© 2014 John Wiley & Sons A/S Published by John Wiley & Sons Ltd.

Xenotransplantation 2014: 21: 465–472 doi: 10.1111/xen.12119

XENOTRANSPLANTATION

Original Article

Identification of HLA-A2-restricted immunogenic peptides derived from a xenogenic porcine major histocompatibility complex Park C-S, Im S-A, Song S, Kim K, Lee C-K. Identification of HLAA2-restricted immunogenic peptides derived from a xenogenic porcine major histocompatibility complex. Xenotransplantation 2014; 21: 465– 472. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. Abstract: Background: Little information is available regarding the precise swine leukocyte antigen (SLA)-derived immunogenic peptides that are presented in the context of human HLA molecules. Here, we identified SLA-derived immunogenic peptides that are presented in association with human HLA-A2 molecule. Methods: The SLA-derived peptides that bind to HLA-A*0201, a representative of the A2 supertype, were predicted using a computerassisted algorithm. The candidate peptides were synthesized, and the stabilities of complexes formed between peptides and HLA-A*0201 were compared using major histocompatibility complex (MHC) stabilization assays. The cytotoxic T lymphocyte (CTL)-inducing activity of the selected peptides was examined in HLA-A*0201-transgenic mice. Results: Among 15 candidate peptides synthesized, two peptides, peptide-35 (YLGPDGLLL) and peptide-43 (TLICHVDSI), were selected to have high affinity and stability with HLA-A*0201. Examination of the CTL-inducing activity of the two peptides in HLA-A*0201-transgenic mice showed that immunization with peptide-35, but not peptide-43, elicited potent CD8-specific CTL responses. The Peptide-35 is present in non-polymorphic a2 domains of 34 SLA-1 alleles, 18 SLA2 alleles, and 1 SLA-3 allele. Conclusion: This study identifies an immunogenic HLA-A*0201restricted epitope derived from the SLA, which may be valuable for the development of epitope-specific immunoregulation strategies.

Introduction

Xenotransplantation offers a potential solution to the problem of the shortage of allografts. Among animal donors, miniature swine offers attractive characteristics such as size, physiologic similarities to humans, and fecundity [1,2]. Immunological rejection of xenografts, however, remains one of the major hurdles to the successful use of xenografts [3–5]. Xenografts are destroyed as a result of hyperacute rejection caused by preformed natural xenoreactive antibodies and complement activation. The development of a-1-3 galactosyltransferase (aGalT)-knockout pigs partly overcomes

Chan-Su Park,1 Sun-A Im,1 Sukgil Song,1 Kyungjae Kim2 and Chong-Kil Lee1 1

College of Pharmacy, Chungbuk National University, Cheongju, 2College of Pharmacy, SahmYook University, Seoul, South Korea

Key words: HLA-A2 – indirect recognition – swine leukocyte antigen – xenorecognition Abbreviations: APC, Antigen-presenting cell; CFA, Complete Freund’s adjuvant; IFA, Incomplete Freund’s adjuvant; HLA, Human leukocyte antigen; MHC, Major histocompatibility complex; SLA, Swine leukocyte antigen; CTL, Cytotoxic T lymphocyte. Address reprint requests to Chong-Kil Lee, College of Pharmacy, Chungbuk National University, Cheongju 361-763, South Korea (E-mail: [email protected]) Received 14 February 2014; Accepted 17 May 2014

hyperacute rejection in non-human primates because of very low levels of cytotoxic non-aGalT natural antibodies [6,7]. However, xenografts may still face destruction mediated by T cells, which represent one of the major cell types that induce delayed xenograft rejection [8–10]. Previous studies have shown that SLA molecules serve as major antigens in human anti-porcine T cell responses [3–5]. Xenografts are rejected by T cells, especially CD8+ cytotoxic T lymphocytes (CTLs), as these cells show cytotoxicity against xenografts by recognition of swine leukocyte antigen (SLA) [11]. T cell-mediated rejection of SLA occurs through two distinct but related 465

