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The Journal of Immunology, 2003, 171: 1984-1993.
Copyright © 2003 by The American Association of Immunologists

Presentation and Binding Affinity of Equine Infectious Anemia Virus CTL Envelope and Matrix Protein Epitopes by an Expressed Equine Classical MHC Class I Molecule1

Travis C. McGuire2, Steven R. Leib, Robert H. Mealey, Darrilyn G. Fraser and David J. Prieur

Department of Veterinary Microbiology and Pathology, Washington State University College of Veterinary Medicine, Pullman, WA 99164


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Control of a naturally occurring lentivirus, equine infectious anemia virus (EIAV), occurs in most infected horses and involves MHC class I-restricted, virus-specific CTL. Two minimal 12-aa epitopes, Env-RW12 and Gag-GW12, were evaluated for presentation by target cells from horses with an equine lymphocyte Ag-A1 (ELA-A1) haplotype. Fifteen of 15 presented Env-RW12 to CTL, whereas 11 of 15 presented Gag-GW12. To determine whether these epitopes were presented by different molecules, MHC class I genes were identified in cDNA clones from Arabian horse A2152, which presented both epitopes. This horse was selected because it is heterozygous for the SCID trait and is used to breed heterozygous females. Offspring with SCID are used as recipients for CTL adoptive transfer, and normal offspring are used for CTL induction. Four classical and three putative nonclassical full-length MHC class I genes were found. Human 721.221 cells transduced with retroviral vectors expressing each gene had equine MHC class I on their surface. Following peptide pulsing, only cells expressing classical MHC class I molecule 7-6 presented Env-RW12 and Gag-GW12 to CTL. Unlabeled peptide inhibition of 125I-labeled Env-RW12 binding to 7-6-transduced cells demonstrated that Env-RW12 affinity was 15-fold higher than Gag-GW12 affinity. Inhibition with truncated Env-RW12 demonstrated that amino acid positions 1 and 12 were necessary for binding, and single substitutions identified positions 2 and 3 as possible primary anchor residues. Since MHC class I 7-6 presented both epitopes, outbred horses with this allele can be immunized with these epitopes to optimize CTL responses and evaluate their effectiveness against lentiviral challenge.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cytotoxic T lympcytes are associated with the control of primary viremia in equine infectious anemia virus (EIAV)3-infected horses, and later in carrier horses. After infection, viremia and associated clinical signs are usually controlled (1, 2), and this requires lymphocyte-specific responses because Arabian foals with SCID do not control the initial viremia, while control occurs in Arabian foals with a normal immune system (3). Further, reconstitution of an SCID foal with immune lymphocytes prevents continuous EIAV replication (4). Recurrence of viremia in infected horses is probably an outcome of antigenic variation, and in fact, antigenic variation of EIAV epitopes recognized by either neutralizing Ab (2, 5) or CTL (6) occurs. Since neutralizing Ab appears some time after the clearance of the initial viremia (1, 7), and CTL can be demonstrated concurrent with clearance (8), it seems that CTL are most important. A similar conclusion has been made in HIV-1-infected humans (9, 10) and SIV-infected rhesus monkeys (11, 12). Moreover, a necessary role for CD8+ lymphocytes in the clearance of the initial SIV viremia in rhesus monkeys and the maintenance of SIV control during chronic infection was determined by mAb depletion of these cells (13). In EIAV carriers, both neutralizing Ab (14) and CTL (14, 15, 16) occur, and drug-induced immunosuppression results in cell-free viremia within 8 days of treatment (17). This result is consistent with CTL restricting virus replication. Therefore, the induction of CD8+ T lymphocytes with cytotoxic activity is a critical component of immunologic control strategies for lentiviruses, including EIAV.

CD8+ CTL effectors that do not require in vitro stimulation are detected in 51Cr release assays using PBMC from EIAV-infected horses taken during initial viremia (8), but are not easily detectable 3 mo after infection (15). Memory CTL that require in vitro stimulation are present in PBMC during the initial viremia and can be detected at least 7 years after infection, the longest time tested (15). These studies detected only MHC class I-restricted CD8+ CTL, because equine kidney (EK) cells, which do not express MHC class II, were used as targets (15). These MHC class I-restricted CTL recognized epitopes in Gag, Pol, Env, Rev, and S2 proteins, with Gag p15 and p26 being the most frequently recognized proteins (18). Peptide epitopes have been defined from several of these proteins, including Gag (19), Env (6), and Rev (6). Identification of MHC class I molecules that present defined EIAV peptides would allow the selection of outbred horses for immunization studies with those defined peptides, and these experiments would help control a major variable in immunizing outbred animals.

