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A NK1.1⁺ Thymocyte-Derived TCR ß-Chain Transgene Promotes Positive Selection of Thymic NK1.1⁺ ß T Cells¹

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

^* Section of Immunobiology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520; Institut National de la Santé et de la Recherche Médicale, Unité 25, Hopital Necker, Paris, France; and Department of Molecular Biology, Princeton University, Princeton, NJ 08540

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
As a consequence of the peptide specificity of intrathymic positive selection, mice transgenic for a rearranged TCR ß-chain derived from conventional ß T lymphocytes frequently carry mature T cells with significant skewing in the repertoire of the companion -chain. To assess the generality of such an influence, we generated transgenic (Tg) mice expressing a ß-chain derived from nonclassical, NK1.1⁺ ß T cells, the thymus-derived, CD1.1-specific DN32H6 T cell hybridoma. Results of the sequence analysis of genomic DNA from developing DN32H6 ß Tg thymocytes revealed that the frequency of the parental -chain sequence, in this instance the V14-J281 canonical -chain, is specifically and in a CD1.1-dependent manner, increased in the postselection thymocyte population. In accordance, we found phenotypic and functional evidence for an increased frequency of thymic, but interestingly not peripheral, NK1.1⁺ ß T cells in DN32H6 ß Tg mice, possibly indicating a thymic determinant-dependent maintenance. Thus, in vivo expression of the rearranged TCR ß-chain from a thymus-derived NK1.1⁺ V14⁺ T cell hybridoma promotes positive selection of thymic NK1.1⁺ ß T cells. These observations indicate that the strong influence of productive ß-chain rearrangements on the TCR sequence and specificity of developing thymocytes, which operates through positive selection on self-determinants, applies to both classical and nonclassical ß T cells and therefore represents a general phenomenon in intrathymic ß T lymphocyte development.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The intrathymic development of classical ß T cells relies on the interaction of the TCR with self-peptide:self-MHC complexes expressed on thymic stromal cells. A minimal interaction is required to rescue immature thymocytes from programmed cell death and allow them to complete their maturation (positive selection), whereas stronger interactions result in the induction of apoptosis to maintain tolerance to most self-determinants expressed on bone marrow-derived cells (negative selection) (1, 2, 3, 4). A recent series of in vivo observations made using MHC class II systems (reviewed in Refs. 5, 6, 7, 8) extended pioneering experiments related to CD8⁺ T cell development (9, 10, 11, 12) and firmly established that positive selection of immature ß thymocytes is self oriented. That is, positive selection relies on the recognition of self-peptide:self-MHC complexes expressed on thymic cortical epithelial cells.

One of the direct demonstrations of the crucial role of self-peptide recognition in positive selection of CD4⁺ T cells took advantage of transgenic mice expressing the rearranged D10 TCR ß-chain (13). It is emphasized that in TCR ß-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 ß Tg mice. The CD3 complex expression by CD4^-CD8^- and CD4⁺CD8⁺ thymocytes is comparable to that of wild-type thymocytes, and -chain selection occurs normally at the appropriate stage (13, 14, 15). In the D10 TCR ß-chain Tg mice, analysis of the D10 TCR VJ rearrangement in mature T cells revealed that a limited set of junctional sequences is selected from a very diverse pool of preselection sequences (13). This restriction was further enforced on the H-2 M^-/- background, which displays a highly limited self-peptide complexity. In H-2 M^-/- mice, the MHC class II expression level is intact, but the invariant chain-derived 81–104 (CLIP) peptide is dominant (16, 17, 18). In a distinct TCR ß-chain Tg system, both V and J gene segment usages of the companion -chain were significantly biased in mature T cells (19). The impact of a TCR ß-chain transgene on the -chain repertoire was also observed in mice expressing a single peptide:MHC class II complex unrelated to the parental TCR; both the V usage and the amino acid composition of the CDR3 loop were significantly influenced (20). Finally, mice transgenic for the ß-chain of the MCC88–103:I-E^k complex-specific 5C.C7 ß TCR were analyzed using MCC88–103:I-E^k tetramers, which stain 95% of 5C.C7 ß TCR Tg thymocytes (15). Virtually all tetramer-positive cells from thymus and lymph node expressed the parental V segment (V11) and displayed the characteristic CDR3 loop length restriction observed among other MCC/I-E^k reactive T cells. Amino acid sequences identical with the 5C.C7 TCR -chain were detected in both tetramer^high and tetramer^low cells, but not among V11⁺/tetramer-negative cells. Collectively, these studies suggest that productive TCR ß-chain rearrangements at the CD44^lowCD25⁺ stage of thymocyte development, and therefore the so-called ß selection process (21, 22, 23), critically impact on the TCR -chain repertoire of mature ß T cells through intrathymic positive selection. However, the fact that the vast majority of these studies used rearranged TCR ß-chains derived from conventional CD4⁺ ß T cells raises the question of the generality of this phenomenon.