Park et al. mechanisms called direct and indirect recognition [12,13]. In the direct pathway, recipient CD8+ CTLs recognize SLA molecules displayed by donor antigen-presenting cells (APCs). The indirect pathway, known as cross-priming, involves the accumulation of recipient CD8+ CTLs that are stimulated by SLA-derived peptides presented by recipient APCs. It is likely that indirect recognition of SLA, as in the allorecognition situation, plays an essential role in xenograft rejection. In allorecognition, cross-primed CD8+ CTLs were shown to mediate destruction of the graft even in the absence of direct recognition of the target organ [14–16]. We identified SLA-derived immunogenic peptides that are presented by human leukocyte antigen (HLA)-DR4 molecules [17]. In the current study, we asked whether we could identify HLAA*0201-restricted CTL epitopes derived from SLA and verify their immunogenicities. For this purpose, we first used computer analysis to select candidate epitopes and then determined their immunogenic properties using HLA-A*0201 transgenic mice.

Material and methods Epitope prediction and synthesis

We used computer analysis to predict SLA-derived peptides with HLA-A*0201-binding motifs with the aid of the SYFPEITHI epitope prediction algorithm and database [18]. The SLA-derived peptides, a positive-control peptide HBcAg18-27 (FLPSDFFPSV), a negative-control peptide OVA257-264 (SIINFEKL) derived from ovalbumin, and the hepatitis B virus (HBV)-derived helper epitope (TPPAYRPPNAPIL) were synthesized by Peptron Co. Ltd. (Taejeon, Korea). Peptide purity was >90% determined using high performance liquid chromatography with a Shiseido Capcell Pak C18 column (Shiseido, Tokyo, Japan). The similarities between the sequence of synthetic peptides and those of the human proteome were determined using the United States National Institutes of Health (NIH) BLAST server [19]. Peptides were dissolved in 100% dimethyl sulfoxide and diluted to the desired concentrations using phosphate-buffered saline. Cell lines and cell culture

The human tumor T2 (174 9 CEM.T2) cell line (deficient in TAP1 and TAP2 transporters) that expresses HLA-A*0201 were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco’s mod466

ified Eagle’s medium (DMEM, Hyclone, Logan, UT, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone), 100 U/ml penicillin, and 100 lg/ml streptomycin (Hyclone) at 37 °C, in an atmosphere containing 5% CO2. Mice

HLA-A*0201/Kb transgenic mice (6–8 weeks of age) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). The mice were housed in specific pathogen-free conditions and fed autoclaved food and water. Mice 8–12 weeks of agematched for gender were used in all experiments. Each experiment included three to five mice per group. Mice were treated according to the institutional protocols approved by the Animal Care Committee of Chungbuk National University. MHC stabilization assay

The ability of peptides to bind HLA- A*0201 molecules was evaluated using an MHC class I stabilization assay [20]. Briefly, T2 cells were incubated with various concentrations of each peptide and 2 lg/ml human b2-microglobulin in serum-free RPMI 1640 medium (Hyclone) at 37 °C for overnight. Then, the cells were washed and stained with an anti-HLA-A2 monoclonal antibody (mAb) conjugated to fluorescein isothiocyanate (FITC; BD Biosciences, San Jose, CA, USA). Flow cytometric analysis was performed using FACSCanto (BD Biosciences). The percentage increase in mean fluorescence index (% MFI) of the HLA-A*0201 molecule was calculated as follows:% MFI increase = [(MFI test peptide MFI of negativecontrol peptide)/(MFI of negative-control peptide)] 9 100. The negative-control peptide was OVA [257–264]. Analysis of the stability of complexes formed between peptides and HLA-A*0201

The stability of peptide-HLA-A*0201 complexes was determined as described previously [21]. Briefly, T2 cells (2 9 106/ml) were incubated at 37 °C overnight in serum-free RPMI 1640 medium (Hyclone) containing 2 lg/ml human b2-microglobulin and 100 lM peptide. The cells were washed with serum-free RPMI 1640 medium to remove unbound peptides and incubated with 10 lg/ml brefeldin A (Sigma-Aldrich, St Louis, MO, USA) to inhibit the expression of newly synthesized HLA-A*0201 molecules on cell surface at 37 °C for 0, 3, 6, or 9 h. The cells were then stained with the

HLA-A2-binding peptides derived from SLA FITC-labeled anti-HLA-A2 mAb to evaluate the expression of HLA-A*0201 molecules. To examine the effect of serum, T2 cells were cultured in media containing 0%, 10%, or 50% FBS at 37 °C for overnight. The cells were then washed and stained with the FITC-labeled anti-HLA-A2 mAb.

permeabilized using a Cytofix/Cytoperm Kit (Pharmingen) and stained with a phycoerythrin (PE)-conjugated anti-IFN-c mAb (Pharmingen). Cells were then analyzed using a FACSCanto flow cytometer (BD Biosciences), and the number of CD8+ cells expressing IFN-c was enumerated using FlowJo software.

