Comparative analysis of the CD8+ T cell repertoires of H-2 class I wild-type/HLA-A2.1 and H-2 class I knockout/HLA-A2.1 transgenic mice
Hüseyin Firat1,2,
Madeleine Cochet2,
Pierre-Simon Rohrlich2,3,
Francisco Garcia-Pons2,
Sylvie Darche4,
Olivier Danos1,
François A. Lemonnier2 and
Pierre Langlade-Demoyen2
1 Généthon III, CNRS URA 1923, 1 bis rue de lInternationale, BP 60, 91002 Evry Cedex, France 2 Unité dImmunité Cellulaire Antivirale, Département SIDA-Rétrovirus, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France 3 Hôpital Robert-Debré, 48 bd Serurier, 75019 Paris, France 4 Unité de Biologie Moléculaire du Gène, INSERM U277, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France
Correspondence to: H. Firat, PCS/ICPP/Pharmacogenomics, WKL-136.182 Novartis Pharma AG, Werk Klybeck, Postfach CH-Basel, Switzerland. E-mail: huesevin.firat{at}pharma.novartis.com
Transmitting editor: C. Martinez-A
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Abstract
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HHD transgenic mice which express HLA-A2.1 monochain molecules in a H-2 class I context have an improved capacity to develop HLA-A2.1-restricted cytotoxic T lymphocyte (CTL) responses as compared with classical A2.1/Kb transgenic mice, which express heterodimeric HLA-A2.1 molecules in a H-2 class I wild-type context. A detailed TCR analysis of HLA-A2.1-restricted CD8+ T cells educated and mobilized in both strains of mice was undertaken. Focusing on TCR ß chains, comparative PCR analysis of naive and immune CD8+ T cell repertoires were performed. In spite of lower cell surface expression of HLA class I molecules and lower overall number of CD8+ T cells, HHD mice educate a qualitatively normally diversified CD8+ T cell repertoire and mobilize a larger variety of CD8+ T cells in response to HLA-A2.1-restricted antigens compared with A2.1/Kb mice. These observations provide the molecular bases accounting for the fact that HHD mice represent the most versatile animal model currently available for preclinical studies of HLA-A2.1-restricted CTL responses.
Keywords: cytotoxic T lymphocyte, HLA-A2.1, MHC, transgenic/knockout
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Introduction
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CD8+ cytotoxic T lymphocytes (CTL) recognize MHC class I molecules complexed with peptides deriving from endogenously processed proteins through their TCR, and further discriminate between peptides of self and non-self origin. In the latter case, CD8+ T cells become activated and lyse abnormal cells, as has been largely documented in viral infections and in some cancers. Soon after the identification of HLA class I genes, several HLA class I transgenic mice were created to facilitate the study of the HLA class I-restricted CTL responses of potential preventive or therapeutic interest in human viral diseases and cancer (13). The first generation of transgenic mice expressing unmodified HLA class I molecules use them inefficiently as restricting elements (4). Improvement was obtained by substituting the third domain of the human class I heavy chain by the homologous mouse domain in order to reinforce the interaction of the mouse CD8 adhesion accessory molecules with the transgenic HLA class I molecules (5,6). Transgenics expressing such chimeric molecules have been widely used for the study of HLA-A2.1 class I-restricted responses (6). However, these mice often failed to develop responses against CTL epitopic peptides known to elicit HLA-A2.1-restricted responses in humans. To further improve the use of HLA class I transgenic molecules by mouse CD8+ T cells, we combined HLA class I transgenesis with H-2 class I gene invalidation. These mice, designated HHD, express a monochain construct in which the C-terminus of human ß2-microglobulin (ß2m) is covalently linked to the N-terminus of the HLA-A2.1 heavy chain in a chimerical (
3 domain of mouse origin) configuration. Since both the H-2Db gene and the ß2m gene of these mice are disrupted, they express a single type of MHC class I molecule on their cell surfaces. Therefore, in such mice, the HLA-A2.1 monochain is the only species of class I molecule available for CD8+ T cell thymic education and subsequent peripheral mobilization in response to antigens (7).
