HLA-B*0702 transgenic, H-2KbDb double-knockout mice: phenotypical and functional characterization in response to influenza virus

Pierre-Simon Rohrlich1,2, Sylvain Cardinaud2, Hüseyin Firat3, Mustapha Lamari4, Pascale Briand5, Nicolas Escriou6 and François A. Lemonnier2

1 Hôpital Robert Debré, 48 Bd Serurier, 75019 Paris, France 2 Unité d‘Immunité Cellulaire Antivirale, Département d‘Immunologie, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France 3 Généthon III, CNRS URA 1922, 1 rue de l’Internationale, 91002 Evry Cedex, France 4 Hôpital Robert Debré, Etablissement Français du Sang, 48 Bd Serurier, 75019 Paris, France 5 Unité de Physiopathologie Infectieuse et Tumorale des Epithelia, Institut Cochin de Génétique Moléculaire, 24 rue du Fbg Saint-Jacques, 75014 Paris Cedex 18, France 6 Unité de Génétique Moléculaire des Virus Respiratoires, Département de Virologie, Institut Pasteur, 27 rue du Dr Roux, 75724 Paris Cedex 15, France

The first two authors contributed equally to the work
Correspondence to: F. Lemonnier; E-mail: flemonn{at}pasteur.fr
Transmitting editor: H. Ploegh


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
HLA-B*0702 transgenic mice (expressing a chimeric heavy chain with a murine {alpha}3 domain: HLA-B7m{alpha}3) in which the H-2Kb and H-2Db class I-a (Cl I-a–/–) genes have been inactivated were compared with H-2KbDb Cl I-a+/+ positive controls. Expression of the HLA-B7m{alpha}3 molecules resulted in a 3- to 4-fold increase in peripheral CD8+ T lymphocyte numbers compared to H-2 Cl I-a–/– knockout mice. These cells show a diversified TCR repertoire. Following influenza infection, a significant improvement in HLA-B0702-restricted cytotoxic T lymphocyte (CTL) responses was observed in HLA-B7m{alpha}3, H-2 Cl I-a–/– compared to HLA-B7m{alpha}3, H-2 Cl I-a+/+ mice. The CTL response of infected HLA-B7m{alpha}3, H-2 Cl I-a–/– mice was directed against the nucleoprotein (NP) 418–426 epitope in which mutations have accumulated. Whereas all NP 418–426 variant peptides induced a CTL response, cross-reactivity to the variants was affected. These NP mutations could have been selected over time in humans for the virus to escape HLA-B0702-restricted CTL responses since a similar response was seen in humans with, as in mice, altered cross-recognition of the NP 418–426 variants. These animals may prove a suitable model to study HLA-B0702-restricted CTL responses.

Keywords: cytotoxic T lymphocyte


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
HLA class I (Cl I) transgenic mice have been developed in several laboratories, in the hope that they would provide an animal model for the study of HLA class I-restricted cytolytic T lymphocyte (CTL) responses of interest in human disease (15). To allow the usage by mouse CTL of the transgenic human class I molecules, it was rapidly realized that the replacement of the HLA class I heavy chain third domain ({alpha}3) with its murine counterpart was necessary, a likely consequence of a more efficient interaction of the mouse accessory CD8 molecules with the chimeric than with the fully human HLA heavy chain (6,7). A further improvement in the use of HLA class I molecules as a restriction antigen by mouse CTL was observed when the HLA-A*0201 transgene was combined with inactivation of H-2 class I genes (8). Using these HLA-A*0201 transgenic H-2 class I knockout mice, we compared and optimized the immunogenicity of cancer- and virus-derived CTL epitopes, identified new epitopes, and analyzed the potential of vaccine formulations (911). Here, we extend this approach to the HLA-B*0702 allele, expressed by 15–20% of individuals in human populations. We characterized the HLA-B7m{alpha}3, H-2 Cl I-a–/– knockout mice and examine their ability to sustain a HLA-B0702-restricted CTL response following infection with influenza. We compared these responses to those seen in HLA-B7+ humans.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
HLA-B7m{alpha}3 transgene and mice
The HLA-B*0702 gene was isolated from a cosmid library from the HLA-B*0702 homozygous HHK lymphoblastoid human cell line. A 1.5-kb EcoRI–KpnI fragment (promoter, exons 1–3) of the HLA-B*0702 gene was ligated to a 4.1-kb KpnI–HindIII fragment (exons 4–8) of the H-2Kd gene. The chimeric HLA-B7m{alpha}3 gene was micro-injected as an EcoRI–HindIII fragment in C57BL/6 x SJL oocytes. Transgenic animals were backcrossed (12 times) on C57BL/6 JIco (H-2b) mice, before derivation of animals homozygous for the transgene. These mice were subsequently intercrossed with H-2KbDb double knockout mice [backcrossed 6 times on C57BL/6 JIco (12,13)] to derive HLA-B7m{alpha}3, Cl I-a–/– mice (backcrossed 6 times on C57BL/6 JIco). Mice were bred in our animal facility and used for experimentation at 6–10 weeks of age.