In the horse there are at least two loci encoding classical, polymorphic MHC class I molecules, although locus-specific assignment for alleles is not yet possible (20, 21, 22, 23). There is evidence for a third classical MHC class I locus (24), and four putative nonclassical MHC class I molecules have been described (22). Serologic reagents identify 17 MHC class I ELA-A haplotypes, and 11 of these were available to type horses (25, 26). Even though typing was incomplete, CTL restriction in previous studies correlated with ELA-A haplotypes (8, 15), except for presentation of a 9-aa CTL epitope from p26 capsid protein (19). In preliminary experiments for this study, presentation of two previously defined, minimal 12-aa CTL epitopes (6), Env-RW12 (Env195–206RW12; RVEDVTNTAEYW) and Gag-GW12 (Gag21–32GW12; GSQKLTTGNCNW), was evaluated. Target cells from fourhorses with an ELA-A1 haplotype presented Env-RW12 to CTL effectors from EIAV-infected horse A2140 (A1/W11), while target cells from only two of these horses presented Gag-GW12. A horse with a W11 haplotype shared by descent, but lacking an A1 haplotype, did not present either peptide. Based on this observation, it was hypothesized that these two peptides were presented by different classical MHC class I molecules associated with the A1 haplotype. This hypothesis was evaluated by identifying cDNA clones with classical MHC class I genes from a horse presenting peptides Env-RW12 and Gag-GW12 to CTL and expressing these genes in target cells for use in CTL assays.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
PBMC stimulation and CTL assay

PBMC were isolated from blood from EIAV-infected horse A2140 and stimulated with peptide-pulsed monocytes (19). Infection of horse A2140 with EIAVWSU5 was described previously (18). EK target cells from 30 uninfected Arabian breed horses, designated by A before the number, or mixed breed ponies, designated by H (Tables I and II), were established from kidney tissue obtained by biopsy (8). The ELA-A haplotype was determined for these horses using a microcytotoxicity assay and described Ab reagents (25, 26, 27). In addition, PCR-single-strand conformation polymorphism analysis of exon 2 was used to type the minimally polymorphic MHC class II DRA locus and the more highly polymorphic DQA locus of some of the horses according to previously published protocols (28, 29). For stimulation with peptides, 40 µM peptide Env-RW12 or Gag-GW12 was added to PBMC in 17% FBS. Peptide and PBMC were incubated for 2 h at 37°C with occasional mixing before centrifugation at 250 x g for 10 min. PBMC were resuspended to 2 x 106/ml in RPMI 1640 medium with 10% FBS, 20 mM HEPES, 10 µg/ml gentamicin, and 10 µM 2-ME. One milliliter was added to each well of a 24-well plate and incubated for 1 wk at 37°C before use in CTL assays.


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  		Table I. Horse A2140 (A1/W11) CTL lysis of peptide-pulsed, ELA-A1 and non-A1 EK target cells

 

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  		Table II. Pedigree of A1 horses and MHC class II alleles linked to ELA-A1 haplotype

 
CTL activity was measured using a 51Cr release assay with a 17-h incubation period using autologous and heterologous EK cell lines. In addition, human B lymphoblastoid 721.221 target cells transduced with retroviral vectors expressing equine MHC class I genes described below were used in assays with a 5-h incubation period. Target cells were pulsed with various amounts of peptide Env-RW12 and Gag-GW12 as indicated in the tables or figures. The formula, % specific lysis = [(E-S)/(M - S)] x 100, was used, where E is the mean of six test wells, S is the mean spontaneous release from six target cell wells without effector cells, and M is the mean maximal release from six target cell wells with 2% Triton X-100 in distilled water (30). The E:T cell ratio was 20:1 in all assays, and each well contained ~30,000 target cells. Only assays with a spontaneous target cell lysis of <30% were used. The SE of percent specific lysis was calculated using a formula that accounts for the variability of E, S, and M (31). Significant lysis was defined in this paper as the percent specific lysis of peptide-pulsed target cells that was >8% and also >3 SE above the nonpulsed target cells or above target cells transduced with control vectors and pulsed with the relevant peptide.

cDNA library construction

PBMC were isolated as previously described (32) from Arabian horse A2152, which had an A1/A4 ELA-A haplotype. PBMC were cultured for 48 h in RPMI 1640 medium with 10% FBS, 2 mM L-glutamine, and 10 µg/ml gentamicin. Con A (40 µg/ml) was added to induce IFN-{gamma} production (33) and up-regulate MHC expression. mRNA (2.5 µg) isolated from these cells was used for first-strand synthesis, which was primed with 1 µg of oligo(dT)12–18 and 50 ng of random hexamers. This reaction was incubated at an initial temperature of 37°C for 15 min, followed by an additional hour at 49°C (34, 35). The second-strand reaction was incubated at 16°C for 2 h using Escherichia coli DNA ligase, polymerase, and RNase H (36). cDNA was blunt-ended with T4 DNA polymerase, EcoRI adapters were added with T4 ligase, and the adapters were phosphorylated with T4 polynucleotide kinase. The cDNA was then size fractionated, and cDNA between 2 and 3 kb was ligated into EcoRI-digested and dephosphorylated pcDNA3 vector (Invitrogen, Carlsbad, CA). E. coli electrocompetent cells (Invitrogen) were transformed with this ligation mixture resulting in a library containing 1.41 x 106 recombinants.

Clone selection and sequencing

Clones were selected from the library by colony lift hybridization with a 32P-labeled HindIII-XbaI fragment from horse MHC class I gene 8/9 (provided by Dr. D. F. Antczak, Cornell University, Ithaca, NY) (20). Positive colonies were isolated, and those with inserts of the correct size were sequenced. Sequencing was performed at the Laboratory for Biotechnology and Bioanalysis, Washington State University, using dye-labeleddideoxynucleotide cycle sequencing.