We previously documented the improved capacity of HHD mice to develop HLA-A2.1-restricted CTL responses as compared to A2.1/Kb transgenic mice which still express their mouse H-2b class I molecules (8), but since these comparisons were based on cytolytic assays, they only provided information on the size of the mobilized CD8+ T cell repertoire. In this study, we additionally identified the variable ß genes (BV gene) and determined for some of them the sequence of the hypervariable CDR3 region which forms the ß chain of the mobilized CD8+ T cells in HHD and A2.1/Kb mice in response to antigens. The results establish that not only the size, but also the diversity, of the CD8+ T cell TCR repertoire which can be mobilized for the development of HLA-A2.1-restricted CTL responses is substantially larger in HHD than in A2.1/Kb transgenic mice.
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Methods
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Mice
HHD homozygous mice were created in our laboratory (7). They express a chimeric monochain (HHD molecule) containing the
1
2 domains of HLA-A2.1, and the
3 and cytoplasmic domains of H-2Db linked at its N-terminus to the C-terminus of human ß2m by a 15-amino-acid peptide linker. They are deprived of serologically detectable cell surface H-2 class I molecules due to the destruction by homologous recombination of both the H-2Db and mouse ß2m genes. Homozygous A2.1/Kb transgenic mice also express HLA-A2.1 chimeric molecules (
1
2 domains of HLA-A2.1 and
3 to cytoplasmic domains of H-2Kb), but in addition they express a full set of H-2b class Ia and Ib molecules (6). These latter mice were obtained from Harlan Sprague-Dawley (Indianapolis, IN). All mice were maintained in our animal facility and used at 68 weeks of age.
HLA A2.1 tetramers loaded with melanoma gp100.154 or human cytomegalovirus (CMV) N9V epitopic peptides
Biotinylated HLA-A2.1human ß2m complexed with gp100.154 (KTWGQYWQV) or control N9V peptide [human cytomegalovirus (CMV)-derived pp65 epitopic peptide, NLVPMVATV] were produced essentially as previously described (9). In brief, purified bacterial inclusion bodies of HLA-A2.1 heavy chains containing a Bir A enzymatic biotinylation site, and human ß2m (Sigma-Aldrich, St Louis, MO) were denatured in 8 M urea and refolded by progressive dilution in the presence of either of the epitopic peptides. The HLA-A2.1ß2mpeptide complexes were isolated by fast protein liquid chromatography and then biotinylated with the Bir A enzyme (a kind gift from Dr Romagné, Immunotech, Marseille, France). Streptavidinphycoerythrin (PE) conjugates (Sigma-Aldrich) were added at a 1:4 molar ratio and the tetrameric products were concentrated to 1 mg/ml using a Millipore (Bedford, MA) filter (exclusion size = 10 kDa). All tetrameric complexes were used at a 20 µg/ml final concentration in cytofluorimetric analyses.