Immunofluorescence assays
Red blood cell-depleted, nylon wool-purified spleen T lymphocytes were analyzed for MHC class I expression in an indirect immunofluorescence assay. First layer mAb [B8-24-3 anti-H-2Kb (14) (ATCC, Manassas, VA), B22.249.R19 anti-H-2 Db (15) and ME.1 anti-HLA-B7 (16)] were incubated at saturating concentrations (30 min, 4°C) with cells. After 3 washes, mAb binding was measured with FITC-conjugated F(ab')2 goat anti-mouse Ig (Caltag, San Francisco, CA), and cells were analyzed by cytofluorometry (FACSCalibur; Becton Dickinson, San Jose, CA). Percentages of CD4+ and CD8+ splenic T lymphocytes were determined by double staining using FITC-conjugated rat anti-mouse CD4 (RM4-5) and phycoerythrin-conjugated rat anti-mouse CD8ß (CT-CD8b) mAb (Caltag). Expression of the different TCR Vß chains was similarly analyzed using purified, FITC-labeled Vß2 (B.20.6) (17), Vß4 (KT.4.10) (18), Vß5.1,.2 (MR.9.4) (19), Vß6 (44.22.1) (20), Vß7 (TR.310) (21), Vß8.1,.2,.3 (F.23.1) (22), Vß9 (MR.10.2) (19), Vß10 (B.21.5) (23), Vß11 (RR.3.15) (24), Vß12 (MR.11.1) (25), Vß13 (MR.12.4) (26), Vß14 (14/2) (27) and Vß17 (KJ.23.288.1) (28) specific mAb. Cells were incubated for 30 min on ice with these mAb and then, after washes, CD8+ T cells were labeled with phycoerythrin-conjugated rat anti-mouse CD8 mAb. Human HLA-B7 phenotyping was performed on Ficoll (Pharmacia, Uppsala, Sweden)-purified peripheral blood mononuclear cells (PBMC) by indirect immunofluorescence as indicated above using ME.1 anti-HLA-B7 antibody.

Peptide stabilization of HLA-B0702 molecules
Peptides at a minimum purity of 80%, purchased from SYNT:EM (Nîmes, France), were dissolved in DMSO (1 mg peptide/20 µl) and subsequently diluted in 1 x PBS (2 mg/ml). Peptides and HLA-B*0702-transfected TAP T2 cells [kindly provided by P. Cresswell (29)] were incubated overnight at 37°C (1 x 106 cells/ml) in FCS-free medium supplemented with 100 ng/ml human ß2-microglobulin (Sigma, St Louis, MO) in the absence (negative control) or presence of either reference human cytomegalovirus (CMV) pp65 265–274 (RPHERNGFTV, R10V) or tested peptides at various final concentrations (100, 10, 1 and 0.1 µM). Following 1 h incubation with Brefeldin A (0.5 µg/ml; Sigma), HLA-B*0702-transfected T2 cells were labeled (30 min, 4°C) with a saturating concentration of ME.1 anti-HLA-B7 mAb, then washed twice and finally stained with FITC-conjugated F(ab')2 goat anti-mouse Ig before cytofluorometry. For each peptide, the concentration needed to reach 20% of the maximal fluorescence (as defined with the R10V peptide) was calculated. Relative affinity is the ratio of the concentrations of the tested and R10V reference peptide needed to reach this value—the lower the relative affinity, the stronger the binding.

Mouse CTL induction, in vitro re-stimulation and cytolytic assay
Ketamine-anesthetized mice were inoculated intranasally with influenza virus (mouse-adapted A/PR/8/34 strain, 20 p.f.u./mouse). Spleen cells were collected 21 days later, depleted of red blood cells and re-stimulated in vitro by syngeneic, {gamma}-irradiated, influenza-infected cells as described before (10). After 6 days, each well was split and assayed for cytolytic activity against 51Cr-labeled (1.5 h at 37°C, 100 µCi/5 x 106 cells) influenza-infected and uninfected HLA-B7m{alpha}3-transfected RMA (H-2b) or P815 (H-2d) target cells. Four experimental mice and one uninfected control mouse were tested in each group. Wells were scored positive when the specific 51Cr release (subtracting for each individual well the background lysis on uninfected targets) exceeded by >3 SD the average spontaneous 51Cr release of 24 control wells without effector cells. CTL-precursor frequency was then calculated using Poisson’s law and GraphPad software.

For peptide immunizations, groups of six mice were injected s.c. at the base of the tail with 50 µg of HLA-B0702-restricted peptide and 140 µg of the Iab-restricted helper peptide [hepatitis B virus core 128–140, TPPAYRPPNAPIL (30)] co-emulsified in 100 µl of incomplete Freund adjuvant (Difco, Detroit, MI). Eight days later, spleen cells were re-stimulated in vitro as described before with peptide-pulsed, LPS-induced, {gamma}-irradiated (25 Gy) syngeneic lymphoblasts (9). On day 6, cultured cells were tested in a 4-h 51Cr-release assay, using experimental or negative control (human CMV pp65–265–274, R10V) peptide-pulsed, 51Cr-labeled HLA-B7m{alpha}3 P815 cells. Specific lysis was calculated as follow: (experimental – spontaneous release)/(total – spontaneous release) x 100, subtracting the background lysis of R10V control peptide-pulsed target cells. Mice were considered as responders when specific lysis >=10% was observed.