In this study we have analyzed mice expressing a rearranged TCR ß-chain transgene derived from V14-J281 expressing NK1.1⁺ T cells, an atypical population of CD4⁺ or CD4^-CD8^- ß T cells that require for both their intrathymic development and their activation, interaction of their semicanonical ß TCR with the TAP-independent, ß₂-microglobulin (ß₂m)-dependent, nonclassical MHC class I molecule CD1.1 (reviewed in Refs. 24, 25, 26). We find that transgenic expression of a rearranged ß-chain isolated from a thymus-derived V14-J281-expressing NK1.1⁺ T hybridoma (DN32H6) promotes positive selection of thymocytes carrying the canonical V14-J281 -chain of NK1.1⁺ T cells. Accordingly, a higher frequency of NK1.1⁺ ß T cells was detected in the thymus, but strikingly not in the periphery, of DN32H6 TCR ß-chain Tg mice. The data identify the critical impact of productive ß-chain rearrangement on the subsequent specificity of developing thymocytes as a general phenomenon in ß T cell development.

	Materials and Methods

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Mice

Mice used were 6–8 wk old and were housed in the Yale Immunobiology Mouse Unit (New Haven, CT). C57BL/6 (B6) and ß₂m-deficient mice (ß₂m^-/-) (27) were obtained from The Jackson Laboratory (Bar Harbor, ME). The CD1.1^-/- mice (B6-129 mixed background) were a gift from Dr. L. Van Kaer (Howard Hughes Medical Institute, Nashville, TN) (28). DN32H6 TCR ß-chain Tg mice were generated by microinjecting the ß-chain transgene into (B6 x SJL)F₂ oocytes (Comparative Medicine Transgenic Facility, Yale University). Transgene integration was assessed by PCR using tail genomic DNA. Founder animals were backcrossed four times to C57BL/6 mice in a specific pathogen-free environment. The study was performed using one Tg line, because the three lines obtained were phenotypically similar. Screening of animals was performed by PCR after extraction of tail genomic DNA using the following oligonucleotide primers: Vß8 specific sense, 5'-GCCTTGTATCGATTCCCCTTCTTGTCTGTA-3'; and Jß1.4-specific antisense, 5'-CCATGACCGAAAAATAATCTTTCGTTGGAC-3'.

A new PCR assay was developed for the identification of TCR ß Tg CD1.1-deficient mice. The sequences of the oligonucleotide primers used were: sense, 5'-AAGCGCAGAAGTCGGAGCCG-3', which is specific for the 5' end of the CD1.1-coding sequence (29); and antisense, 5'-CTTCTCTAGGTCTGACTT-3', which is specific for the end of exon 3 of the CD1.1 gene where the neo-resistance gene was inserted (28). A 1.2-kb band was generated for the CD1.1^+/+ and CD1.1^+/- samples, but not for the CD1.1^-/- samples. Conversely, the neo-resistance gene was detected for both CD1.1^-/- and CD1.1^+/- samples, but not for CD1.1^+/+ samples.

TCR ß-chain construct

The DN32H6 TCR ß-chain transgene was constructed using the rearranged V(D)J sequence from the CD4^-CD8^- DN32H6 T cell hybridoma (Vß8.2-Dß1.1-Jß1.4) (30) and a modified version of the TCR ß shuttle vector (31). The construct was tested for expression in vitro by cotransfection with an -chain construct into the ß-negative 58 T cell line (O. Lantz and A. Bendelac, unpublished observations). For microinjection, the transgene was linearized (SalI/PvuI digest), isolated by electrophoresis on 1% agarose 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 inguinal, and mesenteric) were removed, and single-cell suspensions were prepared. Splenic RBC were lysed using Tris-buffered ammonium chloride. Fluorescence-labeled mAbs were used for multicolor staining. Briefly, 0.2 x 10⁶ cells were incubated in microtiter U-bottom plates with a saturating concentration of labeled mAb in 20 µl for 30 min on ice. Cells were washed twice and analyzed immediately. For two-step staining, cells were incubated first with purified mAbs in PBS with 2% FCS/0.1% NaN₃, followed by a F(ab')₂ of goat anti-mouse Ig-FITC conjugate from Sigma (St. Louis, MO). The mAbs used were anti-Vß8.1-2-FITC (clone MR5-2), anti-Cß-PE (H57-597), anti-V2,3.2,8,11-FITC (B20.1, RR3-16, B21.14, RR8-1) from PharMingen (San Diego, CA), anti-CD8-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-A^b), F23.2 (mouse IgG2, anti Vß8.2), 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-CD3), 20C9 (hamster IgG, anti-heat stable Ag (HSA)), and 14.8 (rat IgG2b, anti-CD45RA/B220) mAbs were affinity purified from hybridoma supernatants using standard procedures.

