Functional analysis of frequently expressed Chinese rhesus macaque MHC class I molecules Mamu-A1*02601 and Mamu-B*08301 reveals HLA-A2 and HLA-A3 supertypic specificities
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The Simian immunodeficiency virus (SIV)-infected Indian rhesus macaque (Macaca mulatta) is the most established model of HIV infection and AIDS-related research, despite the potential that macaques of Chinese origin is a more relevant model. Ongoing efforts to further characterize the Chinese rhesus macaques’ major histocompatibility complex (MHC) for composition and function should facilitate greater utilization of the species. Previous studies have demonstrated that Chinese-origin M. mulatta (Mamu) class I alleles are more polymorphic than their Indian counterparts, perhaps inferring a model more representative of human MHC, human leukocyte antigen (HLA). Furthermore, the Chinese rhesus macaque class I allele Mamu-A1*02201, the most frequent allele thus far identified, has recently been characterized and shown to be an HLA-B7 supertype analog, the most frequent supertype in human populations. In this study, we have characterized two additional alleles expressed with high frequency in Chinese rhesus macaques, Mamu-A1*02601 and Mamu-B*08301. Upon the development of MHC–peptide-binding assays and definition of their associated motifs, we reveal that these Mamu alleles share peptide-binding characteristics with the HLA-A2 and HLA-A3 supertypes, respectively, the next most frequent human supertypes after HLA-B7. These data suggest that Chinese rhesus macaques may indeed be a more representative model of HLA gene diversity and function as compared to the species of Indian origin and therefore a better model for investigating human immune responses.
KeywordsHLA supertype MHC Peptide-binding motif Rhesus macaque
Non-human primates, notably rhesus macaques, are widely used as animal models in biomedical and immunological research. In particular, both the Indian-origin and Chinese-origin rhesus macaque models have contributed invaluable information to the study of disease pathogenesis and novel vaccine evaluation (Desrosiers 1990; Gardner and Luciw 2008; Haigwood 2009; ILAR 2003; Kindt et al. 1992; Persidsky and Fox 2007). However, these two distinct geographic populations have displayed decidedly different outcomes to infection despite their apparent physical similarity (Joag et al. 1994; Otting et al. 2005, 2007), perhaps attributive to genetic profile variations that affect immune responses. Specifically, upon Simian immunodeficiency virus (SIV) infection, Chinese rhesus macaques have a more delayed and prolonged progression to AIDS as compared to the animal populations of Indian origin (Ling et al. 2002).
A number of studies have shown that several macaque major histocompatibility complex (MHC; Mamu) class I alleles, including Mamu-A*01 (Allen et al. 1998), Mamu-B*17 (Mothe et al. 2002), and Mamu-B*01 (Loffredo et al. 2005) among others (Loffredo et al. 2004; Sette et al. 2005), are expressed with high frequency in Indian rhesus macaque populations. Interbreeding of the Indian-origin animals in the USA since 1978, when the exportation of these animals from India was discontinued (Southwick and Siddiqi 1988), has likely played a significant role in this observation. The peptide-binding specificities for several of these and other Indian rhesus MHC allelic forms have been extensively characterized, leading to the identification of specific alleles which influence disease progression (Mothe et al. 2003; O’Connor et al. 2003; Yant et al. 2006; Loffredo et al. 2007) as well as the discovery of viral evasion from cytotoxic T lymphocyte (CTL) responses (Evans et al. 1999; Allen et al. 2000) in the SIV arena. Indeed, Indian rhesus macaques are the model most utilized in HIV- and AIDS-related research studies (Persidsky and Fox 2007; Patterson and Carrion 2005; Gardner and Luciw 2008; Watkins et al. 2008). However, the increased demand for these animals and, more importantly, the rapid progression to disease displayed after SIV infection of the Indian-origin populations (Ling et al. 2002) have underscored the advantages for developing alternative animal models.
Because of their relative accessibility, Chinese rhesus macaques are becoming more widely employed as non-human primate models in infectious disease research. They are utilized for the evaluation of vaccines and the study of immune responses in pathogen systems ranging from Marburg virus, Ebola virus, and influenza virus to the more well-studied SIV (Geisbert et al. 2007; Larsen et al. 2007; Carroll et al. 2008; Degenhardt et al. 2009; Ling et al. 2007, 2002). These animals, however, have not been characterized at the MHC loci to the same extent as their Indian counterparts. Studies to address this disparity have revealed a surprisingly high degree of MHC polymorphism (Otting et al. 2005, 2007, 2008; Karl et al. 2008; Ma et al. 2009; Wiseman et al. 2009; Ouyang et al. 2008). However, it is largely non-overlapping with Indian-origin macaques (Solomon et al. 2010). This polymorphism may be due to the diverse geographic origins from which the animals have been derived, comparable to human population distribution, suggesting that Chinese rhesus macaques may represent human leukocyte antigen (HLA) diversity more effectively than those of Indian origin.
