Cytotoxic T lymphocytes (CTLs) play a central role in the control of persistent human immunodeficiency virus type 1 (HIV-1) infection [1,2]. However, HIV-1 employs several mechanisms for evading the potent CTL response that develops soon after infection . One important mechanism of viral immune evasion is the selective downmodulation of cell surface human leukocyte antigen class I (HLA class I) molecules by HIV-1 nef [4,5], which leads to protection of infected cells from CTL killing. Downregulation is selective for HLA class I A and B molecules, so infected cells continue to express HLA-C, which reduces their vulnerability to natural killer (NK) cell recognition . Evasion also occurs by mutation of the epitopes recognized by CTLs, which can lead to failure of the epitope to be generated by antigen processing, to bind to the HLA class I molecule or to be recognized by the T-cell receptor (TcR) of the responding CTL .
NK cells are a crucial component of the innate immune response to certain tumors and infections. NK cells can recognize HLA class I molecules through killer immunoglobulin-like receptors (KIRs) expressed on the cells surface. The engagement of inhibitory KIRs by their HLA class I ligands can prevent killing by NK cells [7,8]. KIR expression is highly variable on NK cells ; in general, KIR2D molecules tend to interact with HLA-C, whereas KIR3D interact with HLA-A and HLA-B [7,8,10]. We have recently demonstrated that the interaction between KIR3DL1 and its HLA ligands is exquisitely sensitive to variation in the KIR3DL1 allotypes, the HLA molecules and the nature of the bound peptide . This raises the possibility that epitope peptide variants generated in the course of HIV-1 evolution under CTL selective pressure in the infected host could differ in their recognition by specific KIR molecules.
HLA-C-restricted cytotoxic T-cell responses to HIV proteins have been described in HIV-1-infected individuals [12,13] and in some patients, the HLA-Cw4-restricted response to a peptide from gp120 (SF9) may represent the dominant CTL response (Makadzange et al., in preparation). The impact of HIV-1 Nef on HLA-A and B expression may lead to HLA-C-restricted responses playing a particularly important role in HIV-1 infection. Consistent with this hypothesis is the recent observation that a polymorphism associated with the increased expression of HLA-C is associated with delayed disease progression in HIV-1 infection . Alteration of NK cell phenotype and function during the course of HIV infection has been reported [15,16]. HLA-Cw4 can also serve as a ligand for KIR2DL1 [7,8], raising the possibility that variant peptides generated in response to HLA-Cw4-restricted CTL pressure may have altered interaction with the KIR2DL1 ligand. In this study, we evaluated the interaction between KIR2DL1 and the HLA-Cw4-restricted CTL HIV-1 gp120 epitope SFNCGGEFF (SF9), together with its naturally occurring variant SFNCGGEFL (SL9).
Materials and methods
Blood samples were obtained from HIV-1-infected individuals enrolled in an HIV clinic in Oxford, United Kingdom. Human subject approval was given by the Central Oxford Regional Ethics committee. Informed consent was obtained from participants.
Generation of cytotoxic T-lymphocyte clones and natural killer lines
CTL lines and clones were generated as previously described . NK lines were generated as previously described .
Surface plasmon resonance
HLA-Cw4 monomers were made as previously described and folded with the following peptides: QYDDAVYKL (QL9), representing a consensus Cw4-binding peptide , SFNCGGEFF (SF9) and SFNCGGEFL (SL9). Surface plasmon resonance studies were performed using a BIAcoreTM 2000 (BIAcore AB, St Albans, UK) as previously described . Experiments were performed at 25°C. HEPES buffered saline-EP (HBS-EP) buffer was used as the running buffer.
KD values and kinetic measurements were obtained either by Scatchard plots or by curve fitting of the data to the Langmuir binding isotherm. All analysis was done with BIAevaluation 3.2 RCI software (BIAcore AB) and graphs were produced with Origin software (version 5; Microcal Software, Northampton, Massachusetts, USA).