Flow cytometry and cell sorting

For flow cytometry, a FACScan flow cytometer and CellQuest software from Becton Dickinson (Mountain View, CA) were used to collect and analyze the data. Nonviable cells were excluded using forward and side scatter electronic gating or propidium iodide. For cell sorting, freshly isolated thymocytes were triple stained for CD4, CD8, and Vß8. Vß8^low and Vß8^int/high thymocytes were sorted after gating on CD4⁺ cells using a FACStar^Plus station (Becton Dickinson).

Cloning and sequencing of CDR3 regions

Genomic DNA from sorted cells was prepared immediately using a DNA extraction kit (Stratagene, La Jolla, CA) and was resuspended in distilled water overnight. The CDR3 of -chains that use the V14-J281 combination was PCR-amplified (recombinant Taq DNA polymerase, Life Technologies) by a two-step process. The first step used oligonucleotide primers specific for the intronic sequences flanking the V14 and J281 gene segments: V14 Leader-V intron-specific sense, 5'-CTTAGGATTTGGGGGAAAAAT-3'; and post-J281 intron-specific antisense, 5'-TCCGAGGTGCGGCCGCAAAGAAATACTTAAGAAAAATTCTTT-3'. Reaction mixtures were denatured by heating to 94°C for 5 min and then were subjected to 30 cycles of 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, and 72°C for 1.5 min. The final extension was performed at 72°C for 10 min. The second amplification step used 1–5 µl of the primary amplification and oligonucleotide primers specific for the coding sequences of the V14 and J281 gene segments: V14-specific sense, 5'-GATAAGAATTCTAAGCACAGCACGCTGCACA-3'; and J281-specific antisense, 5'-GAAACCGGATCCCCAGGTATGACAATCAGCTGAGTCC-3' (30 cycles as indicated above). Fresh PCR products were cloned into Bluescript II KS phagemid vector (Stratagene) after EcoRI-BamHI double digest (underlined sites) or were cloned directly into the PCR2.1 vector using the TA cloning kit (Invitrogen, San Diego, CA). Transformed DH5 cells (Life Technologies) were plated in the presence of Bluo-gal (Life Technologies) and antibiotic, and recombinant individual colonies were directly subjected to PCR using the M13F(-20) and M13R oligonucleotide primers (25 cycles as indicated above). PCR fragments were purified using silica gel membranes (QIAquick kit, Qiagen, Chatsworth, CA) and directly sequenced with the T7 oligonucleotide primer using the Taq DyeDeoxy Terminator Sequencing kit (Applied Biosystems, Foster City, CA) and an ABI 373A DNA sequencer. Sequence analysis of V2-J4 rearrangements was performed using oligonucleotide primers published previously (13).

Functional assay

For IL-4 production assay, freshly isolated T cell suspensions were prepared and cultured in anti-CD3 (YCD3-1)-coated 24-well plates (Becton Dickinson, Lincoln Park, NJ) overnight at 37°C in Click’s EHAA medium (Irvine Scientific, Santa Ana, CA) supplemented with 5% heat-inactivated FCS (Intergen, Purchase, NY), 5 x 10^-5 M 2-ME (Bio-Rad, Richmond, CA), 2 mM L-glutamine, and 50 µg/ml gentamicin (Life Technologies). IL-4 production was detected using the IL-4-dependent murine CT.4S cells in a proliferation assay. The cells were incubated with supernatants in duplicate wells for 72 h and 1 µCi of [³H]thymidine/well was added to the culture for the last 24 h. The plates were then harvested, and counts per minute were determined using liquid scintillation counting. In some situations cell suspensions were first enriched in NK1.1⁺ T cells by negative selection using magnetic beads (Bio Mag, Advanced Magnetic, Cambridge, MA) and mAbs. Thymocytes were negatively selected using anti-HSA and anti-CD8 mAbs, and ammonium chloride-treated splenocytes were negatively selected using anti-B220, anti-MHC class II, and anti-CD8 mAbs.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Transgenic mice expressing a NK1.1⁺ T cell-derived TCR ß-chain transgene