Generation of CTLs in HLA-A2 transgenic mice

HLA-A*0201 transgenic mice were immunized with 100 lg of each peptide together with 140 lg of an HBV-derived helper epitope (TPPAYRPPNAPIL, 128–140). The peptides were emulsified in complete Freund’s adjuvant (CFA; SigmaAldrich) at a ratio of 1 : 1 and injected subcutaneously. Mice were boosted once with 100 lg of each peptide together with 140 lg of the HBV peptide emulsified in incomplete Freund’s adjuvant (IFA; Sigma-Aldrich). In vivo CTL

Peptide-specific CTL activity was measured using in vivo CTL assays according to published methods [22,23]. Briefly, erythrocyte-depleted spleen cells from naive HLA-A*0201 transgenic mice were resuspended in DMEM and divided into two populations. One population was pulsed with 5 9 10 6 M peptide for 1 h at 37 °C and labeled with a high concentration (10 lM) of CFSE. The other control target population was labeled with a low concentration of CFSE (2 lM). For intravenous (i.v) injection, equal numbers of cells from each population were mixed and injected into immunized recipient mice (1 9 107 cells per mouse). Flow cytometry was used to determine specific in vivo cytotoxicity for spleen cells isolated from recipient mice 18 h after i.v. injection. The ratio of the percentages of uncoated vs. individual peptide-coated cells (CFSElow/CFSEhigh) was calculated to quantitate cytotoxicity. Analysis of intracellular IFN-c expression

Flow cytometry was used according to a published procedure [24] to detect and quantitate peptide-specific T cells that produced IFN-c. Briefly, splenocytes from immunized mice were restimulated with 10 lg/ml solution of peptides in the presence of 1 ll/ml monensin (Golgi Stop; Pharmingen, San Diego, CA, USA) to inhibit intracellular protein transport. After 6 h incubation, the cells were harvested and stained with fluorescein isothiocyanate (FITC)-labeled anti-CD45R mAb, FITC-labeled anti-I-Ab mAb, and an allophycocyanin (APC)conjugated anti-CD8 mAb. The cells were then

Results Prediction of HLA-A*0201-restricted candidate peptides derived from SLA

The HLA-A*0201-restricted CD8+ T cell epitopes (9-mer peptides) derived from the extracellular domains of classical SLA molecules were predicted using the SYFPEITHI epitope prediction algorithm. Fifteen candidate peptides were selected according to the high SYFPEITHI scores (≥25) and the degree of similarity among the sequences encoded by different SLA alleles (Table 1). The peptide sequences in Table 1 reflect those shared by at least five different SLA alleles. Among 15 peptides, five represented class I SLA molecules, and the others represented class II SLA molecules. Binding affinity of candidate peptides for the HLA-A*0201 molecule

The binding affinity of each peptide for HLAA*0201 was evaluated using MHC stabilization assays with TAP1/2-knockout T2 cells expressing HLA-A*0201. T2 cells were incubated with various concentrations of each peptide and 2 lg/ml of human b2-microglobulin in serum-free medium at 37 °C overnight and were then washed and stained with the anti-HLA-A2 mAb. As shown in Table 1, three out of the 15 candidate peptides (Peptide-35, Peptide-43, and Peptide-44) stabilized the expression of HLA-A*0201 on T2 cells. The level of HLA-A*0201 molecules expressed on the surface of T2 cells increased by 300% in the presence of 100 lM each of Peptides-35 and 43, and Peptide-44 increased the MFI value by more than 200%. The stabilizing activity of the three peptides was higher than that of the positive-control peptide, HBcAg18-27 (FLPSDFFPSV). Because a stable peptide-MHC complex is required for the efficient induction of an antigenspecific CTL immune response [25–27], we further investigated the ability of three peptides to stabilize HLA-A*0201 molecules on the cell surface. In this assay, T2 cells were incubated overnight with each peptide in serum-free medium supplemented with human b2-microglobulin, washed, and then treated with 10 lg/ml of brefeldin A to inhibit the 467