CTL clones or NK cell lines were stained with a panel of peptide-major histocompatibility complex (MHC) class I tetramer complexes as previously described . For blocking experiments, cells were incubated on ice with anti-KIR2DL1 (HP3E4) or isotype control antibody for 30 min before adding the tetramers
51Cr release cytotoxicity assay
Target cell lines (721.221 and HLA-Cw4 transfectant) were labeled with 100 μl of Na51CrO4 (Amersham International, Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) for 1 h at 37°C. 721.221-expressing HLA-Cw4 were pulsed with various concentrations of HIV peptides. Effector and target cells were incubated at 37°C for 4 h. Specific lysis was calculated by using the following formula: percentage specific lysis = [(experimental − spontaneous lysis)/(maximum − spontaneous lysis)] × 100%.
The HIV-1 gp120 epitope variant SL9 escapes recognition by human leukocyte antigen-Cw4-restricted cytotoxic T lymphocyte clones
The peptide SFNCGGEFF (SF9) represents the consensus sequence of the HLA-Cw4-restricted HIV-1 gp120 epitope in A, B and D clade strains of HIV-1. The TF9 (TFNCGGEFF) and SL9 (SFNCGGEFL) variants have been identified in HIV-1 isolates listed on the Los Alamos HIV Sequence database (http://www.hiv.lanl.gov). These variants were also found in viral sequences from HIV-1-infected Kenyan individuals in our study (Makadzange et al., in preparation). HLA-Cw4-restricted CTL generated from HIV-1-infected individuals recognized the SF9 peptide with high affinity as shown by their ability to kill target cells pulsed with low peptide concentrations. In contrast, lysis of targets pulsed with the SL9 peptide was only observed at an unphysiologically high peptide concentration (10−5 mol/l). The recognition of variant peptide dramatically decreased at 10−6 mol/l peptide concentration (Fig. 1a and b).
Human leukocyte antigen-Cw4 refolded with the SL9 peptide bind to KIR2DL1 with high affinity
HLA-Cw4 is also recognized by KIR2DL1, an inhibitory receptor on NK cells. We studied the binding of HLA-Cw4 folded with the HIV-1 gp120 peptide to KIR2DL1. The relative importance of class I-associated peptides in recognition differs between TcR and KIR molecules. Specific recognition of MHC peptide by a TcR depends largely on the peptide sequence; in contrast, minimal interactions are observed between the peptide and KIR2DL1 . Nevertheless, KIR recognition of HLA class I molecules does show some dependence on the bound peptide sequence [10,11], but direct contacts are limited to the peptide main chain at its COOH-terminal end. A previous study by Rajagopalan and Long  showed that KIR2DL1 did not interact with HLA-Cw4 bound to the SF9 peptide. Thus, we investigated whether the naturally occurring variants at carboxy terminus, such as SL9, could be recognized by KIR2DL1.
Surface plasmon resonance (SPR) analysis was used to determine equilibrium-binding affinities of HLA-Cw4 to KIR2DL1. HLA-Cw4 monomers folded with three different peptides were used. HLA-Cw4 QL9, HLA-Cw4 SF9 and HLA-Cw4 SL9 were each immobilized in one of four flow cells; the fourth flow cell contained bovine serum albumin (BSA) and served as a control. A range of concentrations from 168 down to 1 μmol/l of KIR2DL1 or KIR2DL3 were passed through all four flow cells at a rate of 10 μl/min. The KD values were determined using equilibrium-binding curves and Scatchard analysis of equilibrium binding. No significant affinity binding of any of the HLA-Cw4 monomers to the KIR2DL3 receptor was observed. HLA-Cw4 molecules expressing the HIV gp120 peptide wild-type epitope SF9 bound to KIR2DL1 with a KD of 23 μmol/l, which is within the range of typical cell–cell recognition events. On the other hand, the KIR2DL1 binding of HLA-Cw4 with the HIV peptide variant SL9 exhibited several times higher affinity (KD = 4.4 μmol/l) than that of SF9, comparable with the Cw4 consensus peptide QL9 (KD = 3.5 μmol/l) (Fig. 2a–d).