The majority of NK1.1⁺ ß T cells are thymus-derived T lymphocytes with unusual features. They are CD4⁺CD8^- or CD4^-CD8^-, express an intermediate TCR level, display an activated/memory cell surface phenotype (CD122⁺, CD44^high, CD62L^low), and coexpress receptors of the NK cell lineage, such as NK1.1 and Ly49. Another major characteristic of NK1.1⁺ T cells is the highly restricted diversity of their ß TCR; the ß-chain uses essentially the Vß8, Vß7, and Vß2 segments along with quite variable VDJ junctions, whereas the -chain is invariant and consists of the V14-J281 rearrangement. The V-J junction has an invariant size and, in most cases, contains the valine (V) 92-glycine (G) 93-aspartate (D) 94-arginine (R) sequence (VGDR) (reviewed in Refs. 24, 25, 26). The TCR repertoire analysis of NK1.1⁺ T cells was facilitated by the generation of T cell hybridomas from sorted CD4⁺ or CD4^-CD8^- mature CD44^high thymocytes (30). One such hybridoma (DN32H6) was used to prepare a TCR ß-chain construct (see Materials and Methods). Mice with germline transmission of the DN32H6 TCR ß-chain transgene (Vß8.2-Jß1.4) were derived and backcrossed to C57BL/6 mice. Expression of the transgenic ß-chain was evaluated using three-color staining and flow cytometric analysis of cell suspensions from lymphoid organs. Compared with that in nontransgenic littermates, expression of the DN32H6 TCR ß-chain transgene resulted in an increase in Vß8⁺ cells in the periphery and thymus, but a nearly normal TCR expression level (Fig. 1) as is usually the case for single TCR ß-chain Tg mice. About 95–97% of peripheral ß⁺ T cells were Vß8.2⁺, indicating that efficient allelic exclusion occurs at the TCR ß-chain locus (not shown). The thymic cellularity was repeatedly comparable to that of transgene-negative littermates, and peripheral DN32H6 TCR ß Tg T cells displayed a virtually normal CD4/CD8 coreceptor distribution (Fig. 1, bottom). Thus, intrathymic ß T cell development appears to operate normally in mice expressing the DN32H6 TCR ß-chain transgene.

View larger version (36K): [in this window] [in a new window]   FIGURE 1. Intrathymic ß T cell development occurs normally in DN32H6 TCR ß Tg mice. Flow cytometric analysis of thymic (top) and splenic (bottom) single-cell suspensions from a 6- to 8-wk-old DN32H6 TCR ß-chain Tg mouse and a transgene-negative littermate. Cell suspensions were triple stained with anti-CD4, anti-CD8, and anti-Vß8.1-2 Abs. Vß8.1-2 expression is shown as a histogram (x-axis, log fluorescence intensity; y-axis, cell number), the splenocyte CD4/CD8 coreceptor distribution is represented as a dot plot without (left) and with (right) electronic gating on Vß8.1-2+ peripheral T cells. Lymph node cells analysis revealed a similar phenotype (not shown). Quadrant statistics are indicated. In this particular analysis, the thymic cellularity was 137.2 x 106 for the DN32H6 ß Tg mouse and 129.9 x 106 for the control littermate.

High frequency of the canonical V14-J281 -chain joint among mature thymocytes from NK1.1⁺ T cell-derived TCR ß-chain Tg mice

The selection of the DN32H6 TCR -chain (the canonical V14-J281 chain of NK1.1⁺ ß T cells) was analyzed by sorting out thymocytes that were CD4⁺Vß8^low or CD4⁺Vß8^int/high and sequencing the junctional region of the V14-J281 rearranged sequences (see Materials and Methods). In the preselection (TCR^low) thymocyte population, the V14-J281 junction was diverse; combining results from two DN32H6 ß Tg mice, a total of 31 sequences contained three -chain canonical junctions (VGDR; 9.6%) encoded by three distinct nucleotide sequences (Fig. 2), 18 out-of-frame sequences (58%), and 10 noncanonical, highly diverse junctions (32.2%) without obvious bias in either length or amino acid composition. In sharp contrast with the preselection population, the postselection thymocyte population showed a high frequency of the canonical junction as well as a severely reduced frequency of out-of-frame sequences; up to 80% (82.8%) of the joints were canonical (15/17 + 14/18 = 29/35 for two mice analyzed) and were encoded in four distinct nucleotide sequences. Such a frequency is higher than that we observed in a non-Tg control mouse when we used stringent sorting conditions; the frequency of the canonical junction was about 65% among CD4⁺Vß8.2⁺ TCR^int thymocytes from a transgene-negative littermate (Fig. 3). Despite the fact that the glycine residue of the canonical junction can be generated from the GGC germline codon (see Fig. 2, top), it frequently results from N additions, because the GGC codon is observed for only a low number of canonical sequences, about 15% of all canonical junctions identified (Figs. 2 and 3). In DN32H6 TCR ß-chain Tg mice, the frequency of out-of-frame V14-J281 rearrangement among postselection thymocytes was clearly reduced; 17% (6 of 35) as opposed to 58% (18 of 31) in the preselection population. These observations indicate that in the DN32H6 TCR ß Tg thymus, the enrichment in the canonical VGDR joint, which occurs at the immature to mature thymocyte transition, is associated with a reduction of the frequency of out-of-frame sequences.