Park et al. Table 1. MHC stabilization assay MFI increase (%) Origin of peptide

Peptide name

Amino acid sequence

Scorea

1 lM

10 lM

100 lM

SLA class I molecules

pep35 pep36 pep37 pep38 pep39 pep40 pep41 pep42 pep43 pep44 pep45 pep46 pep47 pep48 pep49 Control peptide

YLGPDGLLL YLEMGKDTL YLQMGKDTL YLEMGNNTL YLQMGNNTL GALRNIATL VILGQPNTL VMLGQPNTL TLICHVDSI SIFPPVINI YLNGQKEAL ALEQKRAEL ILEQTRAEL LLEQRRAEV ILEDSRASV FLPSDFFPSV

25 25 26 26 27 25 26 26 26 26 26 26 25 25 26 24

39 19 21 0 0 0 0 0 0 0 0 0 0 0 0 0

151 14 14 9 1 0 0 0 31 0 0 0 0 0 0 44

359 0 0 90 6 0 0 3 320 221 0 0 0 0 140 170

SLA class II molecules

HBcAg [18–27]

SLA, swine leukocyte antigen; MFI, mean fluorescence index. Score means the relative binding affinity calculated by the SYFPEITHI program.

a

expression of newly synthesized HLA-A*0201 molecules on the cell surface. HLA-A*0201 molecules complexed with Peptide-35 or Peptide-43 remained on the cell surface for as long as 9 h (Fig. 1A). In contrast, the level of HLA-A*0201Peptide-44 complexes were decreased rapidly (Fig. 1A). We next examined the effect of serum on the formation of peptide-HLA-A*0201 complexes by culturing the T2 cells at 37 °C overnight in the presence of serum-free medium or medium containing 10% FBS. Analysis using the anti-HLA-A2 mAb indicated that the formation of the three HLA-A*0201-peptide complexes was not affected by the presence of 10% serum (Fig. 1B). Unexpectedly, we found that 10% FBS increased the formation of all three HLA-A*0201-peptide complexes.

Analysis of peptide-specific CTL induction in vivo

HLA-A*0201 transgenic mice were immunized with 100 lg of either of the two most active peptides (Peptides 35 or 43) together with 140 lg of an HBV-derived helper epitope emulsified with CFA. Control group of mice were immunized with HBV-derived helper epitope emulsified with CFA. Mice were boosted once using the same respective preparations but with IFA instead of CFA. After 10 days, peptide-specific CTL activity was determined using an in vivo CTL assay (Fig 2A). Immunization with Peptide-35 induced specific killing of Peptide-35-pulsed targets (33%; Fig. 2B). In contrast, induction of CTL activity was undetectable using Peptide-43 (data not shown).

Fig. 1. Analysis of HLA-A*0201 expression on T2 cells in the presence of candidate epitope peptides with and without serum. T2 cells were incubated overnight with each peptide in serum-free medium supplemented with 2 lg/ml human b2-microglobulin, washed, and then treated with 10 lg/ml of brefeldin A. The cells were incubated in serum-free medium at 37 °C, and the changes in the expression levels of HLA-A*0201 molecules were determined at various times (0, 3, 6, and 9 h) (A). To examine the effect of serum on the formation of peptide-HLA-A*0201 complexes, T2 cells were cultured in serum-free, or 10% FBS at 37 °C overnight. The cells were then washed and stained with an anti-HLA-A2 mAb (B). The percentage increase of mean fluorescence index (MFI) was calculated as follows: [(MFI with the test peptide MFI of negative-control peptide)/(MFI of negative-control peptide)] 9 100. The negative-control peptide was OVA [257–264].

468

HLA-A2-binding peptides derived from SLA

Fig. 2. Induction of Peptide-35-specific CTL in HLA-A*0201 transgenic mice. HLA-A*0201 transgenic mice were immunized with Peptide-35 together with an HBV-derived helper epitope. Control group of mice (Ctrl) were immunized with hepatitis B virus (HBV)-derived helper epitope only. Mice were boosted once following initial immunization. After 10 days, Peptide-35-specific CTL activity was determined using an in vivo cytotoxic T lymphocyte (CTL) assay with syngeneic spleen cells that were pulsed with CFSE. (A) Representative histograms showing specific killing of Peptide-35-pulsed target cells. (B) Percentages of Peptide-35-pulsed target cells killed in the spleens. The data are mean  SD of three independent experiments.