Retroviral vectors expressing equine MHC class I genes

Nine retroviral vectors were constructed using the plasmid pLXSN (provided by A. Dusty Miller, Fred Hutchinson Cancer Research Center, Seattle, WA). Each of the nine MHC class I genes was PCR-amplified and ligated into the cloning site of pLXSN downstream of the Moloney murine sarcoma virus long terminal repeat and upstream of the neomycin phosphotransferase gene, which was under the control of the SV40 early promoter (37). The sequences of the inserts and flanking plasmid DNA were determined. To generate vector-producing cell lines, a published procedure was used (38). In brief, an amphotropic packaging cell line, PA317 (CRL-9078; American Type Culture Collection, Manassas, VA) was transfected with each plasmid. Supernatant from PA317 cells was used to transduce amphotropic PG13 packaging cells (CRL-10686; American Type Culture Collection), which were then selected using 750 µg/ml of G-418 sulfate (Life Technologies, Gaithersburg, MD). Vectors were harvested from the selected PG13 cells and used to transduce CTL target cells (38). EK cells and human mutant B lymphoblastoid 721.221 cells (39) were transduced with the retroviral vectors expressing equine MHC class I genes, pulsed with peptides, and used as CTL targets. The 721.221 cells (obtained from Dr. A. Sette, Epimmune, San Diego, CA) express {beta}2-microglobulin, but not HLA-A, -B, or -C class I molecules. These cells were used in similar studies to express human and other primate MHC class I genes for functional studies (40, 41, 42).

Fluorescent flow cytometry

The expression of equine MHC class I genes in transduced 721.221 cells was demonstrated by fluorescent flow cytometry (43). Nontransduced 721.221 cells did not bind previously described mAb H1A and H6A to equine MHC class I molecules (44), and these Abs were used to detect cell surface expression. In addition, increased binding of mAb B2M-01 to human {beta}2-microglobulin (Caltag Laboratories, Burlingame, CA) was used to help verify the expression of the equine MHC class I molecules in the transduced cells. Binding of mAb to cells was detected using fluorochrome-labeled Abs to mouse Igs (Caltag Laboratories). A one-tailed Mann-Whitney U test was used to compare the percentage of positive cells of triplicate assays of 721.221 cells transfected with equine MHC class I genes reacted with each mAb with 721.221 cells transfected with a control retroviral vector that did not contain a class I gene reacted with the same mAb (45).

Live cell, peptide binding assay

Peptide binding to equine MHC class I molecule 7-6 expressed on transduced 721.221 cells was measured as previously described with minor modifications (41, 46, 47). 721.221 cells (2 x 106/ml) transduced with equine classical MHC class I gene 7-6 or with a retroviral vector that did not express an equine gene were preincubated overnight in RPMI with 15% FBS and 3 µg/ml of human {beta}2-microglobulin (The Scripps Clinic, La Jolla, CA) at 27°C. The cells were washed twice in RPMI alone and resuspended to 2 x 107 cells/ml in RPMI containing 2.5% FBS, 3 µg/ml of {beta}2-microglobulin, 1 mg/ml of EDTA, 250 µg/ml of PMSF (Sigma-Aldrich, St. Louis, MO), and 60 µg/ml of N{alpha}-p-tosyl-L-lysine chloromethyl ketone (Sigma-Aldrich). One hundred microliters containing 2 x 106 cells plus 1 µl containing 1.5 x 105 cpm of 125I-labeled Env-RW12 were incubated for 4 h at 22°C. Env-RW12 was synthesized and HPLC-purified by Genmed Synthesis (San Francisco, CA), and 25 µg was labeled with 1 mCi of 125I as previously described (48). Free radioactivity was separated from peptide-bound radioactivity using a Sephadex G-10 matrix (Amersham Pharmacia Biotech, Piscataway NJ) in a 1 x 50-cm column. After the 4-h incubation, cells were washed three times with serum-free medium and then centrifuged through calf serum to remove any remaining unbound radiolabeled peptide. The cell pellet was then counted with a gamma scintillation counter.

Competitive inhibition assays were performed in the same manner as binding assays, except 10 µl containing sufficient unlabeled peptide competitors to result in final concentrations of 1–1000 nM was added to the initial mixture of cells and radiolabeled Env-RW12 peptide. Competing peptides were truncations of Env-RW12 and complete Gag-GW12 synthesized at the Laboratory for Biotechnology and Bioanalysis, Washington State University, and Env-RW12 peptides with single amino acid substitutions of each polar amino acid with leucine and each nonpolar amino acid with lysine synthesized by Genmed Synthesis. The IC50 for competing peptides was calculated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CTL lysis of ELA-A1 target cells from different horses pulsed with Env-RW12 or Gag-GW12

To determine whether target cells from different horses with an ELA-A1 haplotype could present Env-RW12 and Gag-GW12 to CTL that recognized these peptides, peptide-pulsed EK target cells from 15 ELA-A1 horses and 15 non-ELA-A1 horses were evaluated (Table I). CTL effectors were PBMC from EIAV-infected horse A2140 (A1/W11) stimulated with either Env-RW12 or Gag-GW12. Target cells from all 15 horses with an A1 haplotype had significant Env-RW12-specific lysis, while the non-A1, Env-RW12-pulsed targets were not significantly lysed (Table I). The lysis of Env-RW12-pulsed targets was not restricted by molecules associated with the A2140 W11 haplotype because A2145 (A5/W11) and A2140 (A1/W11) inherited W11 from the same sire, and A2145 Env-RW12-pulsed targets were not lysed (Table I). CTL stimulated with peptide Gag-GW12 significantly lysed 11 of 15 Gag-GW12-pulsed A1 targets and none of the non-A1 targets. These data demonstrated that all 15 A1 targets tested presented Env-RW12 peptide to A2140 CTL, whereas peptide Gag-GW12 was not always presented by these A1 targets.