View larger version (20K): [in this window] [in a new window]   FIGURE 2. Enrichment in the canonical VGDR junction and reduced frequency of out-of-frame sequences upon positive selection of DN32H6 TCR ß Tg thymocytes carrying the V14-J281 rearrangement. Nucleotide sequences from genomic DNA and amino acid translations of the V14-J281 CDR3 region from preselection (TCRlow) and postselection (TCRint/high) CD4+ DN32H6 TCR ß Tg thymocytes are represented. Distinct nucleotide sequences encoding identical amino acid sequences are clustered together. The germline sequences encoding the V14 and J281 segments are shown on the top of the figure. Nucleotides resulting from trimming and/or additions are underlined. The sequence frequencies are indicated on the right and are summarized in bold characters for each mouse. The number of different sequences among the out-of-frame sequences is indicated. Data derived from two 6- to 8-wk-old DN32H6 TCR ß-chain Tg mice are shown.

View larger version (20K): [in this window] [in a new window]   FIGURE 3. Sequence analysis of the junction of the V14-J281 rearrangement among TCRlow and TCRint thymocytes from a non-Tg mouse. Nucleotide sequences from genomic DNA and amino acid translations of the V14-J281 CDR3 region from CD4+Vß8.2low and CD4+Vß8.2int thymocytes from a transgene-negative littermate are shown. The F23.2 mAb was used to analyze specifically Vß8.2+ thymocytes. Sequences are represented as described in Fig. 2.

The impact of the DN32H6 TCR ß-chain transgene on the frequency of the canonical V14-J281 -chain is specific

To assess the specificity of the effect we observed, we used developing DN32H6 TCR ß Tg thymocytes to analyze the frequency of a TCR -chain junctional domain that was unrelated to the NK1.1⁺ T cell canonical -chain. To do so, we examined the frequency of the CDR3 segment of the -chain (V2-J4) of the D10.G4.1 (D10) TCR- which also uses the Vß8.2 segment and recognizes a conalbumin peptide in the context of I-A^k (32, 33). The CDR3 of V2-J4 rearranged -chains was previously shown to be highly diverse among preselection thymocytes in D10 TCR ß Tg mice (13). In contrast with the analysis of the NK1.1⁺ T cell canonical CDR3 (Fig. 2), the parental D10 TCR CDR3 was not detected among V2-J4-rearranged sequences derived from the postselection DN32H6 TCR ß Tg thymocyte population (Fig. 4). The CDR3 sequence of V2-J4-rearranged chains is very diverse in both pre- and postselection thymocytes, virtually as diverse as among TCR^low D10 TCR ß Tg thymocytes analyzed previously (13). In addition, the frequency of both in-frame and out-of-frame sequences was virtually unchanged in the postselection population (about 1/3 and 2/3, respectively, in both thymocyte populations). This result indicates that expression of the DN32H6 TCR ß-chain does not promote the selection of a NK1.1⁺ T cell-unrelated Vß8.2⁺ TCR -chain (the CDR3 of the D10 TCR), indicating that its effect on the selection of the NK1.1⁺ T cell invariant -chain is specific.

View larger version (20K): [in this window] [in a new window]   FIGURE 4. The DN32H6 TCR ß-chain does not promote positive selection of thymocytes carrying the NK1.1+ T cell-unrelated D10 TCR CDR3. The sequence of the V2-J4 rearrangement that is used by the -chain of the Vß8.2+ D10 TCR was analyzed in pre- and postselection DN32H6 TCR ß Tg thymocyte populations. The sequencing results are organized as described in Fig. 2. The parental D10 TCR CDR3 junction is indicated (top). Note that in both immature and mature thymocyte populations, the frequency of in-frame and out-of-frame sequences remains stable, and that in-frame sequences are equally diverse. Analysis of a second DN32H6 TCR ß Tg mouse produced similar results (not shown).

The presence of the canonical V14-J281 -chain junction in postselection DN32H6 ß Tg thymocytes is entirely dependent on the expression of the positively selecting ligand CD1.1

Because positive selection of V14-J281-expressing NK1.1⁺ ß T cells is directed by recognition of the nonclassical MHC class I molecule CD1.1 whose expression on CD4⁺CD8⁺ cortical thymocytes is TAP independent and ß₂m dependent (34, 35, 36, 37), we analyzed the V14-J281 -chain junction of thymocytes from DN32H6 TCR ß Tg mice lacking a functional ß₂m gene (27). Sequencing results from two mice (Fig. 5) indicate that in the absence of ß₂m, there is no enrichment in the canonical VGDR joint in postselection thymocytes carrying the V14-J281 -chain rearrangement (0/31), whereas there were 2/37 (5.5%) of these sequences in preselection thymocytes. To obtain more direct evidence of the role of CD1.1, we subsequently repeated this analysis using DN32H6 TCR ß Tg mice on a CD1.1 deficient background when CD1.1^-/- mice became available (28). The Vß8^low thymocyte population analysis (Fig. 6) revealed a sequence profile basically indistinguishable from those obtained from immature DN32H6 TCR ß Tg and DN32H6 TCR ß Tg ß₂m^-/- thymocytes (see Figs. 2 and 5): a low frequency of the canonical junction (3/29 = 10.3%) and a majority of out-of-frame sequences (18/29 = 62%). The postselection thymocyte population analysis confirmed that in the absence CD1.1 molecules, no NK1.1⁺ T cell -chain canonical junction can be detected in thymocytes that are beyond the stage of positive selection (0/28). This indicates that the high frequency of the canonical VGDR junction detected among postselection DN32H6 TCR ß Tg thymocytes (Fig. 2) is entirely ß₂m and CD1.1 dependent and therefore effectively results from intrathymic positive selection on CD1.1.