Analysis of IFN-c-production by peptide-specific T cells

Because CTLs produce the Th1 cytokine IFN-c, Peptide-35-specific T cells were enumerated using intracellular cytokine staining to measure the level of IFN-c. In this experiment, splenocytes from immunized mice were restimulated with peptides in the presence of monensin for 6 h, and the cells were stained with anti-CD45R, anti-I-Ab, and antiCD8 mAbs. The cells were then permeabilized and stained with anti-IFN-c mAb. The cells negative for CD45R and I-Ab were gated and analyzed for CD8 and intracellular IFN-c. CD8+ T cells expressed intracellular IFN-c at a frequency of 0.28% (Fig. 3), which supports the conclusion that Peptide-35 induced a specific CTL response in vivo. Amino acid sequence comparisons of Peptide-35 and SLA alleles

Swine leukocyte antigen alleles containing the Peptide-35 amino acid sequence (YLGPDGLLL) are shown in Table 1. The Peptide-35 amino acid sequences is present in non-polymorphic a2 domains of thirty-four SLA-1 alleles, eighteen SLA-2 alleles, and one SLA-3 allele, suggesting that the Peptide-35 epitope sequence is universally distributed among porcine MHC molecules. Peculiar features of Peptide-35

Ranking the peptides by the Kyte–Doolittle hydrophobicity scale [28] showed that Peptide-35 ranked 3rd among 15 peptides (Figure S1). Peptide-35, however, had a prominent amphiphilic feature, where approximately one half of the molecule was hydrophobic while the other half was hydrophilic (Figure S1). It was previously shown that amphiphilic peptides were more likely to induce potent CD8 T cell responses in vivo [29]. Thus, we are

tempted to speculate that the potent immunogenic activity of Peptide-35 is due to good hydrophobicity and amphiphilicity to associate with HLAA*0201 and induce strong CD8 T cell activation. Discussion

In the present study, we used bioinformatic, biochemical, and immunodetection techniques to identify SLA-derived peptides that are presented to the immune system by HLA-A*0201, which is a representative of the A2 supertype. We found that Peptide-35, YLGPDGLLL, was bound by HLAA*0201 with high binding affinity, formed stable complex with HLA-A*0201 complexes, and induced a CTL response in HLA-A*0201 transgenic mice. The peptide-35 amino acid sequence is distributed among many porcine MHC molecules (Table 2). When Peptide-35 amino acid sequences were analyzed for similarity to sequences of the human proteome using NIH BLAST server [19], we did not identify exact matches with any other human proteome sequence, indicating that peptide-35 is a porcine species-specific peptide. Despite the prominent role of CD4+ T cells in rejection of xenografts, porcine xenografts are eventually rejected by CD8+ T cells [30–33]. CD8+ T cells are present in abundance at sites of graft rejection in CD4+ T cell-deficient mice [34]. However, the mechanism underlying the ability of CD8+ T cells to reject xenografts is unknown. Previous studies have shown that SLA molecules serve as major antigens in human anti-porcine T cell responses [3–5]. It is likely that, in indirect xenorecognition, CD8+ T cells are activated by SLAderived peptides complexed with human MHC molecules expressed by host APCs, as in the allorecognition situation [14–16]. The major finding of the present study is the identification of 469

Park et al.

Fig. 3. Analysis of IFN-c expression by CD8+ T cells. Splenocytes cells were pooled from peptide-immunized mice (n = 3 per peptide) and then stimulated in vitro with 10 lg/ml of Peptide-35. The cells negative for CD45R and I-Ab were gated and analyzed for CD8 and intracellular IFN-c. The left panel shows the results for Peptide-35-stimulated cells, and the right panel shows the results for unstimulated controls.