HLA polymorphism and its function to bind a diverse array of antigenic peptides for CTL scrutiny have been well documented, as has the existence of HLA supertypes, groups of MHC molecules which share similar peptide-binding specificities (Bjorkman and Parham 1990; Maryanski et al. 1986; Parham et al. 1995; Sette and Sidney 1999; Sidney et al. 1995, a, b; Townsend et al. 2006). Previous studies have demonstrated CTL repertoire overlaps between humans and chimpanzees (Bertoni et al. 1998), as well as humans and Indian rhesus macaques (Loffredo et al. 2009), suggesting that HLA binding supertypes may extend to non-human primates. Recently, the peptide-binding specificity associated with the most frequent Chinese-origin allele, Mamu-A1*02201, has been characterized and shown to be an HLA-B7 supertype analog (Solomon et al. 2010), though it is the only one to date. In order for Chinese-origin macaques to become more valuable as animal models of infectious disease research, continued investigation and characterization of their MHC profile is necessary so cellular immunity and immune correlates of protection can be more accurately assessed.
We previously reported that 11 alleles were found in multiple Chinese rhesus animals in different cohorts/colonies, each with a cumulative frequency greater than or equal to 5.6%. Combined, these alleles provide coverage for approximately 68% of all Chinese rhesus animals studied thus far and therefore represent a logical target for further analysis and characterization (Solomon et al. 2010). Accordingly, herein we sought to characterize Mamu-A1*02601 (6.7%) and Mamu-B*08301 (5.8%), two of the most frequently expressed Chinese-origin class I alleles. We report the specific peptide-binding motifs associated with these allelic forms and utilize their respective motifs to map SIV-derived Mamu-A1*02601 and Mamu-B*08301 binding peptides.
Materials and methods
Creation of stable Mamu-A1*02601, Mamu-B*08301 transfectant cell lines
Stable MHC class I transfectants were produced in the MHC class I deficient EBV-transformed B-lymphoblastoid cell line 721.221. An expression construct was created for Mamu-A1*02601 and Mamu-B*08301 by sub-cloning a full-length allele transcript into separate pcDNA 3.1 vectors (Invitrogen). These constructs were then used to transfect MHC class I-null 721.221 cells using an Amaxa Nucleofector II transfection machine (Lonza AG, Walkersville, MD, USA).
To produce secreted Mamu-A1*02601 molecules in the context of endogenous ligand identification, α-chain cDNAs of Mamu-A1*02601 were modified at the 3′ end by PCR mutagenesis to delete codons 5–7 encoding the transmembrane and cytoplasmic domains and to add a 30-bp tail encoding the ten amino acid rat very low density lipoprotein receptor (VLDLr), SVVSTDDDLA, for purification purposes (Hickman et al. 2000). sMHC-VLDLr were cloned into the mammalian expression vector pcDNA3.1 (Invitrogen); 721.221 cells were transfected with sMHC Mamu-A*26TVLDLr by electroporation. After 48 h incubation, cells were plated in 96-well plates (Falcon) in RPMI 1640 containing the antibiotic Geneticin. Transfectants were tested for production of sMHC molecules by a VLDLr-specific ELISA (Hawkins et al. 2008).
Mamu-A1*02601 endogenous ligand determination
Approximately 25 mg of Mamu-A*26TVLDLr molecules from the 721.221 cell line were purified over an affinity column composed of anti-VLDLr antibody (ATCC clone CRL-2197) coupled to CNBr activated Sepharose 4B (GE Healthcare, Piscataway, NJ, USA). sMHC molecules were then eluted in 0.2 N acetic acid, brought up to 10% acetic acid, and heated to 76°C for 10 min. Peptides were separated from heavy and light chains by ultra-filtration in a stirred cell with a 3-kDa molecular weight cutoff cellulose membrane (Millipore, Bedford, MA, USA). The peptide batch was flash frozen and lyophilized. The peptides were then reconstituted in 10% acetic acid.
Following isolation, 10% of the peptide pool was subjected to 14 rounds of N-terminal sequencing by Edman degradation. A motif was generated by calculating the fold increase of each amino acid over the prior round. A hierarchy was then determined based on the amino acid composition at each position (Falk et al. 1991).