Human CTL in vitro re-stimulation and cytolytic assays
Blood samples were obtained following written informed consent from healthy platelet donors tested serologically negative for HIV, HCV and HBV viruses. Nitrogen-frozen HLA-B7+ Ficoll-purified human PBMC were thawed and incubated (4 x 106/well) in 24-well plates in RPMI 1640, 1 mM sodium pyruvate, 100 IU/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES and non-essential amino acids (all from Gibco/BRL, Paisley, UK) supplemented with 10% human serum (Institut Jacques Boy, Reims, France). They were stimulated with influenza-derived peptides at 2 x 10–6 M. On day 3, recombinant human IL-7 (25 ng/ml; kindly provided by Sanofi-Synthelabo, Labège, France) was added and on day 7, human IL-2 was added at 10 IU/ml (Roche, Mannheim, Germany) with fresh medium. On day 16, CD8+ T cells were selected using CD8 Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). CTL lines were subsequently re- stimulated twice monthly using peptide-pulsed Epstein–Barr virus-transformed, {gamma}-irradiated (50 Gy) autologous cells. Cytolytic assays were performed on 51Cr-labeled peptide-pulsed HLA-B*0702-transfected T2 cells. Specific lysis was calculated as follow: (experimental – spontaneous release)/(total – spontaneous release) x 100, subtracting the background lysis (which never exceeded 5%) of HLA-B*0702-transfected target T2 cells pulsed with a HLA-B0702-restricted, HIV1-derived, GP41 (843–851) IPRRIRQGL epitope (31).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
Expression of HLA-B7m{alpha}3 molecules and CD8+ T cell populations
Cell-surface expression of the HLA-B7m{alpha}3 molecules was assessed by indirect immunofluorescence on red blood cell-depleted, T lymphocyte-enriched, splenocytes. Similar levels of expression of HLA-B7m{alpha}3 were observed in H-2 Cl I-a–/– and H-2 Cl I-a+/+ HLA transgenic mice. In the latter case, the transgenic molecule is expressed at a level comparable to that of H-2Kb and Db (Fig. 1). Based on the number of CD8+ cells in H-2 Cl I-a–/– mice (2–3% of total T lymphocytes) and in HLA-B7m{alpha}3 transgenics on the H-2 Cl I-a–/– background (12–20%) (Fig. 2A), we conclude that the transgenic HLA-B7m{alpha}3 molecules contribute to thymic education and peripheral maintenance. However, the number of CD8+ T cells (25–30%) seen in HLA-B7m{alpha}3 on a H-2 Cl I-a+/+ background is larger. These CD8+ T cells are diverse in terms of Vß usage, as seen with a panel of 12 anti-Vß mAb. HLA-B7m{alpha}3 mice on a H-2 Cl I-a–/– background expressed a pattern of Vß genes similar to transgenics on a H-2 Cl I-a+/+ (Fig. 2B) background and to wild-type C57BL/6 [(13) and data not shown]. Thus the transgenic HLA-B7m{alpha}3 molecules participate in the selection of the CD8+ T cells, and elicit a diverse Vß repertoire qualitatively similar to that selected by H-2Kb and Db molecules.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 1. Cell-surface expression of HLA-B0702 on splenocytes of HLA-B7m{alpha}3, H-2 Cl I-a+/+ and HLA-B7m{alpha}3, H-2 Cl I-a–/– mice. (A) HLA-B7m{alpha}3, H-2 Cl I-a+/+ transgenic mice. Purified T lymphocytes were analyzed in an indirect immunofluorescence assay following incubation with either ME.1 (anti-HLA-B7), B-8-24-3 (anti-H-2Kb) or B22.249.R19 (anti-H-2Db) mAb. Abscissa: arbitrary units of fluorescence. (B) HLA-B7m{alpha}3, H-2 Cl I-a–/– knockout mice. Same legend as in (A).

 


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 2. Immunofluorescence analysis of peripheral T lymphocytes of HLA-B7m{alpha}3, H-2 Cl I-a–/– mice. Purified splenic T lymphocytes from four mice of the different mouse strains were individually analyzed for percentages of CD4+ and CD8+ T cells (A), and expression by CD8+ T lymphocytes of the TCR Vß gene families (B). Results (means + SD) are given as percentages of total T lymphocytes (A) or percentages of total CD8+ T cells (B). Mean values of total CD8+ T cell numbers from HLA-B7m{alpha}3, H-2 Cl I-a+/+ and HLA-B7m{alpha}3, H-2 Cl I-a–/– mice were significantly different with P = 0.0209 as calculated by the Mann–Whitney test.

 
Mouse CTL responses to influenza virus
We compared the HLA-B0702-restricted CTL responses of HLA-B7m{alpha}3, H-2 Cl I-a–/– and HLA-B7m{alpha}3, H-2 Cl I-a+/+ mice following intranasal infection with influenza virus (A/PR/8/34 strain) and in vitro re-stimulation with autologous influenza virus-infected splenocytes, under either bulk or limiting dilution culture conditions.

CTL activity against HLA-B7m{alpha}3-transfected, influenza- infected RMA (H-2b) target cells was documented at the bulk effector population level in all infected mice from both strains (Fig. 3A).



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3. Mouse CTL responses to influenza virus. (A) Bulk CTL responses. The splenocytes of four individual mice that were inoculated intranasally 3 weeks earlier with 20 p.f.u./mouse of A/PR/8/34 influenza virus were re-stimulated in vitro with influenza virus-infected syngeneic spleen cells as detailed in Methods. Six days later, the splenocytes were tested for cytolytic activity against HLA-B7m{alpha}3-transfected RMA (H-2b) 51Cr-labeled target cells. Specific lysis was calculated by subtracting the lysis of mock-infected targets. (B) HLA-B0702-restricted influenza-specific CTL-precursor frequencies. Decreasing numbers (from 50,000 to 1850) of responder cells of individual mice in vivo primed as in (A) were seeded per culture well, 24 replicates being set for each concentration, and re-stimulated in vitro with influenza virus-infected, {gamma}-irradiated, syngeneic spleen cells. On day 6, wells were split and tested for cytolytic activity against influenza virus-infected or uninfected HLA-B7m{alpha}3-transfected, allogeneic (H-2d) 51Cr-labeled P815 target cells.