Since it was not clear whether the two peptides were presented by the same or different classical class I molecules associated with the A1 haplotype, horse pedigrees and linkage of ELA-A1 to MHC class II alleles were analyzed (Table II). Among the six Arabian horses listed as A1 targets in Table I, the A1 in A2112 and A2116 was identical by descent, and both EK targets presented both peptides to A2140 CTL. The A1 in A2144 and A2150 was identical by descent and linked to MHC class II DRA*0101/DQA*1901 alleles. EK targets from both horses presented Env-RW12, but not Gag-GW12, to A2140 CTL. Horses 2140, 2148, and 2152 inherited an A1 haplotype from three different dams that was linked to MHC class II DRA*0201/DQA*0901 alleles. EK targets from these horses presented both peptides to A2140 CTL probably with the same or a very similar classical MHC class I molecule. Among the nine mixed breed ponies listed as A1 targets in Table I, H634 inherited an A1 haplotype from its dam H597, and EK targets from both horses presented both peptides (Table II). Progeny studies demonstrated that H504 had two different A1 haplotypes, one linked to MHC class II DQA*0401 that presented Env-RW12, but not Gag-GW12, to A2140 CTL, whereas the other A1 haplotype was linked to MHC class II DQA*1201 and presented both peptides. H521 inherited the A1 haplotype from H504 linked to DQA*0401 and presented Env-RW12, but did not present Gag-GW12. Interestingly, the H603 A1 haplotype was linked to DQA*0401 and presented Env-RW12, but not Gag-GW12. Nothing was known about the pedigrees of H558, H559, H575, or H601, which all presented both peptides. Even though it was clear that those ELA-A1 horses that failed to present Gag-GW12 had different pedigrees and different class II linkages than those that presented both peptides, it was still not clear whether the two peptides were presented by the same or different MHC class I molecules associated with the A1 haplotype in those horses that presented both peptides.

Selection of horse A2152 as a source of mRNA for cloning MHC class I genes

To identify the equine MHC classical class I molecules(s) presenting minimal CTL epitopes Env-RW12 and Gag-GW12, horse A2152 (A1/A4) was selected as a source of mRNA for a cDNA library. Horse A2152 EK target cells pulsed with either Env-RW12 or Gag-GW12 were killed by CTL from horse A2140 (A1/W11), demonstrating that these peptides were presented by one or more A2152 MHC class I molecules (Table I). In addition, horse A2152 is a male carrier of the SCID trait and is being used to breed SCID carrier mares. Foals with SCID are used in studies to determine the mechanisms of lentiviral disease (3, 49) and immune control (4), including evaluating the effectiveness of adoptive transfer of CTL in protection against EIAV challenge.

Classical and putative nonclassical MHC class I genes were cloned from the cDNA library

Clones with MHC class I genes were identified by hybridization with a probe made from a previously cloned equine MHC class I gene (20). One hundred and ten clones were evaluated by sequencing, and seven full-length MHC class I {alpha} genes and two pseudogenes were identified. These genes and their GenBank accession numbers are listed in Table III. Four genes (7-1, 7-4, 7-6, and 8-5) encoded proteins with transmembrane domains of 35 aa and shared other characteristics with equine classical MHC class I molecules described to date (20, 21, 22). These four alleles differed from each other by at least 21 aa in the peptide-binding {alpha}1 and {alpha}2 domains; however, a locus assignment could not be made. Also, it is not known whether these four were the total number of classical MHC class I alleles present in horse A2152. Two genes had the same sequence as two putative nonclassical class I genes previously designated A1 (unrelated to the ELA-A haplotype nomenclature and referred to henceforth as ncA1) and C1 (22). These two putative nonclassical genes contained a 9- to 15-base insertion and/or a 6-base deletion in the sequence encoding the transmembrane domain (22). The finding of ncA1 and C1 genes in horse A2152 further supports, but does not prove, that these are nonclassical MHC class I genes. Another gene, 7-51, was also considered a nonclassical gene based on sequence similarity to putative nonclassical class I gene B3 in group D of a published paper (22). Gene 7-7 resembled classical genes with a transmembrane domain of 35 aa; however, the open reading frame extended 52 aa past the stop codon of the classical genes. Gene 7-7 was considered a pseudogene based on this structural feature and the lack of cell surface expression (see next section). The ninth gene, 7-12, had a transmembrane domain of 27 aa, followed by a stop codon with no cytoplasmic domain and, based on this and the lack of surface expression (see next section), was also considered a pseudogene.


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  		Table III. MHC class I genes derived from a cDNA library from horse A2152

 
Expression of single equine MHC class I molecules on human B lymphoblastoid 721.221 cells