Peptides were reverse-phase HPLC fractionated using a Jupiter Proteo C12 column (Phenomenex, Torrance, CA, USA) on a Paradigm MG4 system (Michrom Bioresources, Auburn, CA, USA). A standard CH3CN gradient was employed to generate approximately 40 peptide-containing fractions. UV absorption was monitored at 215 nm. Peptide fractions were concentrated to dryness and reconstituted in 20 μl of nanospray buffer composed of 50% methanol, 50% H2O, and 0.5% acetic acid. Nano-electrospray capillaries (Proxeon, Denmark) were loaded with 1 μl of each peptide fraction and infused at 1,100 V on a Q-Star Elite quadrupole mass spectrometer with a time of flight detector (Applied Biosystems, Foster City, CA, USA). Ion maps were generated for each fraction in a mass range of 300–1,200 amu. Using independent data acquisition for selection, ions (putative peptides) were fragmented by tandem mass spectrometry (MS/MS). An amino acid sequence was assigned using the publicly available, web-based MASCOT (Matrix Science Ltd., London, UK) and/or de novo sequencing.
Positional scanning combinatorial library and peptide synthesis
Positional scanning combinatorial libraries (PSCL) were synthesized as previously described (Pinilla et al. 1999). In the PSCL, each pool in the library contains randomized 9-mer peptides with one fixed residue at a single position. With each of the 20 naturally occurring residues represented at each position along a 9-mer backbone, the entire library consisted of 180 peptide mixtures.
Peptides utilized in screening studies were purchased as crude or purified material from Mimotopes (Minneapolis, MN, USA/Clayton, Victoria, Australia), Pepscan Systems B.V. (Lelystad, The Netherlands), A&A Labs (San Diego, CA, USA), Genescript Corporation (Piscataway, NJ, USA), or the Biotechnology Center at the University of Wisconsin—Madison (Madison, WI, USA). Peptides synthesized for use as radiolabeled ligands were synthesized by A&A Labs and purified to >95% homogeneity by reverse-phase HPLC. Peptide purity was determined with analytical reverse-phase HPLC and amino acid analysis, sequencing, and/or mass spectrometry. Peptides were radiolabeled utilizing the chloramine T method (Sidney et al. 2001b). Lyophilized peptides were re-suspended at 4–20 mg/ml in 100% DMSO, then diluted to required concentrations in PBS + 0.05% (v/v) nonidet P40 (Fluka Biochemika, Buchs, Switzerland).
SIV peptide sequences were derived from the SIVmac239 sequence, GenBank accession M33262 (Kestler et al. 1990).
MHC purification for peptide-binding assays
HLA and Mamu class I MHC purification was performed by affinity chromatography using the W6/32 and/or B123.2 class I antibodies, as previously described (Sidney et al. 2001b, 2005; Loffredo et al. 2009). Protein purity, concentration, and depletion efficiency steps were monitored by SDS-PAGE.
Quantitative assays for peptide binding to detergent solubilized MHC class I molecules were based on the inhibition of binding of a high-affinity radiolabeled standard probe peptide and performed as detailed in prior studies (Loffredo et al. 2004; Schneidewind et al. 2008; Sidney et al. 2001b, 2005). Peptides were tested at six different concentrations covering a 100,000-fold dose range in three or more independent assays. Control wells to measure non-specific (background) binding were also included. In each experiment, a titration of the unlabeled version of the radiolabeled probe was also tested as a positive control for inhibition.
The radiolabeled peptide utilized for the Mamu-A1*02601 assay was 3317.02 (sequence YLPTQQDVL), representing a sequence identified by Edman degradation and mass spectrometry analysis (described above). For Mamu-B*08301 assays, peptide 3317.04 (KSINKVYGK, an R9->K analog of Vaccinia B13R, an HLA-A3 supertype degenerate binder) was used. For each peptide, the concentration of peptide yielding 50% inhibition of the binding of the radiolabeled probe peptide (IC50) was calculated. Under the conditions used, where [radiolabeled probe] < [MHC] and IC50 ≥ [MHC], the measured IC50 values are reasonable approximations of the true Kd values (Gulukota et al. 1997; Sette et al. 1994a; Cheng and Prusoff 1973).