 
As a first step to evaluate the HLA-B0702-restricted component of the CTL responses developing in both strains, effectors from each well of limiting dilution cultures were tested against HLA-B7m{alpha}3-transfected, influenza-infected H-2d P815 target cells, in order to obscure the component of the CTL response restricted by H-2Kb and/or H-2Db. Influenza-specific CTL-precursor frequencies ranging between 1/32 x 103 and 1/14 x 103 (average 1/19 x 103) were observed in HLA-B7m{alpha}3, H-2 Cl I-a–/– mice, and only between <1/1000 x 103 and 1/189 x 103 in HLA-B7m{alpha}3, H-2 Cl I-a+/+ (Fig. 3B). Taking into account the lower number of CD8+ T cells in HLA-B7m{alpha}3, Cl I-a–/– mice, this suggests a 20-fold relative enrichment in HLA-B0702-restricted CTL-precursor frequency when HLA-B7m{alpha}3 is expressed in a H-2 class I-a–/– context.

To rule out the possibility that the CTL responses could be restricted by H-2 class Ib molecules possibly shared by P815 cells, nine influenza A/PR/8/34-derived peptides with the major anchor sites for HLA-B0702 and capable of binding (32) were used to pulse HLA-B7m{alpha}3-transfected P815 target cells. Most CTL responses of bulk effector populations from influenza-infected mice focused on the nucleoprotein (NP) 418–426 L9M epitope, which is the strongest binder to HLA-B0702 molecules. Stronger responses against this peptide were induced in HLA-B7m{alpha}3, Cl I-a–/– mice than in Cl I-a+/+ transgenics (Table 1). We finally assayed the response to s.c. peptide vaccination using three influenza peptides with a high affinity to HLA-B0702. HLA-B7m{alpha}3, H-2 Cl I-a–/– mice showed stronger CTL responses against these peptides than HLA-B7m{alpha}3, H-2 Cl I-a+/+ transgenics (Table 1).


View this table:
[in this window]
[in a new window]
 
Table 1. Mouse cytolytic responses to influenza-derived peptides following virus infection or peptide immunization
 
Altogether, these results establish that HLA-B7m{alpha}3, H-2 Cl I-a–/– mice develop more frequent and more potent HLA-B0702-restricted, influenza-specific CTL responses than HLA-B7m{alpha}3, H-2 Cl I-a+/+ mice.

Mouse recognition of influenza NP 418–426 variants
The sequences of the NP 416–426 L9M epitope of different influenza strain A isolates (Table 2) show a striking accumulation of sense mutations between the two major anchor residues (proline in position 2, hydrophobic amino acid in position 9) for HLA-B0702 molecules. Could these mutations have been selected in human populations so that the virus might escape HLA-B0702-restricted CTL responses? We therefore assayed individually bulk CTL populations of three A/PR/8/34 infected, HLA-B7m{alpha}3, H-2 Cl I-a–/– mice against HLA-B7m{alpha}3-transfected P815 target cells pulsed with the NP 416–426 peptide variants, following three in vitro re-stimulations with NP 416–426 L9M peptide-pulsed, {gamma}-irradiated, syngeneic splenocytes. Compared to cells pulsed with the index peptide, no significant reduction of lysis was observed for the position 8 (V -> I) peptide variant. By contrast, a marked reduction was observed with peptide with a single position 5 (R -> K) substitution. Lysis was almost totally abrogated at low peptide concentrations for the position 5 and 6 (RT -> KS) or 4–6 (DRT -> ERA and DRT -> EKS) peptide variants (Fig. 4A). These results could either reflect reduced affinity for HLA-B0702 molecules of the variant peptides or alterations in TCR-contact residues. The latter hypothesis was proven to be correct (Table 3). The relative affinities of the variant peptides for HLA-B0702 molecules were similar and all stimulated efficient CTL responses when injected into HLA-B7m{alpha}3, H-2 Cl I-a–/– mice. Cross-testing those bulk CTL populations on peptide-pulsed target cells documented, whichever the immunizing peptide, moderate to profoundly impaired cross-recognition of the other peptide variants.


View this table:
[in this window]
[in a new window]
 
Table 2. NP 418–426 amino acid sequence of several human influenza A virus strain isolates
 


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 4. Cross-recognition of NP 418–426 L9M peptide variants by mouse and human CTL. (A) Bulk CTL populations of influenza-infected mice were tested following three in vitro re-stimulations with NP 416–426 L9M peptide-pulsed, {gamma}-irradiated, syngeneic splenocytes for cytolytic activity against HLA-B7m{alpha}3-transfected, 51Cr-labeled, P815 target cells pulsed (10–6 and 10–8 M) with the different NP 418–426 peptide variants. Means ± SD of specific lysis for the three mouse CTL lines at a 10:1 E:T ratio. (B) CD8+ human CTL from donors 38,590 and 71,320 were re-stimulated for 4 weeks in vitro with the LPFEKSTVM peptide as detailed in Methods. They were tested 10 days after the fourth re-stimulation against 51Cr-labeled HLA-B*0702-transfected TAP T2 cells pulsed (10–9 M) with the different NP 418–426 peptide variants. Specific lysis was calculated by subtracting the lysis of targets pulsed with the HIV1-derived IPRRIRQGL control peptide.

 

View this table:
[in this window]
[in a new window]
 
Table 3. Cross-recognition of influenza NP 418–426 peptide variants
 
HLA-B7-restricted CTL responses to influenza peptides in humans
PBMC of six HLA-B7+ (otherwise randomly selected) human donors without clinical history suggestive of recent influenza infection were first tested in an IFN-{gamma} ELISpot assay (data not shown) to identify those individuals developing NP 418–426-specific responses. Three responders to the LPFEKSTVM variant were identified, with in one case (donor 38590) a moderate response against the A/PR/8/34, LPFDRTTVM peptide. PBMC from two donors were re-stimulated in vitro with the LPFEKSTVM peptide and then tested against peptide-pulsed HLA-B*0702-transfected T2 cells in a 51Cr-release assay. Both donors developed potent CTL responses against the LPFEKSTVM peptide (Fig. 4B) with equal cross-recognition of the LPFERATVM variant and intermediate (LPFDRTTVM and LPFDRTTIM) to marginal (LPFDKTTIM and LPFDKSTVM) cross-recognition of the others.

