-dependent NK1.11 ab T cells (60). In this regard, it isvery interesting that the DN32H6 TCRb-chain was cloned from ahybridoma generated using CD44high mature thymocytes (30).

In conclusion, the molecular, phenotypic and functional resultswe report here show that in vivo expression of a Va14-Ja281-expressing NK1.11 thymocyte-derived TCRb-chain transgenepromotes, in a CD1.1-dependent manner, positive selection of thy-mocytes carrying the NK1.11 T cell Va14-Ja281 canonical TCRa-chain. The increased frequency of thymicab T cells was notobserved in the periphery, possibly reflecting a requirement for athymus-specific Ag for their maintenance. These observations in-dicate that the influence of rearrangedb-chains on the TCR spec-ificity of developing thymocytes applies to a major subset of non-conventionalab T cells, the CD1.1-dependent NK1.11 ab T cells.Thus, the strong impact of productiveb-chain rearrangements onthe matureab TCR repertoire, which operates through positiveselection on self-determinants, appears to be a general principle inthe intrathymic development ofab T lymphocytes.

AcknowledgmentsWe thank L. Van Kaer (Howard Hughes Medical Institute, Vanderbilt Uni-versity School of Medicine, Nashville, TN) for providing us with theCD1.1-deficient mice, R. Medzhitov (Howard Hughes Medical Institute,Yale University) for help with the initiation of the study, D. B. Sant’Angelo(Memorial Sloan-Kettering Cancer Center, New York, NY) for advice, andCharles Annicelli for animal care.

References1. von Boehmer, H. 1994. Positive selection of lymphocytes.Cell 76:219.2. Nossal, G. J. 1994. Negative selection of lymphocytes.Cell 76:229.3. Robey, E., and B. J. Fowlkes. 1994. Selective events in T cell development.

Annu. Rev. Immunol. 12:675.4. Janeway, C. A., Jr. 1994. Thymic selection: two pathways to life and two to

death.Immunity 1:3.5. Bevan, M. J. 1997. In thymic selection, peptide diversity gives and takes away.

Immunity 7:175.6. Viret, C., and C. A. Janeway, Jr. 1999. MHC and T cell development.Rev.

Immunogenet. 1:91.7. Goldrath, A. W., and M. J. Bevan. 1999. Selecting and maintaining a diverse

T-cell repertoire.Nature 402:255.

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8. Janeway, C. A., Jr. 1999. T-cell development: a role for self-peptides in positiveselection.Curr. Biol. 9:342.

9. Nikolic-Zugic, J., and M. J. Bevan. 1990. Role of self-peptides in positivelyselecting the T cell receptor repertoire.Nature 344:65.

10. Sha, W. C., C. A. Nelson, R. D. Newberry, J. K. Pullen, L. R. Pease, J. H. Russell,and D. Y. Loh. 1990. Positive selection of transgenic receptor-bearing thymo-cytes by Kb antigen is altered by Kb mutations that involve peptide binding.Proc.Natl. Acad. Sci. USA 87:6186.

11. Hogquist, K. A., M. A. Gavin, and M. J. Bevan. 1993. Positive selection of CD81

T cells induced by major histocompatibility complex binding peptides in fetalthymic organ culture.J. Exp. Med. 177:1469.

12. Asthon-Rickardt, P. G., L. Van Kaer, T. N. M. Schumacher, H. L. Ploegh, andS. Tonegawa. 1993. Peptide contributes to the specificity of positive selection ofCD81 T cells in the thymus.Cell 73:1041.

13. Sant’Angelo, D. B., G. Waterbury, B. E. Cohen, W. D. Martin, L. Van Kaer,A. C. Hayday, and C. A. Janeway Jr. 1997. The imprint of intrathymic self-peptides on the mature T cell receptor repertoire.Immunity 7:517.

14. Berg, L. J., B. Fazekas de St Groth, A. M. Pullen, and M. M. Davis. 1989.Phenotypic differences betweenab versusb T-cell receptor transgenic mice un-dergoing negative selection.Nature 340:559.

15. Baldwin, K. K., B. P. Trenchack, J. D. Altman, and M. M. Davis. 1999. Negativeselection of T cells occurs throughout thymic development.J. Immunol. 163:689.

16. Martin, W. D., G. G. Hicks, S. K. Mendiratta, H. I. Leva, E. H. Ruley, andL. Van Kaer. 1996. H-2M mutant mice are defective in the peptide loading ofclass II molecules, antigen presentation, and T cell repertoire selection.Cell 84:543.

17. Fung-Leung, W. P., C. D. Surh, M. Liljedahl, J. Pang, D. Leturcq, P. A. Peterson,S. R. Webb, and L. Karlsson. 1996. Antigen presentation and T cell developmentin H2-M deficient mice.Science 271:1278.

18. Miyazaki, T., P. Wolf, S. Tourne, C. Waltzinger, A. Dierich, N. Barois,H. Ploegh, C. Benoist, and D. Mathis. 1996. Mice lacking H2-M complexes,enigmatic elements of the MHC class Ii peptide loading pathway.Cell 84:531.