Cytofluorometric analyses of peripheral T lymphocytes
Cytofluorometric studies were performed on wheat germ agglutinin T lymphocyte-enriched splenocytes as described (10). Expression of H-2 class Ia (H-2Kb, H-2Db) or HLA-A2.1 molecules was analyzed by indirect immunofluorescence using B8.24 3 (anti-H-2Kb), B22.249.R.19 (anti-H-2Db) and BB7.2 (anti-HLA-A2) unlabeled mAb and FITC-conjugated goat IgG F(ab')2 anti-mouse Ig. The percentage of CD4+ and CD8+ T lymphocytes was determined by double staining using PE-labeled anti-mouse CD4 (CT-CD4) and FITC-labeled anti-mouse CD8 (CT-CD8) (Caltag, South San Francisco, CA). Expression of the different BV TCR was similarly analyzed using PE-labeled anti-CD8 mAb (Caltag) and purified, FITC-labeled BV2 (B.20.6), BV4 (KT.10.4), BV5.1, 5.2 (MR.9.4), BV6 (44.22), BV7 (TR 130), BV 8.1, 8.2, 8.3 (F.23.1), BV9 (MR.10.2) BV10 (B.21.5), BV11 (RR3.15), BV12 (MR11.1), BV13 (MR12.4), BV14 (14/2) and BV17 (KJ.23.288.1) specific mAb. Cells were incubated for 30 min on ice with the first layer mAb at saturating concentrations and, following 2 washings, incubated with the conjugates (4°C, 30 min), washed, twice, fixed with Paraformaldehyde and analyzed by a FACSCalibur (Becton Dickinson, Bedford, MA).
Tetramer staining was performed on live cells purified by centrifugation on Ficoll-Hypaque (Amersham Pharmacia Biotech, Little Chalfont, UK). Cells were washed twice with PBS, 1% BSA and 4 mM EDTA, and incubated with the PE-labeled tetramers for 40 min at 37°C. FITC-labeled anti-CD8 mAb was then added for 20 min on ice. Cells were subsequently washed once and immediately analyzed with a FACSCalibur (Becton Dickinson). gp100.154 tetramer+, CD8+ T cells were sorted using an Epics Elite ESP (Beckman Coulter, Brea, CA).
Immunoscope analyses and CDR3 sequence determination
The TCR repertoire of CD8+ T cells from naive and immunized transgenic mice was analyzed for BV usage and in some cases for BVJ CDR3 region diversity as described elsewhere (11,12), using oligonucleotides specific for each family of BV segments (13). Briefly, CD8+ cells were positively selected on MiniMacs separation columns (Miltenyi Biotec, Bergisch Gladbach, Germany) or by cell sorting of tetramer- and CD8 double-positive T cells. FACS analyses were performed to evaluate contaminant CD4+ cells. A small fraction of MiniMacs-selected cells (3.4 ± 2.7%) was CD4+, while no CD4+ cells were detected after cell sorting. RNA was prepared by the guanidium isothiocyanate procedure and used for cDNA synthesis. A 1/20 fraction of the product was then amplified by 40 cycles of PCR (annealing at 60°C) in 25 µl, in the presence of BV-specific primers and subjected to an elongation reaction (run-off) with internal BC or BJ fluorescent-tagged primers. The labeled products were then loaded on a 6% acrylamide/8 M urea gel and separated by a run of 7 h at 35 W on a 373 DNA sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA). Data were analyzed as described previously (11) using Immunoscope software.
When necessary, PCR products of CDR3ß DNA were cloned into TOPO II vectors (Invitrogen, Carlsbad, CA) and sequenced according to the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit protocol (Perkin-Elmer Applied Biosystems), and run on a 377A ABI DNA sequencer.
Influenza immunization
A2.1/Kb and HHD transgenic mice were i.p. infected with 1000 HAU of influenza virus A/PR/8/34 strains. Two weeks later, 2.5 x 107 splenocytes were re-stimulated in vitro in complete RPMI medium (10% FCS, 2 mM L-glutamine, 5 x 105 M 2-mercaptoethanol, 50 U/ml penicillin and 50 µg/ml streptomycin) with an equal amount of syngeneic splenocytes infected for 1.5 h with influenza A virus (10 HAU/106 cells) in FCS-free otherwise complete RPMI medium.
Responder mice were individually tested in a 4 h 51Cr-release assay, using V-bottomed 96-well plates containing 5 x 103 HHD-transfected EL4 S3 Rob target cells/well which were either uninfected, infected with virus or pulsed with influenza A virus matrix peptide 5866-(106 M, Inf.m.58, GILGFVFTL; Neosystem, Strasbourg, France). Tests were performed in the presence of effector cells from bulk cultures at different E:T ratios. Precursor frequencies of influenza virus-specific CTL were estimated by limiting dilution analysis as described previously (14).