View larger version (18K): [in this window] [in a new window]   FIGURE 5. The high frequency of the canonical VGDRG joint among postselection DN32H6 TCR ß Tg thymocytes is entirely ß2m dependent. DN32H6 TCR ß Tg ß2m-deficient mice were generated by breeding and were analyzed at 6–8 wk of age. Sequencing data derived from two mice are represented as described in Fig. 2.

View larger version (18K): [in this window] [in a new window]   FIGURE 6. The high frequency of the canonical VGDRG joint among postselection DN32H6 TCR ß Tg thymocytes is entirely dependent on the expression of CD1.1. CD1.1-/- mice were used to derive DN32H6 TCR ß Tg CD1.1-deficient mice. Genomic DNA-derived sequences from two mice are represented as described in Fig. 2.

Thymic, but not peripheral, increase in NK1.1⁺ ß T cells in DN32H6 TCR ß Tg mice

Besides restrictions in the V-J junction (38, 39), several studies on TCR ß-chain Tg mice reported skewing toward Ag-specific mature T cells (40, 41, 42). In addition, mice carrying a ß-chain transgene derived from autoreactive T cells showed accelerated onset of disease in the case of both diabetes (43) and collagen-induced arthritis (44). In the case of the DN32H6 TCR ß Tg mice, we tried to determine whether the sequence analysis of the V14-J281 junction correlates with a detectable phenotype. We first looked at the frequency of NK1.1⁺ T cells in thymus and spleen. A simple NK1.1 staining repeatedly revealed a slight increase in thymic NK1.1⁺ cells in DN32H6 TCR ß Tg mice compared with transgene-negative littermates. Such an increase was not detected among splenocytes (Fig. 7A, left panels). To obtain better resolution, we repeated the NK1.1 staining after enrichment of the cell suspensions in NK1.1⁺ T cells; thymocytes were depleted of CD8⁺ and immature (HSA⁺) cells, and splenocytes were depleted of CD8⁺ T cells and B cells. The enriched populations confirmed an increased frequency of NK1.1⁺ cells in the thymic, but not the splenic, compartment (Fig. 7A, right panels). A NK1.1/Cß double staining indicated more precisely that DN32H6 TCR ß Tg mice have an increased frequency of NK1.1⁺ ß T cells in the thymus (Fig. 7B). A similar phenotypic analysis of both unmanipulated and NK T cell-enriched cell suspensions from bone marrow of DN32H6 TCR ß Tg mice and control littermates revealed no significant differences in the frequency of NK1.1⁺ ß T cells (data not shown). Because the thymic cellularity is virtually unmodified in DN32H6 TCR ß Tg mice (see Figs. 1 and 7), this increased frequency corresponds to a significant increase in the absolute number of thymic NK1.1⁺ ß T cells. In addition, the fraction of V(2-3.2-8-11)⁺ cells was reduced among Vß8.2^int thymocytes in the presence of the DN32H6 TCR ß-chain transgene (data not shown). We then searched for a functional reflection of this observation. It is well documented that NK1.1⁺ ß T cells are able to produce various types of cytokines. Anti-CD3 stimulation causes rapid and massive secretion of IL-4, but also significant amounts of IFN- and TNF-ß (45, 46, 47, 48). This potential is further extended by the ability to elicit IFN- secretion upon stimulation through NK1.1 (49) or the IL-12R (50, 51). To determine whether DN32H6 TCR ß Tg lymphocytes have a modified capacity to produce IL-4, thymocytes and splenocytes from transgenic and control mice were stimulated overnight using plate-bound anti-CD3 (YCD3-1) mAb, and IL-4 secretion was measured by the IL-4-specific CT.4S cell-based bioassay. Under these conditions, no obvious difference was observed among splenocytes, whereas transgenic thymocytes revealed a slight increase in IL-4 production (Fig. 8A). Stimulation of NK1.1⁺ T cell-enriched thymic and splenic populations confirmed this result (Fig. 8B). These observations are consistent with the sequence analysis of genomic DNA (Fig. 2) and indicate that in the presence of the NK1.1⁺ T cell-derived DN32H6 TCR ß transgene, the number of NK1.1⁺ ß T cells 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 of NK1.1⁺ ß T cells does not occur or, alternatively, is not sustained in the periphery of DN32H6 TCR ß-chain Tg mice.