19. Burns, R. P., Jr., K. Natarajan, N. J. LoCascio, D. P. O’Brien, J. A. Kobori,N. Shastri, and R. K. Barth. 1998. Molecular analysis of skewed Tcra-V gene usein T-cell receptorb-chain transgenic mice.Immunogenetics 47:107.

20. Fukui, Y., O. Hashimoto, A. Inayoshi, T. Gyotoku, T. Sano, T. Koga,T. Gushima, and T. Sasazuki. 1998. Highly restricted T cell repertoire shaped bya single major histocompatibility complex-peptide ligand in the presence of asingle rearranged T cell receptorb chain.J. Exp. Med. 188:897.

21. Mallick, C. A., E. C. Dudley, J. L. Viney, M. J. Owen, and A. C. Hayday. 1993.Rearrangement and diversity of T cell receptorb chain genes in thymocytes: acritical role for theb chain in development.Cell 3:513.

22. Dudley, E. C., H. T. Petrie, L. M. Shah, M. J. Owen, and A. C. Hayday. 1994.T cell receptorb chain gene rearrangement and selection during thymocyte de-velopment in adult mice.Immunity 2:83.

23. Hoffman, E. S., L. Passoni, T. Crompton, T. M. Leu, D. G. Schatz, A. Koff,M. J. Owen, and A. C. Hayday. 1996. Productive T-cell receptorb-chain generearrangement: coincident regulation of cell cycle and clonality during develop-ment in vivo.Genes Dev. 10:948.

24. MacDonald, H. R. 1995. T cell receptora/b cells: new clues to their origin,specificity and function.J. Exp. Med. 182:633.

25. Vicari, A. P., and A. Zlotnik. 1996. Mouse NK1.11 T cells: a new family of Tcells. Immunol. Today 17:71.

26. Bendelac, A., M. N. Rivera, S. H. Park, and J. H. Roark. 1997. Mouse CD1-specific NK1 T cells: development, specificity, and function.Annu. Rev. Immu-nol. 15:535.

27. Zijlstra, M., M. Bix, N. E. Simister, J. M. Loring, D. H. Raulet, and R. Jaenisch.1990.b2-microglobulin deficient mice lack CD42CD81 cytolytic T cells.Nature344:742.

28. Mendiratta, S. K., W. D. Martin, S. Hong, A. Boesteanu, S. Joyce, andL. Van Kaer. 1997. CD1d1 mutant mice are deficient in natural T cells thatpromptly produce IL-4.Immunity 6:469.

29. Park, S. H., J. H. Roark, and A. Bendelac. 1998. Tissue-specific recognition ofmouse CD1 molecules.J. Immunol. 160:3128.

30. Lantz, O., and A. Bendelac. 1994. An invariant T cell receptora chain is used bya unique subset of major histocompatibility complex class I-specific CD41 andCD4282 T cells in mice and humans.J. Exp. Med. 180:1097.

31. Groth, B., J. L. Jorgensen, and M. M. Davis. 1993. Transfer of putative comple-mentarity-determining region loops of T cell receptor V domains confers toxinreactivity but not peptide/MHC specificity.J. Immunol. 150:2281.

32. Hong, S. C., A. Chelouche, R. H. Lin, D. Shaywitz, N. S. Braunstein,L. Glimcher, and C. A. Janeway, Jr. 1992. An MHC interaction site maps to theamino-terminal half of the T cell receptora chain variable domain.Cell 69:999.

33. Sant’Angelo, D. B., G. Waterbury, P. Preston-Hurlburt, T. S. Yoon,R. Medzhitov, S. C. Hong, and C. A. Janeway, Jr. 1996. The specificity andorientation of a TCR to its peptide-MHC class II ligands.Immunity 4:367.

34. Bix, M., M. Coles, and D. H. Raulet. 1993. Positive selection of Vb81 CD4282

thymocytes by class I molecules expressed by hematopoietic cells.J. Exp. Med.178:901.

35. Ohteki, T., and H. R. MacDonald. 1994. Major histocompatibility complex classI related molecules control the development of CD4182 subset of natural killer1.11 T cell receptor-a/b1 cells in the liver of mice.J. Exp. Med. 180:699.

36. Bendelac, A. 1995. Positive selection of mouse NK11 T cells by CD1-expressingcortical thymocytes.J. Exp. Med. 182:2091.

37. Coles, M. C., and D. H. Raulet. 2000. NK1.11 T cells in the liver arise in thethymus and are selected by interactions with class I molecules on CD41CD81

cells.J. Immunol. 164:2412.38. Merkenschlager, M., C. Benoist, and D. Mathis. 1994. MHC control of the naive

TCR a-chain repertoire.J. Immunol. 153:3005.39. Hsu, B. L., D. L. Donermeyer, and P. M. Allen. 1996. TCR recognition of the

Hb(64–76)/I-Ek determinant: single conservative amino acid changes in thecomplementarity-determining region 3 dramatically alter antigen fine specificity.J. Immunol. 157:2291.