Table 2. SLA alleles containing Peptide-35 sequence SLA-1 alleles SLA-1*0101 SLA-1*0102 SLA-1*01rh28 SLA-1*0201 SLA-1*0202 SLA-1*02we02 SLA-1*0401 SLA-1*04gx01 SLA-1*04gz01 SLA-1*04we01 SLA-1*0501 SLA-1*0601

SLA-1*06an04 SLA-1*0801 SLA-1*08Lw02 SLA-1*08an03 SLA-1*08 ms05 SLA-1*08pt13 SLA-1*08sk11 SLA-1*08sm08 SLA-1*08sy01 SLA-1*1101 SLA-1*11jh01 SLA-1*1201

SLA-2 alleles SLA-1*12Lw01 SLA-1*12hy01 SLA-1*1301 SLA-1*13 ms21 SLA-1*an01 SLA-1*cs02 SLA-1*es11 SLA-1*es12 SLA-1*rh03 SLA-1*st11

SLA-2*0301 SLA-2*0302 SLA-2*03gz01 SLA-2*0501 SLA-2*0502 SLA-2*05rh03 SLA-2*05rh07 SLA-2*05rh34 SLA-2*05sy01 SLA-2*1001 SLA-2*1002 SLA-2*10es21

SLA-3 alleles SLA-2*10sk21 SLA-2*10sm01 SLA-2*11so01 SLA-2*1201 SLA-2*12Lw01 SLA-2*es22

SLA-3*hm22

SLA, swine leukocyte antigen

SLA-derived peptide that is involved in indirect xenorecognition of SLA molecule. In xenotransplantation scenarios, the porcine graft cells express porcine class I but not human class I MHC molecules on their cell surface. Thus, although human recipient CD8+ T cell may be primed by human recipient APCs expressing human recipient class I MHC molecules complexed with SLA-derived peptides, cross-primed human CD8+ T cells cannot directly recognize porcine graft cells within the transplanted organ. The mechanisms underlying this process may be 470

explained by findings that cross-primed CD8+ T cells mediate graft rejection through recognition of recipient non-professional APCs such as vascular endothelial cells that express donor-derived antigens via cross-presentation [16,35,36]. Immunization of HLA-A*0201-transgenic mice with Peptide-35 together with an HBV-derived helper epitope elicited efficient CD8+ CTL cell responses. However, immunization of Peptide-35 without the HBV-derived helper epitope did not induce Peptide-35-specific CTL cell responses. These data establish that the activation of CD4+

HLA-A2-binding peptides derived from SLA T cells by the HBV-derived helper epitope is required for the induction of Peptide-35-specific CD8+ T cells. Because CTLs produce the Th1 cytokine IFN-c, Peptide-35-specific T cells were enumerated using intracellular cytokine staining of IFN-c. We found that CD8+ T cells expressed intracellular IFN-c at a frequency of 0.29% among CD8 T cells (Fig. 3). It is noteworthy that the increase of the frequency to 0.29% is meaningful, because we examined the frequency of IFN-c-producing CD8+ T cells that are specific for only one epitope, Peptide-35. In summary, we identified here SLA class I-derived porcine species-specific peptide that elicited a CTL response. Our results may facilitate the design of strategies to mitigate human CD8+ T cell-mediated xenorejection. For instance, peptide-35 may be used to develop peptide-35-specific CD8+ T cell immune tolerance using the methods described earlier [37,38]. Immunization of immature dendritic cells pulsed with immunogenic peptide was shown to induce peptide-specific inhibition of effector T cells [37]. Rapamycin-treated dendritic cells pulsed with alloantigen were also shown to induce alloantigen-specific T cell suppression in vivo [38]. Therefore, our findings have implications not only for the identification of an immunogenic indirect epitope shared by diverse SLA alleles, but also for the future development of epitope-specific immunoregulation strategies. Acknowledgement

This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ009619) and Rural Development Administration, Republic of Korea. Author contributions

Chan-Su Park: Performed the experiments, drafting article. Sun-A Im: Involved in the animal experiments. Sukgil Song: Data interpretation. Kyungjae Kim: Research design. Chong-Kil Lee: Concept and design, article writing. Conflict of interest

The authors of this manuscript have no conflict of interests to disclose. References 1. SACHS DH, LEIGHT G, CONE J et al. Transplantation in miniature swine. I. Fixation of the major histocompatibility complex. Transplantation 1976; 22: 559–567.

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Supporting Information

Additional Supporting Information may be found in the online version of this article: Figure S1. Hydrophobicity and amphiphilicity analysis of the synthetic peptides.