Human leukocyte antigen-Cw4-SL9 tetramers also bind to KIR2DL1 on an natural killer cell line
To confirm the binding of the HLA-Cw4 tetramer assembled with the SL9 peptide variants to KIR2DL1 naturally expressed on NK cells, an NK cell line was generated and stained with HLA-Cw4 refolded with either the SL9 or SF9 peptides. An HLA-Cw4-SF9-restricted CTL clone was also stained with the same SF9 and SL9 tetramers. As expected, the CTL clone showed better recognition of the SF9 compared with the SL9 tetramer (Fig. 2e), in keeping with the CTL lysis assays in which very low concentrations of the SF9 peptide could sensitize targets for lysis by HLA-Cw4-SF9-restricted CTL clones.
The expression of KIR2DL1 on the NK line was determined by staining with an antibody to KIR2DL1 (HP3E4), as shown in Fig. 2f. Similar to the results from the BIAcore study, the HLA-Cw4-SL9 tetramer could bind to KIR2DL1 on NK cell lines and the binding was abrogated by specific antibody. In contrast, only very weak binding was observed when the NK cell line was stained with the HLA-Cw4-SF9 index peptide tetramer (Fig. 2g).
Our data demonstrated that while HLA-Cw4-restricted T cells respond most efficiently to the index sequence of the gp120 SF9 peptide, the T-cell escape variant forms a better ligand for KIR2DL1 when bound to HLA-Cw4.
The selective downregulation of HLA class I by HIV-1 regulatory proteins has been shown to confer some resistance to HLA-A and HLA-B-restricted CTL lysis for the infected cell . A recent study by Adnan et al.  indicated that recognition of infected cells by HLA-C-restricted CTLs was unaffected by HIV-1 Nef expression, consistent with the ability of Nef to downregulate cell-surface HLA-A and HLA-B but not HLA-C molecules. As a consequence, HLA-C-restricted CTL responses may play a particularly important role in HIV-1 infection. Consistent with this hypothesis is the recent report that a single nucleotide polymorphism, which is known to lead to increased HLA-C expression on the cell surface, is associated with delayed disease progression in HIV-1 infection . We have observed the HLA-Cw4-restricted response to the gp120 SF9 peptide to be immunodominant in both European and African participants and noted that this was the only CTL response that could be detected in one long-term nonprogressor (LTNP) donor (Makadzange et al., in preparation). Taken together, selective downregulation of HLA class I A and B molecules provides a mechanism by which HIV-1-infected cells are able to maintain resistance to lysis by the majority of CTLs and NK cells, and hence establish a pool of long-lived infected cells in chronic HIV-1 infection.
HLA-C-restricted CTLs are, therefore, to provide strong selection pressure on the virus that would be likely to lead to the emergence of variants that escape CTL recognition. In this study, we show that the naturally occurring variant of the HLA-Cw4-restricted HIV-1 gp120 epitope SL9 represents an escape variant for wild-type SF9-specific HLA-Cw4-restricted CTL clones; this variant has been detected in patients with HLA-Cw4 (Makadzange et al., in preparation). However, this variant apparently provides the virus with an additional selective advantage, through its ability to bind significantly more strongly to the NK inhibitory receptor KIR2DL1. It is plausible that this peptide variant has been selected during viral evolution because it enables infected cells to escape simultaneously from CTL and NK recognition – a phenomenon of ‘double escape’.
We are very grateful to the donors who gave blood for this study. We thank Drs Peter Parham and Andrew McMichael for helpful discussion. We also thank Yuko Fukunaga for technical assistance. H.T., S.R.J. and T.D. prepared the manuscript. H.T., T.M., S.R.J. and T.D. designed the experiment. H.T., T.M., K.M., K.K. and Y.C.P. performed the experiment. C.C. provided clinical sample.
The present work was funded by the Medical Research Council, UK and the Ministry of Education, Culture, Sports, Science and Technology of Japan. H.T. was funded by the Royal Thai Government.
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