Peptide-specific CTL
Each mouse was injected s.c. at the base of the tail with 100 µg of the HLA-A2.1-restricted melanoma epitopic peptide, with or without 140 µg of a I-Ab-restricted helper peptide from the hepatitis B core protein (HBVc.128140 TPPAYRPPNAPIL; Neosystem), co-emulsified (v/v) in incomplete Freunds adjuvant (Difco, Detroit, MI). Eleven days later, spleen cells were re-stimulated in vitro with peptides (10 µg/ml) for 6 days. Cultured cells were then tested for cytolytic activity against HHD-transfected RMA-S (TAP-deficient) targets loaded with relevant or negative control peptide.
Immunization with DNA encoding a melanoma polyepitope
A melanoma polyepitopic sequence was inserted into the pre-S2 segment of the hepatitis B virus surface (HBVs) middle protein using a pCMV-B10 mammalian cell-expression vector (15). Recombinant plasmids were purified on lipopolysaccharide-free Qiagen columns (Qiagen, Hilden, Germany). Mice were injected in the thigh i.m. with 10 µM cardiotoxin (Latoxan, Rosans, France) in 50 µl PBS once and, 5 days later, with 50 µg of pCMV-B10 DNA for a 21-day priming.
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Results
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HLA-A2.1 cell surface expression and number of peripheral CD8+ T cells
The cell surface expression of HLA-A2.1 molecules and percentages of CD8+ T cells in the periphery were comparatively evaluated by indirect immunofluorescence analysis of T cell-enriched splenocytes from HHD and A2.1/Kb transgenic mice.
As illustrated in Fig. 1,
5-fold lower cell surface expression of HLA-A2.1 molecules, as detected with HLA-A2.1-specific BB7.2 mAb, was observed in HHD compared with A2.1/Kb mice. For the A2.1/Kb mice, a subpopulation of cells without expression of the transgenic molecules was observed in all mice tested. The variable size of this subpopulation between mice (1030% of total T cells) and the fact that it was not restricted to lymphocytes are suggestive of a variegation phenomenon (14). Whereas the number of peripheral CD8+ T cells (31% ± 7%) in A2.1/Kb transgenics was comparable to wild-type C57Bl/6 mice, HHD mice only contain 5 ± 4% of such cells, with considerable interindividual differences, even in mice from the same litter.

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Fig. 1. Phenotypic characterization of A2.1/Kb and HHD transgenic mice. Expression of HLA-A2.1 molecules was detected by indirect immunostaining on naive purified T cells from A2.1/Kb (A) and HHD (B) transgenic mice using anti-HLA-A2 BB7.2 and control (GAP/3, anti-HLA-A3) mAb. Percentage of CD8+ T lymphocytes is indicated in the upper right of each panel. For HLA-A2.1 expression, results are expressed in arbitrary fluorescence intensity units.
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BV peripheral repertoire of CD8+ T cells
Expression of the various BV families was next studied on CD8+ purified T cells by indirect immunofluorescence with 13 BV-specific mAb (Fig. 2A) and RT-PCR-based immunoscope assay (Fig. 2B and C). All BV families and subfamilies were represented in peripheral CD8+ T cells from both strains, with no significant differences either in percentage expression as measured by indirect immunofluorescence or in CDR3ß size diversity (normal Gaussian-like distribution) when compared to wild-type C57Bl/6 mice.