Thus, in HLA-B7+ humans, the NP 418–426 peptide is as potent an epitope as it is in mice. The preferential recognition by both donors of LPFEKSTVM must be compared with the fact that this peptide has been repeatedly identified since 1980 in the H3N2 influenza A isolates collected worldwide. Impaired cross-recognition of several NP 418–426 variants is reminiscent of the results seen in mice. The potent cross-recognition of the LPFERATVM variant is both surprising and interesting, since this sequence, characteristic of the swine influenza viruses, has never been found in virus strains responsible for influenza outbreaks in humans, but was documented only in infected humans in close contact with swine (33,34). The absence of a detectable HLA-B7-restricted response in three donors could be explained in different ways. Most likely they might have developed immunodominant influenza-specific CTL responses restricted by other HLA class I molecules, which could have pre-empted the development of a HLA-B7-restricted response.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
We documented in this report the improved capacity of HLA-B7m{alpha}3, H-2 Cl I-a–/– animals to develop HLA-B0702-restricted responses, as compared to similar transgenics on a H-2 Cl I-a+/+ background. In the absence of significant differences in the levels of expression of the transgenic molecules, the absence of H-2 class I-a molecules improves the ability of mouse CD8+ T cells to utilize the HLA class I molecule as a restriction element. It seems likely that this observation will hold true as well for mice expressing other HLA class I alleles.

The limited usage of HLA class I molecules as restriction elements in H-2 Cl I-a+/+ mice is still not understood. Competition between HLA-B0702 and mouse H-2Kb H-2Db molecules for peptide capture is unlikely to be significant in view of the differences of their peptide binding motifs (35). Similarly, the structural diversity of the Vß and V{alpha} complementary-determining regions (CDR) 3 and the absence of species-specific structural features of the CDR1 and CDR2 variable subregions (36) argue against a strong bias of the mouse TCR repertoire in favor of H-2 class I molecules. We rather favor the possibilities that the interactions in the endoplasmic reticulum with the various chaperones that assist histocompatibility class I molecules in assembly and peptide loading and/or the interaction on the cell surface with the mouse CD8 accessory molecules (despite the {alpha}3 domain substitution) could account for the preferential usage of the H-2 class I molecules in HLA-B7m{alpha}3, H-2 Cl I-a+/+ mice. These two possibilities are not mutually exclusive and their respective importance could vary, depending on the HLA class I allele expressed. In fact, the improved usage of the HLA-B0702 molecule we observed in HLA-B7m{alpha}3, H-2 Cl I-a–/– mice compared to their H-2 Cl I-a+/+ counterparts was less pronounced than that observed with HLA-A*0201 transgenics (9). This suggests that the HLA-B7m{alpha}3 molecule could develop more efficient interactions than the HLA-A2m{alpha}3 one with some of the mouse molecules associated with the histocompatibility class I antigen-processing/presentation pathway. Such interallelic functional polymorphism in a xenogeneic setting was not unexpected, since it has already been documented for both HLA and H-2 class I molecules expressed in their natural cellular environment (37).

Expression of the chimeric HLA-B7m{alpha}3 molecules in a H-2 Cl I-a–/– context significantly increased the peripheral CD8+ T cell number (16% of total T cells versus 3% in non-transgenic H-2 Cl I-a–/– mice). Therefore, some 80% of peripheral CD8+ T lymphocytes in HLA-B7m{alpha}3, H-2 Cl I-a–/– must have been selected and maintained by the HLA-B7m{alpha}3 molecules. No major distortion of Vß usage in mice combining an HLAm{alpha}3 Cl I-a transgene with a H-2 Cl I-a deficiency was observed [this report and (10)]. From such observations and more detailed analysis of CDR3 in HLA-A*0201 transgenic H-2 Cl I–/– mice (38), we infer that CD8+ T cell selection driven by chimeric HLA-B7m{alpha}3 molecule results in a rather normal TCR repertoire. In fact, an efficient HLA-B0702-restricted CD8+ T cell response was seen in response to influenza virus in HLA-B7m{alpha}3, H-2 Cl I-a–/– mice even if the average frequency of CTL-precursors (1/9500), corrected for the number of CD8+ T cells, is slightly lower than that (1/3000) mobilized in C57Bl/6 mice under similar conditions (39).

Infection of HLA-B7m{alpha}3, H-2 Cl I-a–/– mice with influenza A/PR/8/34 virus consistently resulted in an immunodominant CTL response specific for the NP 418–426 L9M epitope. Numerous influenza A type isolates that arose since 1934 have been sequenced. Mutations of the major anchor residues to HLA-B0702 molecules (proline in position 2, methionine in position 9) were never observed. By contrast, many isolates from the H1N1 and H3N2 subtypes (responsible for the majority of influenza outbreaks in human populations) bear mutations in the central part of the NP 418–426 epitope that can be distinguished by TCR. Since the NP 418–426 sequence also appears to be a potent epitope for HLA-B7+ humans, we assume that the NP 418–426 variants have been over time immunoselected in HLA-B7+ individuals. Indeed, the 418–426 region of the influenza NP is a hot spot of structural variability (Table 2). Few other hot spots can be identified in the NP sequence. Interestingly, several of them concern sequences that encompass the canonical major anchor residues for other HLA class I molecules prevalent in human populations [NP 89–101, HLA-A68.1 (40); NP 188–198, HLA-A3 and HLA-A11 (32,41); NP 335–351, HLA-B8, B37, B44 and A11 (4244); NP 383–391, HLA-B27 (45)].