We performed analysis of the PSCL data as described previously (Sidney et al. 2008). Briefly, IC50 (nanomolars) values for each residue/position mixture were standardized as a ratio to the geometric mean IC50 (nanomolars) value of the entire set of 180 mixtures and further normalized to the average of libraries tested at each position. To identify predicted binders, all possible 9-mer peptides in SIVmac239 sequences were scored using the matrix values derived from the PSCL analyses of Mamu-A1*02601 and Mamu-B*08301. The final score for each peptide represents the product of the corresponding matrix values for each peptide residue–position pair. Peptides scoring among the top 3.0% (n = 100) were selected for binding analysis.
We assembled representative MHC class I sequences from humans, Chinese, and Indian rhesus macaques. Sequences were normalized to 1,068 nucleotides in length and aligned using the ClustalX program (Thompson et al. 2002). A phylogenetic tree was built with the neighbor-joining method (Saitou and Nei 1987) using the Tamura three-parameter distance model (Tamura 1992). One thousand bootstrap samples were analyzed to ensure reliable clustering.
Determination of natural Mamu-A1*02601 ligands
Endogenous Mamu-A1*02601 ligands identified by MS/MS sequencing analysis
Next, peptides were aligned and at each position, the frequency of each residue was tabulated (Supplemental Table 1, online resource). At position 2, leucine (L) was found in 15 of the 30 ligands, and other aliphatic residues valine (V) and isoleucine (I) were present in ten and three ligands, respectively. The amide residue glutamine (Q) was found in 15 peptides at position 6, and related residues glutamic acid (E) or asparagine (N) were found in seven ligands. At the C terminus, L was dominant (26 out of 30 peptides). The related aliphatic residues methionine (M) and I were also present at the C terminus. This information presents a preliminary motif with positions 2 and 6 and the C terminus, tentatively assigned as the main anchor positions based on ≥50% residue frequency.
Establishment of peptide-binding assays for Mamu-A1*02601 and Mamu-B*08301
Endogenous ligands or defined epitopes for the Mamu-B*08301 molecule have not been reported. However, previous studies demonstrated that HLA supertype ligands also bind to MHC molecules expressed in other species, such as chimpanzees (Pan troglodytes), mice (Mus musculus; McKinney et al. 2000; Sette et al. 2005; Sidney et al. 2006), and, recently, Chinese rhesus macaques (Solomon et al. 2010), where the most frequent allele Mamu-A1*02201 was shown to be an analog of the HLA-B7 supertype. Hierarchical clustering analysis performed by our group (data not shown) predicted that Mamu-B*08301 might be associated with an HLA-A3 supertypic peptide-binding specificity. Experiments examining direct binding for a panel of HLA-A3-supertype peptides to Mamu-B*08301 were performed. An analog of a Vaccinia-derived HLA-A3 supertype ligand (KSINKVYGK, peptide 3317.04) bound Mamu-B*08301 with significant counts and showed specificity, displaying negligible binding to Mamu-A1*02601 (Fig. 1b).
Furthermore, the binding for both alleles was specific at the level of inhibition by unlabeled ligands. Mamu-A1*02601 binding to the radiolabeled YLPTQQDVL ligand could be inhibited by an excess of unlabeled 3317.02 peptide with an IC50 value of approximately 0.2 nM. Mamu-B*08301 binding to the radiolabeled KSINKVYGK ligand could be inhibited by an excess of unlabeled 3317.04 with an IC50 value of approximately 2.0 nM (Fig. 1c). In conclusion, these results demonstrate that the binding assays are specific for the purified MHC molecule and thus enable detailed investigation of the peptide-binding specificity for both Mamu-A1*02601 and Mamu-B*08301.
Definition of Mamu-A1*02601 peptide-binding motif
Identification of Mamu-A1*02601 binding SIV-derived peptides
Next, we determined if the PSCL-based motifs could be used to identify Mamu-A1*02601 binding peptides. With Chinese rhesus macaques becoming an increasingly utilized animal model in SIV pathogenesis studies, we targeted the SIV proteome for these experiments (Joag et al. 1994; Ling et al. 2002; Trichel et al. 2002; Burdo et al. 2005; Monceaux et al. 2007; Degenhardt et al. 2009). The PSCL matrix was used to score all 9-mer peptides in the SIVmac239 proteome. Peptides scoring in the upper 3.0% range (n = 100) were tested for binding capacity (Supplemental Table 2, online resource). Additionally, a set of 35 control peptides, ranging from 3.1% to 70% in rank (five peptides randomly selected within each 10% demarcation), was also tested.