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Fig. 2. BV family representation in the CD8+ T cell peripheral population. (A) Immunofluorescence analysis. Double stainings were performed on unstimulated purified T splenocytes with FITC-labeled BV-specific and PE-labeled anti-CD8 mAb. Results are the means + SD of five to six mice tested individually and are expressed as the percentage of total CD8+ T cells. (B and C) Immunoscope analysis of CDR3ß chain size distribution among CD8+ T cells from A2.1/Kb (B) and HHD (C) mice. Mouse splenic CD8+ T cells were selected on a MiniMacs column and processed as described in Methods by Immunoscope analysis with a set of 22 primers, including BV17 which corresponds in the C57Bl/6 mouse to a pseudogene. The CDR3 size is expressed in number of amino acids.
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Thus, even in HHD mice which express a single type of human class I molecule, thymus education and peripheral maintenance resulted in a BV-diversified CD8+ T cell repertoire.
HLA-A2.1-restricted anti-influenza CTL responses
A2.1/Kb and HHD mice were injected i.p. with influenza virus and 15 days later their splenocytes re-stimulated in vitro either in bulk or limiting dilution culture conditions with either influenza virus infected or Inf.m.58 peptide-loaded, irradiated autologous splenocytes. Five days later, effector cells were tested in 51Cr-release assays against influenza virus-infected, Inf.m.58 peptide-pulsed HHD-transfected EL4 S3 Rob target and control cells.
As already reported (7), HHD mice developed more potent influenza virus-specific CTL responses than A2.1/Kb mice as illustrated with bulk effector cells in Fig. 3. The CTL responses were directed for the most part at the Inf.m.58 peptide, which is also the immunodominant one in HLA-A2.1 human individuals. Frequencies (mean ± SEM) of HLA-A2.1-restricted CTL specific for influenza virus and Inf.m.58 peptide were evaluated assaying effectors re-stimulated under limiting culture conditions (not shown) and were respectively 1/5856 ± 253 and 1/5677 ± 846 in HHD mice, and 1/17883 ± 2080 and 1/22164 ± 3298 in A2.1/Kb mice. Thus, taking into account the 5- to 10-fold reduction of CD8+ T cell numbers in HHD mice, the relative frequency of HLA-A2.1-restricted influenza virus-specific CTL among CD8+ T cells was estimated to be
20- to 30-fold higher in HHD than in A2.1/Kb transgenic mice.

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Fig. 3. Influenza virus-specific CTL responses of A2.1/Kb and HHD transgenic mice. Two weeks after i.p. infection with influenza A viruses, splenocytes from six HHD (left panel) and six A2.1/Kb (right panel) mice were re-stimulated in vitro for 1 week with influenza-infected syngeneic irradiated spleen cells. Pooled effectors were tested against non-infected, influenza-infected or peptide-pulsed EL4 S3 Rob cells expressing or not HHD molecules.
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CTL responses against HLA-A2.1-restricted melanoma epitopic peptides
The low HLA-A2.1-restricted response of A2.1/Kb transgenics against influenza virus could have resulted from the preferential and simultaneous development of H-2 class I-restricted CTL responses which, to some extent, could have pre-empted the induction of the HLA-A2.1-restricted responses (16). To avoid such experimental bias, the CTL responses of both strains of mice to HLA-A2.1-restricted synthetic melanoma epitopic peptides were compared. CD8+ peptides emulsified in incomplete Freunds adjuvant were co-injected with a I-Ab-restricted epitopic peptide (HBVc.128140) which was previously shown to substantially improve peptide-induced CTL responses (8).