The relevance of observations made in HLA class I transgenic H-2 class I knockout mice for the human CTL repertoire is still a matter of debate. The peptides bound by HLA-B0702 molecules terminate with a hydrophobic residue, and thus are transported efficiently by mouse and human TAP molecules (46). Therefore, the only functional difference as yet documented between mouse and human class I processing machineries, i.e. impaired transport of peptides with a positively charged C-terminus by mouse TAP, should not reduce the cytolytic potential of HLA-B*0702 transgenic mice. More comparative studies are required to evaluate precisely the overlap between the HLA-B0702-restricted CTL responses of transgenic mice and humans. However, in view of the results reported here and from additional data we are collecting analyzing CTL responses to HIV 1 antigens (S. Cardinaud, unpublished results), we are confident that these mice will prove a reliable model for the study of HLA-B0702-restricted CTL responses developing in humans.


    Note added in proof
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
During the editing process, similar observations were published by Cheuk et al.: Cheuk, E., D’Souza, C., Hu, N., Liu, Y., Lang, H. and Chamberlain, J. W. 2002. Human MHC class I transgenic mice deficient for H2 class I expression facilitate identification and characterization of new HLA class I-restricted viral T cell epitopes. J. Immunol. 169:5571.


    Acknowledgements
 
This work was funded by the Institut Pasteur, the Ligue Nationale contre le Cancer, Comité de Paris, the Agence Nationale de Recherche sur le SIDA, the Association pour la Recherche sur le Cancer (grant 5129) and the Association pour la Recherche Médicale. P.-S. R. was supported by the Assistance Publique-Hôpitaux de Paris and S. C. by a fellowship from the Caisse Nationale d’Assurance Maladie et Maternité des Travailleurs Non Salariés des Professions Non Agricoles. We thank Pr Philippe Bierling for providing the human blood samples (Etablissement Français du Sang, Ile de France), Dr Soline Vigneau for help with database analysis and Nicole Sauzet for secretarial assistance.