The efficiency of PSCL matrices in predicting Mamu-A1*02601 binders
Mamu-A1*02601 binding affinity (IC50, nM)
Mamu-A1*02601 and the HLA-A2 MHC molecules share overlapping binding repertoires
HLA-A*0202 displayed the highest degree of cross-reactivity with Mamu-A1*02601. Of the 27 Mamu-A1*02601 binders that bound an A2-like allele, 23 (85%) bound HLA-A*0202. Additionally, 17 of the 18 promiscuous binders (94%) bound HLA-A*0202. HLA-A*0201 and HLA-A*0203 were also highly cross-reactive, as both alleles bound 18 Mamu-A1*02601 binders (67%); 16 of these cross-reactive peptides were shared between the two alleles.
Interestingly, the amino acid present at position 6 greatly influences cross-reactivity. More specifically, while Q at position 6 is strongly preferred for Mamu-A1*02601 binding, peptides possessing Q at position 6 are less likely to cross-react with HLA-A2 supertype molecules. Indeed, only 14% (three out of 21) of Mamu-A1*02601 binders with Q at position 6 were promiscuous HLA-A2 supertype binders, while 63% (15 out of 24) of Mamu-A1*02601 binders that did not have Q at p6 bound three or more HLA-A2 supertype molecules.
Definition of Mamu-B*08301 peptide-binding motif
Identification of Mamu-B*08301 binding SIV-derived peptides
Repeating the exercise performed for Mamu-A1*02601, all 9-mer peptides in the SIVmac239 proteome were scored using the Mamu-B*08301 PSCL matrix. Peptides scoring in the upper 3.0% range (n = 100) were tested for binding capacity (Supplemental Table 3, online resource), and a set of control peptides was included.
The efficiency of PSCL matrices in predicting Mamu-B*08301 binders
Mamu-B*08301 binding affinity (IC50, nM)
Mamu-B*08301 and the HLA-A3 MHC molecules share overlapping binding repertoires
HLA-A*3101 displayed the highest degree of cross-reactivity with Mamu-B*08301. Of the 52 Mamu-B*08301 binders that bound an A3-like allele, 39 (75%) bound HLA-A*3101. HLA-A*0301 and HLA-A*6801 also displayed good cross-reactivity, as both alleles bound at least 26 Mamu-B*08301 binders. Additionally, 41% of Mamu-B*08301 binders (26 out of 64) were promiscuous HLA-A3 supertype binders, a percentage that is comparable to that seen between Mamu-A1*02601 and HLA-A2 (40%).
Evolutionary origin of Chinese rhesus macaque HLA-A2 and HLA-A3 functional analogy
The two common Chinese rhesus class I alleles described above are associated with HLA-A2 and HLA-A3 supertype specificities, which have not been detected in Indian rhesus macaques, although functional analysis of many of the most common alleles expressed in Indian-origin animals has been performed. To investigate the evolutionary origin of the functional analogy between the HLA and Chinese rhesus macaque MHC class I alleles in this study, we built a phylogenetic tree using representative MHC allele sequences.
For humans, we included one HLA allele (HLA-A*01010101, HLA-A*02010101, HLA-A*03010101, HLA-A*24020101, HLA-B*070201, HLA-B*080101, HLA-B*270202, HLA-B*44020101, HLA-B*580101, and HLA-B*15010101) from each of the known supertypes (A1, A2, A3, A24, B7, B8, B27, B44, B58, and B62 respectively) with an additional four HLA-B7 supertype alleles (HLA-B*35010101, HLA-B*510101, HLA-B*530101, and HLA-B*5401) and the four additional HLA-A2 supertype alleles (HLA-A*0202, HLA-A*020301, HLA-A*020601, and HLA-A*68020101) and HLA-A3 supertype alleles (HLA-A*110101, HLA-A*310102, HLA-A*330101, and HLA-A*680101) that were used in this study. For Indian rhesus macaques, we selected 14 sequences represented among the most common specificities (Boyson et al. 1996; Kaizu et al. 2007; Knapp et al. 1997; Loffredo et al. 2007; Voss and Letvin 1996). Similarly, the 14 Chinese rhesus macaque sequences included in the analysis were selected from the most frequent alleles (Solomon et al. 2010).
Herein we report the peptide-binding motifs associated with the common Chinese-origin rhesus macaque class I molecules Mamu-A1*02601 and Mamu-B*08301. This is the first description of a motif for Chinese-origin Mamu-B alleles and only the second motif for Mamu-A alleles, since until now Mamu-A1*02201 was the only Chinese-origin MHC molecule for which a peptide-binding motif had been described (Solomon et al. 2010). Thus, our study triples the number of alleles for which peptide-binding motifs are available.
