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Characterization of MHC class I alleles in sooty mangabeys as a tool for evaluating cellular immunity in natural hosts of SIV infection.

ABSTRACT: Although immune pressure exerted by MHC class I-restricted cytotoxic T lymphocytes (CTL) are an important determinant of outcome in pathogenic HIV and SIV infection, lack of data on MHC class I genes has hampered study of its role in natural hosts with nonpathogenic SIV infection. In this study, we cloned and characterized full-length MHC class I genes derived from the cDNA library of two unrelated naturally infected sooty mangabeys (Cercocebus atys) in whom SIV-specific CTL epitopes were previously mapped. Twenty one full-length MHC class I alleles consisting of five MHC-A (Ceat-A), 13 MHC-B (Ceat-B), and three MHC-E (Ceat-E) alleles were identified. Sequence-specific primers (SSP) for high-throughput screening of genomic DNA by PCR were developed for 16 of the 18 Ceat-A and Ceat-B alleles. Screening of 62 SIV-negative and 123 SIV-infected sooty mangabeys at the Yerkes National Primate Research Center (YNPRC) revealed the presence of up to four MHC-A and eight MHC-B alleles in individual mangabeys, indicating that similar to macaque species, mangabeys have at least two duplications of the MHC-A locus and four duplications of the MHC-B locus in the absence of an MHC-C locus. Using stable transfectants of Ceat MHC Class I alleles in the MHC-null 721.221 cell line, we identified Ceat-B*12:01 as the restricting allele of a previously reported Nef20-28 CTL epitope. Ceat-B*1201/Nef20-28 tetramers identified tetramer-positive CD8+ T lymphocytes in Ceat-B*1201-positive SIV-infected mangabeys. This study has laid the groundwork for comprehensive analysis of CTL and SIV evolution in a natural host of SIV infection.

SUBMITTER: Wang Z

PROVIDER: S-EPMC4536953 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Characterization of MHC class I alleles in sooty mangabeys as a tool for evaluating cellular immunity in natural hosts of SIV infection.

Wang Zichun Z Metcalf Benjamin B Kasheta Melissa M Kasala-Hallinan Caitlin C Tran Dollnovan D Johnson R Paul RP Else James G JG Karl Julie J O'Connor David D Apetrei Cristian C Kaur Amitinder A

Immunogenetics 20150701 8

Although immune pressure exerted by MHC class I-restricted cytotoxic T lymphocytes (CTL) are an important determinant of outcome in pathogenic HIV and SIV infection, lack of data on MHC class I genes has hampered study of its role in natural hosts with nonpathogenic SIV infection. In this study, we cloned and characterized full-length MHC class I genes derived from the cDNA library of two unrelated naturally infected sooty mangabeys (Cercocebus atys) in whom SIV-specific CTL epitopes were previo ...[more]