Considering both the number of responder mice and the levels of lysis, a significant advantage was documented for HHD mice which responded to all peptides, whereas A2.1/Kb transgenic mice responded only to three out of the 10 epitopic peptides tested (Table 1). HHD and A2.1/Kb mice were then immunized once i.m. with a highly immunogenic recombinant pCMV-B10 DNA encoding all of the 10 melanoma-derived epitopic peptides (8). After one in vitro re-stimulation of pooled splenocytes from six mice of each strain, detectable specific CTL responses were induced against only three and five out of 10 epitopes in A2.1/Kb and HHD mice respectively (Fig. 4A and C). After three in vitro re-stimulations, CTL specific for most of the melanoma epitopic peptides could be detected in both strains, but these were of lower magnitude in A2.1/Kb than in HHD mice (Fig.4B and D). Thus, challenging A2.1/Kb and HHD mice with epitopic peptides which only bind to HLA-A2.1 molecules and therefore selectively stimulate the HLA-A2.1-educated CD8+ T cells in both mice, a clear quantitative advantage in the capacity to develop HLA-A2.1-restricted responses was documented in HHD mice which solely express, in terms of MHC class I molecules, the transgenic HLA-A2.1 molecules. The gp100.154 epitopic peptide which induces the most potent CTL responses in both strains was selected for more detailed comparative analysis of the TCR of the CD8+ cells stimulated in these two strains of mice by this epitopic peptide.

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Fig. 4. CTL response of HHD and A2.1/Kb mice after immunization with a melanoma polyepitope. Mice were immunized with the pCMV-B10 plasmid encoding the recombinant middle size hepatitis B virus glycoprotein, the melanoma polyepitope being inserted in its pre-S2 segment. Spleen cells were harvested 3 weeks later and re-stimulated in vitro with the corresponding melanoma synthetic peptides. Six days after, culture cells from A2.1/Kb (A) and HHD (C) mice were harvested and tested for their ability to lyse HHD-transfected RMA-S cells in a 4-h 51Cr-release assay. (B and D) Results obtained after two rounds of in vitro re-stimulations for A2.1/Kb and HHD mice respectively. Specific lysis was calculated as indicated in Methods. Values refer to specific lysis for each peptide, calculated for three E:T ratios (60:1, 20:1 and 6:1).
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Frequency of specific CTL for the gp100.154 epitope in naive and peptide-immunized A2.1/Kb and HHD mice
Frequencies of CTL precursors specific for gp100.154 were evaluated in naive HHD and A2.1/Kb mice after 10 days in vitro concanavalin A stimulation of their splenocytes or following in vivo peptide immunization and in vitro re-stimulation (14). CTL frequencies were determined using limiting dilution analysis and, additionally (immune mice), gp100.154/HLA-A2.1 tetramer staining.
Frequencies of CTL precursors specific for gp100.154 were 3- to 4-fold higher in HHD mice than in A2.1/Kb mice using limiting dilution analysis whether naive (Fig. 5A) or immune (Fig. 5B) mice were tested. The latter result was confirmed by the specific gp100.154/HLA-A2.1 tetramer staining of in vitro stimulated splenocytes from six immunized mice: 67 ± 5.6 and 27 ± 10% of CD8+ T cells in HHD and A2.1/Kb mice respectively (Fig. 5C and D). Negative control N9V/HLA-A2.1 tetramers stained <0.5% of CD8 cells in all experiments.

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Fig. 5. Frequencies of CTL specific for the gp100.154 epitopic peptide in naive and immunized A2.1/Kb and HHD transgenic mice. (A) Spleen cells from naive transgenic mice were in vitro stimulated with concanavalin A for 7 days in the presence of T cell growth factor, and precursor frequency evaluated for A2.1/Kb (filled symbols) and HHD (open symbols) transgenic mice in a limiting dilution assay using gp100.154 peptide-pulsed HHD-transfected EL4 S3 Rob target cells. (B) CTL frequency of immunized mice was similarly determined 11 days post-immunization following in vitro re-stimulation for 7 days with gp100.154 epitopic peptide in the presence of T cell growth factor (same symbols as in A). (C and D) gp100.154/HLA-A2.1 tetramer staining of CD8+ cells from in vitro re-stimulated spleen cells of immunized HHD and A2.1/Kb mice.
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Peripheral CD8+ T cell repertoire of HHD and A2.1/Kb mice specific for the gp100.154 epitopic peptide
BV diversity of the CD8+ T cells mobilized in A2.1/Kb and HHD mice following immunization by the melanoma gp100.154 epitopic peptide was then analyzed by testing the mice individually for all BV families by the Immunoscope assay. Control mice which had only been injected with the helper peptide were similarly analyzed.