    Abbreviations
 
CDR—complementary-determining region

Cl I—histocompatibility class I

CTL—cytotoxic T lymphocyte

CMV—cytomegalovirus

NP—nucleoprotein

PBMC—peripheral blood mononuclear cells


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 

  1. Chamberlain, J. W., Nolan, J. A., Conrad, P. J., Vasavada, H. A., Vasavada, H. H., Ploegh, H. L., Ganguly, S., Janeway, C. A., Jr and Weissman, S. M. 1988. Tissue-specific and cell surface expression of human major histocompatibility complex class I heavy (HLA-B7) and light (beta 2-microglobulin) chain genes in transgenic mice [published erratum appears in Proc. Natl Acad. Sci. USA 1989;86:3792]. Proc. Natl Acad. Sci. USA 85:7690.[Abstract]
  2. Barra, C., Perarnau, B., Gerlinger, P., Lemeur, M., Gillet, A., Gibier, P. and Lemonnier, F. A. 1989. Analysis of the HLA-Cw3-specific cytotoxic T lymphocyte response of HLA-B7 x human beta 2m double transgenic mice. J. Immunol. 143:3117.[Abstract/Free Full Text]
  3. Arnold, B. and Hammerling, G. J. 1991. MHC class-I transgenic mice. Annu. Rev. Immunol. 9:297.[CrossRef][ISI][Medline]
  4. Wentworth, P. A., Vitiello, A., Sidney, J., Keogh, E., Chesnut, R. W., Grey, H. and Sette, A. 1996. Differences and similarities in the A2.1-restricted cytotoxic T cell repertoire in humans and human leukocyte antigen-transgenic mice. Eur. J. Immunol. 26:97.[ISI][Medline]
  5. Kuon, W., Lauster, R., Bottcher, U., Koroknay, A., Ulbrecht, M., Hartmann, M., Grolms, M., Ugrinovic, S., Braun, J., Weiss, E. H. and Sieper, J. 1997. Recognition of chlamydial antigen by HLA-B27-restricted cytotoxic T cells in HLA-B*2705 transgenic CBA (H-2k) mice. Arthritis Rheum. 40:945.[ISI][Medline]
  6. Kalinke, U., Arnold, B. and Hammerling, G. J. 1990. Strong xenogeneic HLA response in transgenic mice after introducing an alpha 3 domain into HLA B27. Nature 348:642.[CrossRef][ISI][Medline]
  7. Vitiello, A., Marchesini, D., Furze, J., Sherman, L. A. and Chesnut, R. W. 1991. Analysis of the HLA-restricted influenza-specific cytotoxic T lymphocyte response in transgenic mice carrying a chimeric human-mouse class I major histocompatibility complex. J. Exp. Med. 173:1007.[Abstract]
  8. Pascolo, S., Bervas, N., Ure, J. M., Smith, A. G., Lemonnier, F. A. and Perarnau, B. 1997. HLA-A2.1-restricted education and cytolytic activity of CD8+ T lymphocytes from beta2 microglobulin (beta2m) HLA-A2.1 monochain transgenic H-2Db beta2m double knockout mice. J. Exp. Med. 185:2043.[Abstract/Free Full Text]
  9. Firat, H., Garcia-Pons, F., Tourdot, S., Pascolo, S., Scardino, A., Garcia, Z., Michel, M. L., Jack, R. W., Jung, G., Kosmatopoulos, K., Mateo, L., Suhrbier, A., Lemonnier, F. A. and Langlade-Demoyen, P. 1999. H-2 class I knockout, HLA-A2.1-transgenic mice: a versatile animal model for preclinical evaluation of antitumor immunotherapeutic strategies. Eur. J. Immunol. 29:3112.[CrossRef][ISI][Medline]
  10. Ureta-Vidal, A., Firat, H., Perarnau, B. and Lemonnier, F. A. 1999. Phenotypical and functional characterization of the CD8+ T cell repertoire of HLA-A2.1 transgenic, H-2KbnullDbnull double knockout mice. J. Immunol. 163:2555.[Abstract/Free Full Text]
  11. Tourdot, S., Scardino, A., Saloustrou, E., Gross, D., Pascolo, S., Cordopatis, P., Lemonnier, F. and Kosmatopoulos, K. 2000. A general strategy to enhance immunogenicity of low HLA A2.1 affinity peptides: implication in the identification of cryptic tumor epitopes. Eur. J. Immunol. 30:3411.[CrossRef][ISI][Medline]
  12. Vugmeyster, Y., Glas, R., Perarnau, B., Lemonnier, F. A., Eisen, H. and Ploegh, H. 1998. Major histocompatibility complex (MHC) class I KbDb –/– deficient mice possess functional CD8+ T cells and natural killer cells. Proc. Natl Acad. Sci. USA 95:12492.[Abstract/Free Full Text]
  13. Perarnau, B., Saron, M. F., San Martin, B. R., Bervas, N., Ong, H., Soloski, M. J., Smith, A. G., Ure, J. M., Gairin, J. E. and Lemonnier, F. A. 1999. Single H2Kb, H2Db and double H2KbDb knockout mice: peripheral CD8+ T cell repertoire and anti-lymphocytic choriomeningitis virus cytolytic responses. Eur. J. Immunol. 29:1243.[CrossRef][ISI][Medline]
  14. Kohler, G. 1981. Characterization of a monoclonal anti-H-2Kb antibody. Immune Syst. 2:202.
  15. Klein, J., Huang, H., Lemke, H., Hämmerling, G. and Hämmerling, U. 1979. Serological analysis of H-2 and Ia molecules with monoclonal antibodies. Immunogenetics 8:419.[ISI]
  16. Ellis, S. A., Taylor, C. and McMichael, A. 1982. Recognition of HLA-B27 and related antigen by a monoclonal antibody. Hum. Immunol. 5:49.[CrossRef][ISI][Medline]
  17. Gregoire, C., Rebai, N., Schweisguth, F., Necker, A., Mazza, G., Auphan, N., Millward, A., Schmitt-Verhulst, A. M. and Malissen, B. 1991. Engineered secreted T-cell receptor alpha beta heterodimers. Proc. Natl Acad. Sci. USA 88:8077.[Abstract]
  18. Tomonari, K., Lovering, E. and Spencer, S. 1990. Correlation between the V beta 4+ CD8+ T-cell population and the H-2d haplotype. Immunogenetics 31:333.[ISI][Medline]
  19. Utsunomiya, Y., Kosaka, H. and Kanagawa, O. 1991. Differential reactivity of V beta 9 T cells to minor lymphocyte stimulating antigen in vitro and in vivo. Eur. J. Immunol. 21:1007.[ISI][Medline]
  20. Acha-Orbea, H., Zinkernagel, R. M. and Hengartner, H. 1985. Cytotoxic T cell clone-specific monoclonal antibodies used to select clonotypic antigen-specific cytotoxic T cells. Eur. J. Immunol. 15:31.[ISI][Medline]
  21. Okada, C. Y., Holzmann, B., Guidos, C., Palmer, E. and Weissman, I. L. 1990. Characterization of a rat monoclonal antibody specific for a determinant encoded by the V beta 7 gene segment. Depletion of V beta 7+ T cells in mice with Mls-1a haplotype. J. Immunol. 144:3473.[Abstract/Free Full Text]