The human B lymphoblastoid cell line 721.221 does not express HLA-A, -B, and -C {alpha}-chains (39) and was employed to evaluate the surface expression of equine MHC class I {alpha}-chains. Retroviral vectors expressing the nine equine {alpha}-chains (Table III) were made, and 721.221 cells were transduced with each vector. Following antibiotic selection, the transcription of each gene was demonstrated by RT-PCR. Cell surface expression was evaluated by flow cytometry using mAb to equine MHC class I and to human {beta}2-microglobulin, and the results of one construct with controls are presented in Fig. 1. Cell lines transcribing the four classical and three putative nonclassical MHC class I genes had significant binding of either one or both mAb to equine MHC class I with the mean percent positive ranging from 27–73% (Fig. 2). Cell lines transduced with pseudogenes 7-7 and 7-12 did not bind either of the mAb. For the data in Fig. 2, the percentage of cells of each transduced cell line binding a mAb was determined in triplicate by comparing binding to control 721.221 cells transduced with a retroviral vector lacking an equine MHC class I gene. The gate for these control cells incubated with each mAb was adjusted so that <3% of the total cells were positive. Significant binding was determined by comparing the percentage of positive cells of each transfected cell line with the cells transduced with pseudogene 7-7 or the control vector. Surface expression was confirmed by detecting significant binding of the mAb to human {beta}2-microglobulin with the mean percent positive ranging from 28–56% on six of the equine MHC class I-positive cell lines, whereas the seventh (8-5) had 8%. These results indicated that the expression of equine MHC class I protein on the cell surface resulted in additional human {beta}2-microglobulin on the cell surface. Cells transduced with pseudogenes 7-7 and 7-12 did not have an increase in {beta}2-microglobulin on their surface (Fig. 2). It was concluded that 721.221 cells transduced with the four classical and three nonclassical MHC class I genes expressed class I proteins on the cell surface, while those transduced with the two pseudogenes did not.



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  		FIGURE 1. Fluorescent flow cytometric evaluation of equine MHC class I 7-6 expression on 721.221 cells. Cells in the top row were transduced with a retroviral vector with MHC class I 7-6, whereas cells in the bottom row were transduced with a vector lacking a class I gene (CV). Cells were reacted with a control mAb, mAb H6A to equine MHC class I (class I), and mAb to human {beta}2-microglobulin ({beta}2M).

 


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  		FIGURE 2. The percentage of transfected 721.221 cells expressing different equine MHC class I molecules and {beta}2-microglobulin. mAb to human {beta}2-microglobulin ({beta}2M) and to equine MHC class I molecules (H1A and H6A) were reacted with 721.221 cells transduced with class I genes, including four classical (C), three nonclassical (NC), and two pseudogenes (P). The columns are the mean percentage of positive cells from three different assays, and the bars represent 1 SD. The increase in the percentage of positive cells in cell line 8-5C reacted with mAb to {beta}2M was statistically significant as were the other columns with higher means than 8-5C reacted with mAb to {beta}2M.

 
Presentation of peptide Env-RW12 to CTL was restricted by equine MHC class I 7-6

To identify the A2152 (A1/A4) MHC class I molecule presenting the minimal 12-aa peptide epitope Env-RW12 to A2140 (A1/W11) CTL, 721.221 cells transduced with the seven MHC class I genes and two pseudogenes (Table III) were pulsed with Env-RW12 and used as target cells. PBMC from EIAV-infected horse A2140 were stimulated with peptide Env-RW12 and used as effector cells. Only cells transduced with equine classical MHC class I gene 7-6 and pulsed with peptide Env-RW12 were significantly lysed by stimulated CTL (Fig. 3). The specific lysis of the 7-6 targets was 71% compared with <1% for cells transduced with the other eight targets and a vector control (Fig. 3). In the absence of peptide, the specific lysis of all nine transduced target cells in CTL assays was <2% (data not shown).



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  		FIGURE 3. 721.221 cells expressing equine MHC class I 7-6 presented peptide Env-RW12 to CTL. 721.221 cells transduced with different MHC class I genes were pulsed with 10 µM peptide Env-RW12 and used as targets in a CTL assay with Env-RW12-stimulated PBMC from horse A2140 (A1/W11). CV, Cells transduced with a control vector lacking a class I gene. The SE for the percent specific lysis was <2 for each transduced target. *, Significant lysis.

 
The presentation of peptide Env-RW12 by equine MHC class I 7-6-expressing human target cells to CTL was verified in equine target cells. EK cells from ELA-A-mismatched horse 585 (A6), which did not present peptide Env-RW12 to Env-RW12-stimulated CTL from horse A2140 (A1/W11), were transduced with the retroviral vector expressing 7-6 and seven other MHC class I genes. Those EK cells transduced with gene 7-6, Env-RW12-pulsed, and incubated with Env-RW12-stimulated CTL from horse A2140 were significantly lysed (68%), whereas those transduced with seven other genes were not (Fig. 4). EK cells transduced with 7-6 and not pulsed with peptide had 6% specific lysis (data not shown). Therefore, only equine classical MHC class I 7-6 expressed in either human or equine target cells presented peptide Env-RW12 to CTL.



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  		FIGURE 4. Equine target cells expressing MHC class I 7-6 presented peptide Env-RW12 to CTL. EK cells from horse 585 (A6) transduced with different MHC class I genes and pulsed with 10 µM peptide Env-RW12 were used as CTL targets with Env-RW12-stimulated PBMC from horse A2140 (A1/W11). The SE for the percent specific lysis was 4.1 for 7-6 targets and <2 for all others. *, Significant lysis.