As illustrated in Fig. 6, oligoclonal expansions were observed in all immunized mice tested, with more diversity (both in terms of BV segments and number of expansions for a given BV) in HHD (BV2, 4, 6, 8.1 and 10) than in A2.1/Kb (BV6, 11 and 13) mice. To better characterize these expansions we used HLA-A2.1 tetramers to purify the CD8+ T cells specific for gp100.154 peptide. After sorting of tetramer+/CD8+ double-labeled cells, BV diversity was analyzed by an Immunoscope. As shown with individual mice, HHD mice mobilized a more diversified pattern of BV genes than did A2.1/Kb mice (Fig. 7A and B respectively). Among the tetramer+/CD8+ sorted cells, we observed expansions of TCR-BV6 cells again in both strains of transgenics. BV6+BC rearrangements from isolated CTL clones or from tetramer+/CD8+ cells were selected and their TCRßCDR3 hypervariable subregions sequenced. Whereas a diversity in amino acid usage was observed in both strains, CDR3 subregions of different length were only documented in HHD mice (Table 2).

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Fig. 6. BV repertoire of CD8+ splenocytes from HHD and A2.1/Kb mice immunized with gp100.154 peptide. CDR3ß chain size profiles were determined for each mouse 11 days post-immunization with gp100.154 peptide followed by 5 day in vitro re-stimulation with the same peptide. CD8+ T cells selected on MiniMacs column were treated for Immunoscope analysis as described in Methods. Only BV clonal expansions resulting in non-Gaussian distribution of CDR3ß size for each of the tested mice are presented.
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Fig. 7. BV repertoire of tetramer+/CD8+ splenocytes from A2.1/Kb and HHD mice immunized with gp100.154 peptide. A2.1/Kb and HHD mice were immunized with the gp100.154 peptide s.c. in incomplete Freunds adjuvant for 11 days. After 5 days of in vitro re-stimulation, splenocytes were stained with gp100.154/HLA-A2.1 tetramers and anti-CD8 mAb. Tetramer+/CD8+ double-labeled cells were sorted and the diversity of BV usage was analyzed by Immunoscope analysis for A2.1/Kb (A) and HHD (B) mice.
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Thus, focusing the analysis on the gp100.154, the most immunogenic peptide in A2.1/Kb mice, and further restricting the comparative analysis to the BV6+ gp100.154-specific CTL clones efficiently mobilized in the two strains, more diversity was always observed in HHD than in A2.1/Kb mice.
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Discussion
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The CD8+ T cell repertoires of A2.1/Kb and HHD transgenic mice which do or do not co-express H-2 class I molecules have been quantitatively and qualitatively compared. In spite of significant reduction in the overall number of peripheral CD8+ T lymphocytes, HHD mice mobilized a larger and, in BV terms, more diversified set of HLA-A2.1-restricted CD8+ T cells in response to influenza virus or melanoma-derived epitopic peptides than A2.1/Kb mice. This was even verified in response to the melanoma-derived gp100.154 epitopic peptide, the most immunogenic for A2.1/Kb mice of the peptides tested and therefore relatively the least advantageous for HHD mice.