View larger version (25K): [in this window] [in a new window]   FIGURE 7. Increased frequency of thymic, but not splenic, NK1.1+ ß T cells in DN32H6 TCR ß-chain Tg mice. A, Expression of the NK1.1 receptor by total and NK1.1+ T cell-enriched thymocyte and splenocyte suspensions from 8-wk-old DN32H6 TCR ß Tg and littermate control mice. NK1.1 is detected using the PK136 mAb. x-axis, log fluorescence intensity; y-axis, cell number. Histogram statistics are indicated. For enrichment, thymocytes were negatively selected using magnetic beads and anti-HSA (20C9) and anti-CD8 (53-6.72) mAbs, and ammonium chloride-treated splenocytes were negatively selected using anti-B220 (14.8), anti-I-Ab (Y3JP), and anti-CD8 (53-6.72) mAbs. B, NK1.1/ß TCR distribution of total and NK1.1+ T cell-enriched thymic cell suspensions from DN32H6 TCR ß Tg and transgene-negative littermates. ß T cells were detected using the anti-Cß H57 mAb. The frequency of NK1.1+ ß T cells is indicated after electronic gating on NK1.1+/ß TCRint thymocytes. In this experiment the thymic cellularity was: DN32H6 TCR ß Tg, 165 x 106; and control littermate, 171.2 x 106. Results are representative of three experiments.

View larger version (16K): [in this window] [in a new window]   FIGURE 8. Increased IL-4 production by freshly isolated DN32H6 TCR ß Tg thymocytes exposed to anti-CD3 mAb. A, Total thymocytes and splenocytes from DN32H6 TCR ß Tg mice (ß Tg) and transgene-negative littermate (B6) were exposed overnight to polystyrene-immobilized anti-CD3 mAb (YCD3- 1). Detection of IL-4 production was assessed by incubating supernatants with the IL-4-dependent murine CT.4S cells. B, Anti-CD3 stimulation of NK1.1+ T cell-enriched thymocyte and splenocyte suspensions from DN32H6 TCR ß Tg and wild-type littermates. Cell suspensions were enriched in NK1.1+ T cells as described in Fig. 7. IL-4 production was detected as described in A.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the course of the analysis of V14-J281-rearranged sequences, we observed that among the preselection repertoire, a VADR junction was detected twice (Fig. 2). Because a VVDR junction has been reported for hybridomas derived from mature CD44^high NK1.1⁺ thymocytes (30) and given that V and A are both small and hydrophobic amino acids, the usage of the VADR junction by NK1.1⁺ ß T cells is theoretically possible. In line with this possibility, the VADR junction is indeed observed among CD4⁺ Vß8.2⁺ TCR^int thymocytes from a nontransgenic control mouse (Fig. 3). However, the confirmation of the usage of a VADR junction by the V14-J281 -chain of NK1.1⁺ T cell TCR would require a functional analysis, for instance using transfectants.

With respect to NK1.1⁺ ß T cell development, both a committed precursor model and a mainstream precursor model have been considered (reviewed in Ref. 26). The first model postulates the existence of a precursor committed to become an NK1.1⁺ ß T cell and implies that the canonical V14-J281 rearrangement is genetically programmed. Important developmental differences appear to exist between NK1.1⁺ ß T cells and ß T cells. For instance, the expression of pT has been shown to be absolutely required for NK1.1⁺ ß T cell development (53). In addition, the Src protein kinase Fyn is mandatory for the CD1-dependent development of NK1.1⁺ ß T cells, but not for the development of conventional T cells (54, 55). However, the occurrence of random rearrangements at the second locus of NK1.1⁺ ß T cells (30, 56), the diversity of nucleotide sequences encoding the canonical -chain amino acid sequence (30) and the lack of evidence for directed V14-J281 rearrangements among thymic NK1.1⁺ ß T cells (57) argue against such a committed precursor model. This suggests, rather, that the V14-J281 rearrangement is stochastic in nature and that positive selection on CD1.1 rescues the rare mainstream ß thymocytes carrying the canonical -chain and a permissive Vß segment (mainstream precursor model) (26). The repertoire analysis we performed here using genomic DNA from immature DN32H6 TCR ß Tg and wild-type thymocytes indicates that despite the germline encoded sequence of the canonical joint, about 50–65% of the V14-J281 rearrangements are not productive, and about 25–40% produce noncanonical joints. Such proportions are drastically reduced in the postselection thymocyte population, which, in the presence of CD1.1, is dominated by the canonical VGDR joint selected at the amino acid level. Our observations are best explained by a strong ligand-mediated selection and directly support the mainstream precursor model of NK1.1⁺ ß T cell development. Sequences derived from immature thymocytes also suggest that under the experimental conditions used here, the probability of observing the canonical VGDR junction among the rearranged V14-J281 -chains ranges from 1/15 to 1/19 in most mice analyzed (five of seven; see Figs. 2, 3, 5, and 6).