 
Peptide Gag-GW12 was also restricted by MHC class I 7-6

To identify which MHC class I protein presented the other minimal 12-aa epitope, Gag-GW12, the same transduced 721.221 cells used in Fig. 3 were pulsed with Gag-GW12 and used as targets in CTL assays. Significant killing of 721.221 target cells transduced with gene 7-6 and pulsed with peptide Gag-GW12 occurred with horse A2140 CTL stimulated with peptide Gag-GW12, and the percent specific lysis (±SE) was 22 ± 1.4% (Fig. 5). Among the other nine transduced and peptide-pulsed 721.221 targets, the maximum lysis was 6.6 ± 0.5% (Fig. 5), and this was not considered significant as defined in Materials and Methods. In addition, there was no significant lysis of the nonpeptide-pulsed cells expressing gene 7-6 (not shown). This result was verified in EK target cells by transducing ELA-A-mismatched horse 585 (A6), which did not present peptide Gag-GW12 to stimulated CTL from horse A2140 (A1/W11), with the retroviral vector expressing gene 7-6. There was significant lysis of these cells pulsed with peptide Gag-GW12 (33 ± 1.9%) compared with peptide-pulsed EK cells transduced with a retroviral vector lacking an MHC class I gene (9 ± 1.0%). Nonpulsed EK cells expressing 7-6 had specific lysis of 5 ± 1.0%. Thus, the classical MHC class I 7-6 molecule that presented peptide Env-RW12 also presented peptide Gag-GW12 to CTL.



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  		FIGURE 5. Peptide Gag-GW12 was presented by 721.221 cells expressing equine MHC class I 7-6. 721.221 cells transduced with different MHC class I genes were pulsed with 10 µM Gag-GW12 and used as targets in a CTL assay with Gag-GW12-stimulated PBMC from horse A2140 (A1/W11). The bars on each column are 1 SE. *, Significant lysis.

 
Peptides Env-RW12 and Gag-GW12 were recognized by different CTL populations

To verify that killing of 721.221 target cells expressing equine MHC class I 7-6 and pulsed with peptide Env-RW12 or Gag-GW12 was caused by different CTL populations, horse A2140 PBMC were divided and stimulated with individual peptides. Env-RW12-stimulated CTL lysed Env-RW12-pulsed targets beginning at 100 nM peptide pulse, with maximum lysis occurring at 102–105 nM (Fig. 6). The amount of Env-RW12 peptide required to cause 50% maximum lysis of pulsed target cells was 1.8 nM. The Env-RW12-stimulated effectors did not lyse Gag-GW12-pulsed targets at any peptide concentration tested (Fig. 6). Similarly, Gag-GW12-stimulated CTL lysed Gag-GW12-pulsed targets, and there was no specific lysis of Env-RW12 pulsed targets. The amount of Gag-GW12 peptide required to cause 50% maximum lysis of pulsed target cells was 126 nM. These data demonstrated that even though the two peptides were presented by the same classical MHC class I molecule, they were recognized by different CTL populations. In addition, the amount of Gag-GW12 peptide required for 50% maximum lysis of pulsed target cells expressing classical equine MHC class I 7-6 was 70 times more than that required for Env-RW12.



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  		FIGURE 6. Peptide Env-RW12-stimulated CTL were peptide specific. Env-RW12:Env-RW12, CTL were stimulated, and 721.221 targets were pulsed with Env-RW12; Env-RW12:Gag-GW12, the same CTL, but targets were pulsed with Gag-GW12. The error bars for percent specific lysis are 1 SE.

 
Identification of amino acid residues for optimal peptide binding to MHC class I 7-6

To identify amino acid residues for optimal peptide binding, a live cell, peptide binding assay described by others (44, 45) was used, and 125I-labeled peptide Env-RW12 binding to 721.221 cells expressing classical equine MHC class I 7-6 occurred. This binding was inhibited in a dose-dependent manner by both unlabeled Env-RW12 and Gag-GW12 peptides (Fig. 7). The affinity of Env-RW12 binding was 15 times greater than that of Gag-GW12 based on an IC50 of 39 nM for Env-RW12 and 605 nM for Gag-GW12 (Table IV). A previously described natural antigenic variant of Env-RW12, Env-HW12V (6), had an IC50 of 880 nM in this study (Table IV), indicating that part of the lack of recognition of this peptide by CTL that recognized Env-RW12 was due to reduced binding to MHC class I 7-6. However, this did not explain the complete lack of recognition observed in previous CTL assays (6), indicating that there was also decreased TCR recognition.



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  		FIGURE 7. Unlabeled Env-RW12 and Gag-GW12 peptides inhibited 125I-labeled Env-RW12 binding to live 721.221 cells expressing MHC class I 7-6. The Env-RW12 sequence was RVEDVTNTAEYW, and the Gag-GW12 sequence was GSQKLTTGNCNW.

 

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  		Table IV. IC50 of peptide Env-RW12 truncations, Gag-GW12, and a natural variant of Env-RW12.

 
In a previous study (6) epitope mapping using truncations of Env-RW12 and Gag-GW12 in CTL assays demonstrated the critical importance of both the N-terminal position 1 and the C-terminal position 12 residues for lysis of peptide-pulsed cells by CTL. The lack of lysis with these truncated peptides was probably due to lack of binding to MHC class I 7-6, because an 11-aa version of Env-RW12 lacking position 1 caused 0% inhibition at 1000 nM (Table IV). This 11-aa peptide also served as a negative control peptide for the inhibition assay. A 12-aa version of Env-RW12 extending from positions -1 to 11 caused only 21% inhibition at 1000 nM (Table IV). Therefore, positions 1 and 12 were necessary residues for optimal binding to MHC class I 7-6.