40. Dillon, S. R, C. Jameson, and P. J. Fink. 1994. Vb51 T cell receptors skewtoward OVA1H-2Kb recognition.J. Immunol. 152:1790.

41. Hsu, B. L., B. D. Evavold, and P. M. Allen. 1995. Modulation of T cell devel-opment by an endogenous altered peptide ligand.J. Exp. Med. 181:805.

42. Sant’Angelo, D. B., B. Lucas, G. Waterbury, B. E. Cohen, T. Brabb,J. Goverman, R. N. Germain, and C. A. Janeway, Jr. 1998. A molecular map ofT cell development.Immunity 9:179.

43. Verdaguer, J., J. W. Yoon, B. Anderson, N. Averill, T. Utsugi, B. J. Park, andP. Santamaria. 1996. Acceleration of spontaneous diabetes in TCR-b-transgenicnonobese diabetic mice byb-cell cytotoxic CD81 T cells expressing identicalendogenous TCR-a chains.J. Immunol. 157:4726.

44. Mori, L., H. Loetscher, K. Kakimoto, H. Bluethmann, and M. Steinmetz. 1992.Expression of a transgenic T cell receptorb chain enhances collagen-inducedarthritis.J. Exp. Med. 176:381.

45. Bendelac, A., and R. H. Schwartz. 1991. CD41 and CD81 T cells acquire spe-cific lymphokine secretion potentials during thymic maturation.Nature 353:68.

46. Hayakawa, K, B. T. Lin, and R. R. Hardy. 1992. Murine thymic CD41 T cellsubsets: a subset (Thy0) that secretes diverse cytokines and overexpresses theVb8 T cell receptor gene family.J. Exp. Med. 176:269.

47. Yoshimoto, T., and W. E. Paul. 1994. CD4pos, NK1.1pos T cells promptly pro-duce interleukin 4 in response to in vivo challenge with anti-CD3.J. Exp. Med.179:1285.

48. Chen, H., and W. E Paul. 1997. Cultured NK1.11 CD41 T cells produce largeamounts of IL-4 and IFN-g upon activation by anti-CD3 or CD1.J. Immunol.159:2240.

49. Arase, H., N. Arase, and T. Saito. 1996. Interferong production by natural killer(NK) cells and NK1.11 T cells upon NKR-P1 cross-linking.J. Exp. Med. 183:2391.

50. Cui, J., T. Shin, T. Kawano, H. Sato, E. Kondo, I. Toura, Y. Kaneko, H. Koseki,M. Kanno, and M. Taniguchi. 1997. Requirement for Va14 NKT cells in IL-12-mediated rejection of tumors.Science 278:1623.

51. Kawamura, T., K. Takeda, S. K. Mendiratta, S. K. Kawamura, L. Van Kaer,H. Yagita, T. Abo, and K. Okumura. 1998. Critical role of NK11 T cells inIL-12-induced immune responses in vivo.J. Immunol. 160:16.

52. Porcelli, S. A., and R. L. Modlin. 1999. The CD1 system: antigen-presentingmolecules for T cell recognition of lipids and glycolipids.Annu. Rev. Immunol.17:297.

53. Eberl, G., H. J. Fehling, H. von Boehmer, and H. R. MacDonald. 1999. Absoluterequirement for the pre-T cell receptora chain during NK1.11 TCR a-b celldevelopment.Eur. J. Immunol. 29:1966.

54. Eberl, G., B. Lowin-Kropf, and H. R. MacDonald. 1999. NK T cell developmentis selectively impaired in Fyn-deficient mice.J. Immunol. 163:4091.

55. Gadue, P., N. Morton, and P. L. Stein. 1999. The Src family tyrosine kinase Fynregulates natural killer T cell development.J. Exp. Med. 190:1189.

56. Barbo, J. V., J. E. McCormack, J. W. Moorhead, and R. L. Fairchild. 1995.Reconstitution of TCRa-chain expression in deletion mutants restores dinitro-phenyl-specific/class I MHC-restricted suppressor molecule production.J. Im-munol. 154:1551.

57. Shimamura, M., T. Ohteki, U. Beutner, and H. R. MacDonald. 1997. Lack ofdirected Va14-Ja281 rearrangements in NK11 T cells. Eur. J. Immunol. 27:1576.

58. Bendelac, A., R. D. Hunziker, and O. Lantz. 1996. Increased interleukin-4 andimmunoglobulin E production in transgenic mice overexpressing NK1 T cells.J. Exp. Med. 184:1285.

59. Cardell, S., S. Tangri, S. Chan, M. Kronenberg, C. Benoist, and D. Mathis. 1995.CD1-restricted CD41 T cells in major histocompatibility complex class II-defi-cient mice.J. Exp. Med. 182:993.

60. Eberl, G., R. Lees, S.T. Smiley, M. Taniguchi, M. J. Grusby, andH. R. MacDonald. 1999. Tissue-specific segregation of CD1d-dependent andCD1d-independent NK T cells.J. Immunol. 162:6410.

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