Several hypotheses could be put forward to account for the reduced CD8+ T cell number of HHD mice. The one we considered in the first place, i.e. the low cell surface expression of HHD molecules, has to be discardedafter initiation of this work, a second line of HHD transgenics was created with
5 times more cell surface expression of HHD molecules, but without sizable change in peripheral CD8+ T cell number (H. Firat, unpublished data). Presentation by HHD molecules of a reduced diversity of peptides at the thymus level for CD8+ T cell education could also have been considered as a possible result of its monochain configuration which might alter its capacity to interact with chaperones in the endoplasmic reticulum (1720). This hypothesis appears unlikely since vaccination of HHD mice with two different polyepitope DNA constructs [this study and (21)] resulted in multiepitopic CTL responses in all injected mice. Therefore, efficient presentation of the 23 different epitopic peptides present in these two constructs is insured by HHD molecules. For these reasons, we rather favor the possibility that, despite the heavy chain
3 domain substitution, interaction with mouse CD8+ accessory molecules might remain suboptimal. Crystallographic studies have documented that whereas mouse CD8 molecules are only in contact with the mouse H-2Kd heavy chain
3 domain, human CD8 molecules also interact with the HLA-A2.1 heavy chain
2 domain (22,23). It is therefore conceivable that HHD mice mainly select a reduced subset of CD8+ T cells expressing TCR of intrinsic high affinity at the thymus level. However, inasmuch as HHD mouse CTL responses reflect quite satisfactorily human HLA-A2.1-restricted CTL responses, improvement of the CD8-mediated interaction is not considered, for this precise strain of mice, as an absolute priority.
The weakness of the HLA-A2.1-restricted responses in influenza-infected A2.1/Kb mice could have been the consequence of the parallel development of immunodominant, influenza virus-specific, H-2 class I-restricted CTL responses. This argument cannot apply to CTL responses induced by synthetic peptides which only bind to HLA-A2.1 molecules and therefore selectively stimulate HLA-A2.1-educated CD8+ T cells. In response to such peptides, as documented in this report, HHD mice constantly develop more potent and more diversified CTL responses than A2.1/Kb mice in BV terms. Therefore, we conclude that, although HHD mice have a lower cell surface expression of the transgenic molecules, due to the absence of H-2 class I molecules, they educate and/or maintain HLA-A2.1-restricted CD8+ cells both quantitatively and qualitatively more efficiently than A2.1/Kb mice. In vivo competition between populations of T lymphocytes expressing each monoclonally distinct TCR has been documented and regulates the final size of these populations in lymphoid tissues (24). Such competition between HLA-A2.1- and H-2 class I-educated populations of lymphocytes would provide a reasonable explanation for the reduced size of the HLA-A2.1-educated population, as compared to HHD mice, in A2.1/Kb transgenics. Since not only the magnitude of the elicited CTL responses but also the diversity of BV chains used to mount these responses is reduced in A2.1/Kb compared to HHD mice, it appears likely that the competition process already begins at the thymic educational stage. As previously suggested to account for the reduced overall number of CD8+ T cells in HHD mice, suboptimal interaction of mouse CD8 molecules with chimeric A2.1/Kb molecules, recurrently occurring at all steps of the lymphocyte life cycle (thymic education, peripheral maintenance and antigen-driven final mobilization), could also account for the reduced size of the HLA-A2.1-educated repertoire in mice co-expressing H-2 class I molecules.
Since we recently documented that H-2KbDb knockout/HLA-B7.2 transgenic mice also develop more potent HLA-B7.2-restricted CD8+ CTL responses than their transgenic counterparts expressing H-2 class I molecules (P. Rohrlich et al., submitted), we anticipate that the improved usage of HLA class I molecules when expressed in a H-2 class I knockout context will also apply to other HLA class I alleles and we are currently developing these additional HLA class I transgenic H-2 class I knockout mice.
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Acknowledgements
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The authors thank Dr J. C. Manuguerra for providing influenza virus, M. Henry for expert technical assistance, and Dr P. van Endert and Dr S. Cure for critical reading of the manuscript. This work was funded by the Ligue Nationale contre le Cancer (Comité de Paris), the Association pour la Recherche contre le Cancer (grant 5129), the Société Française dExpérimentation Animale and the Institut Pasteur.
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Abbreviations
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ß2mß2-microglobulin
BVß variable
CMVcytomegalovirus
CTLcytotoxic T lymphocyte
HBVhepatitis B virus
PEphycoerythrin
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References
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