Possible primary anchor residues were identified by substituting each polar amino acid of Env-RW12 with leucine and each nonpolar amino acid with lysine (Table V). Only substitutions at positions 2 and 3 resulted in a >10-fold reduction in inhibitory capacity, indicating that these were primary anchor residues (40). To verify that these are anchor residues, additional substitutions are needed to determine whether the majority result in a similar reduction in inhibitory capacity. The substitutions tested at positions 1 and 12 resulted in 4.9- and 2.8-fold reductions in inhibitory capacity, respectively, making it unclear whether these necessary positions were primary or secondary anchor residues. Substitution of position 6 reduced the inhibitory capacity by 4.3-fold, while only substitutions at positions 9 and 11 had no effect. The results presented in Fig. 6 and Tables IV and V demonstrated that the live cell, peptide binding assay using 721.221 cells expressing equine MHC class I gene 7-6 was specific and could be used for further determination of primary and secondary anchor residues of binding peptides.


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  		Table V. IC50 of peptide Env-RW12 with single amino acid substitutions

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These experiments defined the first equine classical MHC class I molecule that can present peptides to CTL. This was demonstrated by sequencing cDNA clones containing MHC class I genes and identifying four classical and three putative nonclassical sequences. This classification was based on published sequences and criteria, including the presence of a 35-aa transmembrane domain of equine classical MHC class I molecules, in contrast to some of the putative nonclassical MHC class I molecules that have a 9- to 15-base insertion and/or a 6-base deletion in the coding sequence of the transmembrane domain (21, 22). Two of the cloned nonclassical genes were exactly the same as two putative nonclassical MHC class I genes designated ncA1 and C1, while gene 7-51 was similar to a third putative nonclassical gene B3 (22). To evaluate peptide presentation to CTL, the classical and nonclassical genes obtained were inserted into retroviral vectors that were used individually to transduce the human B lymphoblastoid cell line 721.221. All these transduced cells expressed equine MHC class I on their cell surface; however, only the one expressing equine classical MHC class I 7-6 presented EIAV peptides Env-RW12 and Gag-GW12 to CTL.

Cell surface expression of single equine MHC class I molecules was demonstrated on transduced 721.221 cells that do not express HLA-A, -B, and -C {alpha}-chains (39). This was done by detecting binding of mAb H1A and H6A, which recognize equine MHC class I molecules (44), and finding increased binding of a mAb to human {beta}2-microglobulin by flow cytometry. All four of the classical MHC class I molecules and the three putative nonclassical class I molecules were expressed on the cell surface, but two transcribed pseudogenes were not. However, the expression of equine MHC class I genes on these cells may not have been optimal because of the absence of equine {beta}2-microglobulin, and this may have affected the expression of some genes more than others. mAbs H1A and H6A did not bind to all the same expressed molecules, indicating that they did not bind a shared epitope among the seven equine MHC class I molecules from A2152. However, surface expression of equine MHC class I molecules on 721.221 cells allowed further evaluation of their ability to present peptides to CTL.

The product of a single classical MHC class I gene, 7-6, clearly presented the two different minimal 12-aa epitopes, Env-RW12 and Gag-GW12, to CTL. None of the other expressed classical and putative nonclassical molecules presented these peptides. Thus, the hypothesis that Env-RW12 and Gag-GW12 were presented by different MHC class I molecules was incorrect. This hypothesis was based on the preliminary observation that target cells from four horses with an ELA-A1 haplotype determined by serology presented Env-RW12 to CTL from A2140 (A1/W11), which recognized both Env-RW12 and Gag-GW12, while target cells from only two of these horses presented Gag-GW12 to the CTL. These results were expanded in this study to EK target cells from a total of 30 horses. Similar to the data in preliminary studies, target cell presentation of peptide Env-RW12 to A2140 CTL occurred with targets from 15 horses with an ELA-A1 haplotype, but not with target cells from 15 non-ELA-A1 horses. However, only 11 of the 15 target cells from horses with an ELA-A1 haplotype presented peptide Gag-GW12. Whether the target cells from these four horses would fail to present Gag-GW12 under all assay conditions is not known; however, the relative difference between presentation by these four and the other 11 target cells was significant. Since both Env-RW12 and Gag-GW12 were presented by a single MHC class I molecule (7-6), the explanation for the lack of Gag-GW12 presentation by the EK targets from four horses that still presented Env-RW12 is probably that the presenting MHC class I molecule was structurally different from the molecule from the other 11 horses that presented both peptides. This possibility was supported by pedigree data and linkage data demonstrating that some horses with similar responses have identical ELA-A1 haplotypes by descent or have ELA-A1 haplotypes linked to the same MHC class II alleles (Table II). Presentation of Env-RW12 by apparently different equine classical MHC class I molecules may be similar to the degeneracy of peptide binding noted by HLA classical class I molecules within a superfamily (50, 51). Further definition of the observation in horses requires strict identification of the gene presenting peptide Env-RW12, but not Gag-GW12 in the other horses, so that comparison with 7-6 can be made.

The concentration of Env-RW12 required for pulsing 721.221 target cells expressing equine MHC class I molecule 7-6 to obtain 50% maximum lysis by Env-RW12-stimulated CTL was 1.8 nM, while the concentration of Gag-GW12 required for 50% maximum lysis by Gag-GW12-stimulated CTL was 126 nM (a 70-fold difference). The concentrations of peptides needed for 50% maximum lysis of 721.221 targets were determined in this study with PBMC stimulated with only one relatively high concentration of peptide (40 µM), whereas in a previous study using EK cell targets (6), the functional avidity of the two peptides was determined using a range of peptide concentrations for PBMC stimulation and was expressed as a geometric mean of 0.21 nM for Env-RW12 and 155 nM for Gag-GW12 (6). Thus, 721.221 cells expressing the 7-6 class I molecule presented Env-RW12 and Gag-GW12 peptides in the same way as autologous EK target cells in CTL assays, and this conclusion was verified by obtaining similar results with heterologous EK cells transduced with the MHC class 1 gene 7-6 as targets in CTL assays. The concentration of peptide required for pulsing target cells to obtain 50% maximum lysis has been referred to as the functional avidity and is used to describe the effectiveness of peptide-specific CTL lysis (52). This functional avidity represents the affinity of the peptide for the MHC class I molecule, for the TCR, or for both.