  22. Staerz, U. D., Rammensee, H. G., Benedetto, J. D. and Bevan, M. J. 1985. Characterization of a murine monoclonal antibody specific for an allotypic determinant on T cell antigen receptor. J. Immunol. 134:3994.[Abstract/Free Full Text]
  23. Necker, A., Rebai, N., Matthes, M., Jouvin-Marche, E., Cazenave, P. A., Swarnworawong, P., Palmer, E., MacDonald, H. R. and Malissen, B. 1991. Monoclonal antibodies raised against engineered soluble mouse T cell receptors and specific for V alpha 8-, V beta 2- or V beta 10-bearing T cells. Eur. J. Immunol. 21:3035.[ISI][Medline]
  24. Bill, J., Kanagawa, O., Woodland, D. L. and Palmer, E. 1989. The MHC molecule I-E is necessary but not sufficient for the clonal deletion of V beta 11-bearing T cells. J. Exp. Med. 169:1405.[Abstract]
  25. Kanagawa, O., Nussrallah, B. A., Wiebenga, M. E., Murphy, K. M., Morse, H. C., III and Carbone, F. R. 1992. Murine AIDS superantigen reactivity of the T cells bearing V beta 5 T cell antigen receptor. J. Immunol. 149:9.[Abstract/Free Full Text]
  26. Bill, J., Kanagawa, O., Linten, J., Utsunomiya, Y. and Palmer, E. 1990. Class I and class II MHC gene products differentially affect the fate of V beta 5 bearing thymocytes. J. Mol. Cell Immunol. 4:269.
  27. Liao, N. S., Maltzman, J. and Raulet, D. H. 1989. Positive selection determines T cell receptor V beta 14 gene usage by CD8+ T cells. J. Exp. Med. 170:135.[Abstract]
  28. Kappler, J. W., Wade, T., White, J., Kushnir, E., Blackman, M., Bill, J., Roehm, N. and Marrack, P. 1987. A T cell receptor V beta segment that imparts reactivity to a class II major histocompatibility complex product. Cell 49:263.[ISI][Medline]
  29. Smith, K. D. and Lutz, C. T. 1996. Peptide-dependent expression of HLA-B7 on antigen processing-deficient T2 cells. J. Immunol. 156:3755.[Abstract]
  30. Milich, D. R., Hughes, J. L., McLachlan, A., Thornton, G. B. and Moriarty, A. 1988. Hepatitis B synthetic immunogen comprised of nucleocapsid T-cell sites and an envelope B-cell epitope. Proc. Natl Acad. Sci. USA 85:1610.[Abstract]
  31. Korber, B., Barton Haynes, C. B., Koup, R., Moore, J. and Walker, B., eds. 1997. HIV Molecular Immunology Database. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM.
  32. Gianfrani, C., Oseroff, C., Sidney, J., Chesnut, R. W. and Sette, A. 2000. Human memory CTL response specific for influenza A virus is broad and multispecific. Hum. Immunol. 61:438.[CrossRef][ISI][Medline]
  33. Wentworth, D. E., Thompson, B. L., Xu, X., Regnery, H. L., Cooley, A. J., McGregor, M. W., Cox, N. J. and Hinshaw, V. S. 1994. An influenza A (H1N1) virus, closely related to swine influenza virus, responsible for a fatal case of human influenza. J. Virol. 68:2051.[Abstract]
  34. Altmuller, A., Kunerl, M., Muller, K., Hinshaw, V. S., Fitch, W. M. and Scholtissek, C. 1992. Genetic relatedness of the nucleoprotein (NP) of recent swine, turkey, and human influenza A virus (H1N1) isolates. Virus Res. 22:79.[CrossRef][ISI][Medline]
  35. Falk, K., Rotzschke, O., Stevanovic, S., Jung, G. and Rammensee, H. G. 1991. Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules. Nature 351:290.[CrossRef][ISI][Medline]
  36. Wilson, R. K., Lai, E., Concannon, P., Barth, R. K. and Hood, L. E. 1988. Structure, organization and polymorphism of murine and human T-cell receptor alpha and beta chain gene families. Immunol. Rev. 101:149.[ISI][Medline]
  37. Neisig, A., Wubbolts, R., Zang, X., Melief, C. and Neefjes, J. 1996. Allele-specific differences in the interaction of MHC class I molecules with transporters associated with antigen processing. J. Immunol. 156:3196.[Abstract]
  38. Firat, H., Cochet, M., Rohrlich, P., Garcia-Pons, F., Darche, S., Danos, O., Lemonnier, F. and Langlade-Demoyen, P. 2002. Comparative analysis of the CD8+ T cell repertoire of H-2 class I wild-type/and H-2 class I knock-out/HLA-A2.1 transgenic mice. Int. Immunol. 14:925.[Abstract/Free Full Text]
  39. Flynn, K. J., Belz, G. T., Altman, J. D., Ahmed, R., Woodland, D. L. and Doherty, P. C. 1998. Virus-specific CD8+ T cells in primary and secondary influenza pneumonia. Immunity 8:683.[ISI][Medline]
  40. Cerundolo, V., Tse, A. G., Salter, R. D., Parham, P. and Townsend, A. 1991. CD8 independence and specificity of cytotoxic T lymphocytes restricted by HLA-Aw68.1. Proc. R. Soc. Lond. B Biol. Sci. 244:169.[ISI][Medline]
  41. DiBrino, M., Tsuchida, T., Turner, R. V., Parker, K. C., Coligan, J. E. and Biddison, W. E. 1993. HLA-A1 and HLA-A3 T cell epitopes derived from influenza virus proteins predicted from peptide binding motifs. J. Immunol. 151:5930.[Abstract/Free Full Text]
  42. McMichael, A. J., Gotch, F. M. and Rothbard, J. 1986. HLA-B37 determines an influenza A virus nucleoprotein epitope recognized by cytotoxic T lymphocytes. J. Exp. Med. 164:1397.[Abstract]
  43. DiBrino, M., Parker, K. C., Margulies, D. H., Shiloach, J., Turner, R. V., Biddison, W. E. and Coligan, J. E. 1995. Identification of the peptide binding motif for HLA-B44, one of the most common HLA-B alleles in the Caucasian population. Biochemistry 34:10130.[ISI][Medline]
  44. Carreno, B. M., Anderson, R. W., Coligan, J. E. and Biddison, W. E. 1990. HLA-B37 and HLA-A2.1 molecules bind largely nonoverlapping sets of peptides. Proc. Natl Acad. Sci. USA 87:3420.[Abstract]
  45. Voeten, J. T., Bestebroer, T. M., Nieuwkoop, N. J., Fouchier, R. A., Osterhaus, A. D. and Rimmelzwaan, G. F. 2000. Antigenic drift in the influenza A virus (H3N2) nucleoprotein and escape from recognition by cytotoxic T lymphocytes. J. Virol. 74:6800.[Abstract/Free Full Text]
  46. Engelhard, V. H., Appella, E., Benjamin, D. C., Bodnar, W. M., Cox, A. L., Chen, Y., Henderson, R. A., Huczko, E. L., Michel, H., Sakaguichi, K., et al. 1993. Mass spectrometric analysis of peptides associated with the human class I MHC molecules HLA-A2.1 and HLA-B7 and identification of structural features that determine binding. Chem. Immunol. 57:39.[Medline]