PMID: 26129855

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Project description:Transcriptional Profiling Reveals Distinguishing Features of Immune Activation in the Lymphatic Tissues of Sooty Mangabeys and Rhesus Macaques in Early SIV Infection Immune activation in the chronic stages of HIV-1 and SIV infection of rhesus macaques is thought to play a critical role in CD4+ T cell depletion and progression to AIDS. Conversely, the lack of immune activation in the chronic stages of SIV infection of sooty mangabeys is thought to protect this species from a pathogenic outcome. This critical difference in immune activation in rhesus macaques and sooty mangabeys has recently been shown to be established in the early stages of SIV infection where there is immune activation in both species, but in sooty mangabeys immune activation is subsequently resolved. Here we report the results of a microarray analysis of early SIV infection in the lymphatic tissues of rhesus macaques and sooty mangabeys that provides a comprehensive view of the distinguishing features of immune activation and host defenses in the two species. We confirm immune activation in sooty mangabeys, but show that it is less robust than in rhesus macaques, where positive feedback loops involving cytosolic pattern recognition receptors, and chemokines and their ligands amplify the response to early SIV infection. We confirm resolution of immune activation in sooty mangabeys, but show it to be partial and selective, identifying the interferon system and CD38 as key correlates of outcome. We also identify potentially novel immunoregulatory mechanisms that mediate resolution, and defensins as host defenses sooty mangabeys employ as expression of other innate immune system effectors declines. The studies we report here were undertaken in a collaborative longitudinal analysis of early SIV infection in SMs described in Bosinger et al and previously reported studies (Estes et al., J Immunol 1008, 180, 6798-6807.). Briefly, SMs were inoculated i.v. with 1 ml of plasma from an experimentally SIVsmm-infected SM sampled at day 11 post infection, with a viral load 107 copies/ml. In our studies we analyzed axillary or inguinal lymph node biopsies that had been obtained from two SMs prior to infection; two SMs at the peak of viral replication at 14 dpi; and two SMs at 30 dpi as viral loads were decreasing to set point. We analyzed axillary or inguinal LNs from 4 RMs without SIV infection and LNs from four RMs infected intravaginally with 2x105 TCID50 of SIVmac239 and sacrificed at 14 dpi the peak of viral replication from two cross-sectional studies described in (Miller et al., The Journal of Virology 2005, 79, 9217-9227.). For lymph node (LN) biopsies, animals were anesthetized with Ketamine or Telazol; the skin over the axillary or inguinal region was clipped and surgically prepped. An incision was made over the LN, which was exposed by blunt dissection and excised over clamps. A portion of the lymph node biopsy for microarray analysis was snap frozen in liquid N2. All animals were housed and cared for at the Yerkes National Primate Research Center in Atlanta, Georgia in accordance with the regulations of the American Association of Accreditation of Laboratory Animal Care standards. These studies were approved by the Emory University and University of Pennsylvania Institutional Animal Care and Usage Committees (IACUC). Microarray Analysis RNA extractions, synthesis of biotin-labeled cRNA probes, microarray hybridization, and data analysis followed previously published procedures (Li et al., The Journal of infectious diseases 2004, 189, 572-582; Li et al., J Immunol 2009, 2009, 183: 1975–1982.). Briefly, snap-frozen lymph node was homogenized in TRIzol. Total RNA was isolated and further purified. Double stranded cDNA and biotin-labeled cRNA probes were synthesized, column purified and fragmented. Fifteen micrograms of fragmented cRNA was hybridized to an Affymetrix GeneChip® Rhesus Macaque Genome Array. After hybridization, chips were washed, stained with streptavidin-phycoerythrin, and scanned with GeneChip Operating Software at the Biomedical Genomics Center at the University of Minnesota. Preparation of cRNA probes and microarray hybridizations were done in duplicate for each RNA sample. Cel. files were uploaded into the Expressionist program (Genedata, Pro version 5.1) and the expression level for each of the 47,000 transcripts in the arrays were analyzed using the RMA algorithm. The expression levels from duplicate microarrays of the same animal’s RNA were correlated and averaged. Tests for differences between the before and after infection at various time points were conducted using the 2-sample Wilcoxon signed-rank test. Fold differences in the level of gene expression between after infection and before infection were calculated with the ratio of the means. After statistical analysis, data was sorted based on these transcript cutoffs: p-value of < 0.05 and fold change ≥ 2.0. Significantly changed genes and transcripts were uploaded into NetAffix Analysis Center (http://www.affymetrix.com/analysis/index.affx) to query gene ontology information and into Ingenuity Pathways Analysis (Ingenuity® Systems, www.ingenuity.com) for gene annotation and pathway analysis. Hierarchical clustering analysis was carried out by using Spotfire for DecisionSite for Functional Genomics.

Dataset Information

Characterization of MHC class I alleles in sooty mangabeys as a tool for evaluating cellular immunity in natural hosts of SIV infection.

Publications

Characterization of MHC class I alleles in sooty mangabeys as a tool for evaluating cellular immunity in natural hosts of SIV infection.

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