Using immunostaining and functional assay, we observed an increased frequency of NK1.1⁺ ß T cells in the thymus, but, strikingly, not in the spleen and bone marrow of DN32H6 TCR ß-chain Tg mice. This increase of about 2-fold appears moderate and could be regarded as the reflection of a rather inefficient positive selection of V14-J281/DN32H6 TCR ß Tg thymocytes. However, in C--deficient mice expressing a canonical V14-J281 chain transgene, that is, in mice in which any permissive ß-chain rearrangement allows the assembly of a V14-J281-expressing NK1.1⁺ T cell TCR, the increase in NK1.1⁺ ß T cells observed was about 7-fold (58). These increases are thus characterized by a similar order of magnitude. This may suggest that limiting factors control positive selection of V14-J281-expressing NK1.1⁺ ß T cells. Possibly, the abundance of appropriate CD1.1-presented glycolipidic self-Ags may have a limiting effect. The reason for the thymus-restricted increase in NK1.1⁺ ß T cells in DN32H6 TCR ß-chain Tg mice remains unclear. One possibility is that a mechanism exists that tightly regulates the number of NK1.1⁺ ß T cells in vivo. Thus, although expression of the DN32H6 TCR ß transgene seems to promote positive selection of NK1.1⁺ ß T cells, their number could be rapidly reduced to normal before or concomitantly with their departure from the thymus. Such a homeostatic hypothesis is supported by the observation that in MHC class II-deficient (I-Aß^b-/-) mice that lack most conventional CD4⁺ ß T cells, the absolute number of CD4⁺ NK1.1⁺ T cells is virtually unchanged (59). However, this possibility appears in conflict with the increased number of NK1.1⁺ ß T cells in the periphery of V14-J281 Tg mice (58). An alternative possibility can be considered in light of the documented tissue-specific recognition of CD1.1. It is known that CD1.1-specific T hybridomas respond differently to CD1 presentation by thymic and splenic CD1.1⁺ cells and that further differences are seen when analyzing distinct CD1.1⁺ cell types derived from the spleen. Indeed, a careful functional analysis of 12 hybridomas revealed that no spleen-specific hybridoma was found among thymus-derived hybridomas, and that no thymus-specific hybridoma was identified among spleen-derived hybridomas (29). This observation supports a model in which tissue-resident CD1.1-specific NK1.1⁺ ß T cells recognize CD1.1 locally, that is CD1.1 associated with tissue-specific coligands that may only partially overlap. According to this model, DN32H6 TCR ß Tg NK1.1⁺ T cells could recognize a thymus-specific ligand in the context of CD1.1. This would lead to their maintenance in the thymic microenvironment, but not in the periphery. Such a scenario appears consistent with the idea that the thymus, but not the spleen, is highly enriched in CD1.1-dependent NK1.1⁺ ß T cells (60). In this regard, it is very interesting that the DN32H6 TCR ß-chain was cloned from a hybridoma generated using CD44^high mature thymocytes (30).

In conclusion, the molecular, phenotypic and functional results we report here show that in vivo expression of a V14-J281-expressing NK1.1⁺ thymocyte-derived TCR ß-chain transgene promotes, in a CD1.1-dependent manner, positive selection of thymocytes carrying the NK1.1⁺ T cell V14-J281 canonical TCR -chain. The increased frequency of thymic ß T cells was not observed in the periphery, possibly reflecting a requirement for a thymus-specific Ag for their maintenance. These observations indicate that the influence of rearranged ß-chains on the TCR specificity of developing thymocytes applies to a major subset of nonconventional ß T cells, the CD1.1-dependent NK1.1⁺ ß T cells. Thus, the strong impact of productive ß-chain rearrangements on the mature ß TCR repertoire, which operates through positive selection on self-determinants, appears to be a general principle in the intrathymic development of ß T lymphocytes.

	Acknowledgments

 
We thank L. Van Kaer (Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, TN) for providing us with the CD1.1-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, and Charles Annicelli for animal care.

	Footnotes

 
¹ This work was supported in part by the Howard Hughes Medical Institute and by Grant AI14579 (to C.A.J.).

² Address correspondence and reprint requests to Dr. Charles A. Janeway, Jr., Yale University School of Medicine. Section of Immunobiology, LH 416, 310 Cedar Street, New Haven, CT 06520-8011.

³ C.A.J. is an investigator with the Howard Hughes Medical Institute.

⁴ Abbreviations used in this paper: Tg, transgenic; ß₂m, ß₂-microglobulin; HSA, heat-stable Ag; int, intermediate; CDR, complementarity-determining region.

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

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