Having live 721.221 target cells expressing an equine MHC class I molecule allowed direct measurement of the affinity of peptide binding to this molecule using a previously described assay (46, 47). The IC50 of each peptide was determined using 125I-labeled Env-RW12, and the concentration of Env-RW12 required was 15-fold less than that of Gag-GW12. Since this 15-fold difference in affinity for MHC class I molecule 7-6 did not account for all the 70-fold or greater difference in the concentrations of the two peptides required to pulse target cells for 50% maximum CTL lysis, it seems that Gag-GW12 also had less affinity for the TCR of Gag-GW12-stimulated CTL than Env-RW12 had for the TCR of Env-RW12-stimulated CTL. In mice, affinity differences in TCR-peptide binding correlated with the ability of peptide-specific CTL clones and lines to clear experimental infection, and increasing the number of transferred CTL did not compensate for decreased function (52). Whether there are in vivo functional differences in the CTL to Env-RW12 and Gag-GW12 is not known, but this could be determined by transfer of CTL clones or lines to MHC class I 7-6-matched SCID foals, followed by EIAV challenge (4).

Determining the IC50 for truncations of Env-RW12 in this study by inhibiting radiolabeled Env-RW12 binding to live cells expressing MHC class I 7-6 extended the previous peptide mapping using the CTL assay (6). That peptide mapping demonstrated that positions 1 and 12 were critical for CTL killing, while the inhibition results demonstrated here that these residues were necessary for binding to the MHC class I molecule. It was expected that single substitutions of the nonpolar amino acids in positions 1 and 12 of Env-RW12 with lysine would cause at least a 10-fold reduction in the IC50, which would indicate that these were primary anchor residues (41); however, the result was 4.9- and 2.8-fold reductions, respectively. Therefore, the IC50 needs to be determined with additional amino acid substitutions at these positions to determine their status as a primary or secondary anchor residue. Positions 2 and 3 were identified as possible primary anchor residues based on the single substitutions evaluated. Further definition of primary and secondary anchors may identify a 7-6 molecule binding motif that can be used to predict possible CTL epitopes presented by this molecule. That the two known optimal peptides that bind to equine classical MHC class I 7-6 molecules were 12 aa is not unique, because one other equine molecule is known that binds 12-aa peptides (S. Ellis, personal communication). However, not all equine classical MHC class I molecules preferentially present 12-aa peptides, because we have defined optimal CTL epitopes that have 9 (19), 10 (6), and 11 (6) aa.

In summary, the availability of expressed classical and putative nonclassical MHC class I genes from horse A2152 provides a system to identify the presenting class I molecule for any epitope that horse A2152 targets can present to CTL from any other horse sharing alleles with A2152. Horse A2152 is a breeding stallion and will not be infected with EIAV; however, these MHC class I genes will still be valuable, because we have EIAV-infected horses that are half-siblings of A2152, and future A2152 offspring can also be used for EIAV experiments. In addition, horse A2152 is a carrier of the autosomal recessive SCID defect and is being used to breed SCID carrier mares. Therefore, SCID foals with the identified MHC class I alleles will be available for CTL functional studies using adoptive transfer of clones and lines. Tetramers can be made with the identified MHC class I molecule 7-6 and Env-RW12 and Gag-GW12 for determining the frequency of CD8+ T lymphocytes recognizing these peptides. The four classical MHC class I genes identified add to the small number of equine sequences available and provide additional information for the development of sequence-based typing procedures. The hypothesis that different classical MHC class I molecules presented the two different minimal 12-aa CTL epitopes, Env-RW12 and Gag-GW12, was proven incorrect. That the MHC class I 7-6 molecule presented both epitopes is a favorable outcome because it enables immunization of horses expressing this molecule with at least two CTL epitopes to optimize epitope-specific CTL responses in outbred horses and to evaluate their effectiveness against lentiviral challenge.


   Acknowledgments
 
The important technical assistance of Eldon Libstaff, Matt Littke, and Emma Karel is acknowledged. The contributions of Dr. Shirley Ellis, including sharing sequence information on equine MHC class I molecules and discussion on classification of these molecules, are also acknowledged, as well as the help of Dr. Alessandro Sette with the live cell, peptide binding assay.


   Footnotes
 
1 This work was supported in part by U.S. Public Health Service, National Institutes of Health Grants AI24291, AI47660, and AI01575 and Morris Animal Foundation Grant D01EQ-09. Back

2 Address correspondence and reprint requests to Dr. Travis C. McGuire, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040. E-mail address: mcguiret{at}vetmed.wsu.edu Back

3 Abbreviations used in this paper: EIAV, equine infectious anemia virus; EK, equine kidney; ELA, equine lymphocyte Ag. Back

Received for publication February 20, 2003. Accepted for publication June 5, 2003.


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