The significance of our study is underscored by the fact that, while Mamu-A1*02201 is the most frequently expressed class I allele in Chinese rhesus macaques, Mamu-A1*02601 is the second most frequent allele and Mamu-B*08301 is the most frequent Mamu-B allele, a distinction it shares with six other molecules (Solomon et al. 2010). These three alleles combined allow for potential coverage of approximately 20% of Chinese rhesus macaques used in biomedical research, irrespective of the geographical origin from China, thus significantly enhancing our knowledge of the functional MHC profile of these experimental animals.
Unexpectedly, we found that both of these molecules are associated with motifs that are overlapping with well-known HLA supermotifs. Specifically, Mamu-A1*02601 shares a high degree of cross-reactivity with the HLA-A2 supertype allele HLA-A*0202, as well as HLA-A*0201 and HLA-A*0203. Mamu-B*08301 is highly cross-reactive with HLA-A3 supertype alleles HLA-A*3101, HLA-A*0301, and HLA-A*6801. These results are even more remarkable in the context of the recently described HLA-B7 supertype specificity similarity of Mamu-A*02201 (Solomon et al. 2010). HLA-B7, HLA-A3, and HLA-A2 are the three most abundant supertypes in the human population. Separately, each of these three supertypes has a phenotypic frequency greater than 42% averaged across various ethnic groups. When these three supertypes are combined, over 86% of the human population is covered (Sette and Sidney 1999). Thus, each of the three common Chinese Mamu class I alleles thus far investigated are associated with a motif corresponding to one of the three most common HLA supertypes expressed in humans. It is possible that cross-reactivity with additional HLA or Mamu alleles will be identified, thus increasing the relevance and utility of the assays characterized herein for studies directed toward epitope discovery and characterization of the immune response to specific pathogens.
These observed similarities of Mamu with HLA could be explained by either common ancestry or convergent evolution (Sette et al. 2003). Previous studies have detected functional similarities between Mamu and HLA class I molecules, such as Mamu-B*08 with HLA-B*27 molecules. Notably, other similarities like Mamu-A*11 with HLA-B*44 and Mamu-A*22 with HLA-B*07 (Dzuris et al. 2000; Sette et al. 2005; Loffredo et al. 2009; Solomon et al. 2010) spanned across loci, arguing against the common ancestry hypothesis. In this study, we show that a Mamu molecule encoded by the B locus shares overlapping binding characteristics with HLA-A alleles, also arguing against common ancestry. This point was further and more formally demonstrated by phylogenetic analysis. Indeed, prior to this study, Mamu class I alleles, regardless of loci, have been purported to be functional HLA-B analogs only (Hickman-Miller et al. 2005), with rare associations linked to HLA-A thus far reported, even at the level of binding specificity. Our results present evidence of two Mamu class I molecules that share overlapping binding characteristics with HLA-A alleles.
The class I loci for both Indian- and Chinese-origin rhesus macaques are highly polymorphic, though Chinese alleles appear to be more polymorphic given the allele frequencies that have emerged in this population (Solomon et al. 2010). Additionally, the allelic variants are largely non-overlapping, possibly because of the geographical distance between India and China resulting in low probability of genetic exchange as demonstrated by mitochondrial DNA analysis of captive rhesus macaques (Kanthaswamy and Smith 2004; Satkoski et al. 2008). In this respect, the absence of the HLA-A2, HLA-A3, and HLA-B7 specificities from the seven common Indian rhesus macaque MHC class I alleles characterized to date (Loffredo et al. 2007; Knapp et al. 1997; Kaizu et al. 2007) is intriguing. This occurrence is likely the result of a combination of factors, including but not limited to a probable founder effect following the 1978 moratorium on importation of Indian-origin animals and the consequent breeding in the USA of captive populations.
Regardless of the evolutionary ramifications of MHC polymorphism and function, our findings have important practical implications because of the role of Chinese-origin rhesus macaques in biomedical research. The presence and identification of key HLA-like specificities in macaque populations of Chinese origin provides the scientific community valuable tools to evaluate disease pathogenesis and vaccine concepts in a setting more reflective of the global community, with broader human population coverage implications.
We would like to thank A. Steen, S. Ngo, and C. Moore for MHC purification and binding assay assistance. This research is supported by the National Institutes of Health (grants R01 AI070902-01A2 to Alessandro Sette and Bianca R. Mothé and R15 AI064175-01 to Bianca R. Mothé).
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
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