Dataset Information

TCR affinity associated with functional differences between dominant and subdominant SIV epitope-specific CD8+ T cells in Mamu-A*01+ rhesus monkeys.

ABSTRACT: Many of the factors that contribute to CD8+ T cell immunodominance hierarchies during viral infection are known. However, the functional differences that exist between dominant and subdominant epitope-specific CD8+ T cells remain poorly understood. In this study, we characterized the phenotypic and functional differences between dominant and subdominant simian immunodeficiency virus (SIV) epitope-specific CD8+ T cells restricted by the major histocompatibility complex (MHC) class I allele Mamu-A*01 during acute and chronic SIV infection. Whole genome expression analyses during acute infection revealed that dominant SIV epitope-specific CD8+ T cells had a gene expression profile consistent with greater maturity and higher cytotoxic potential than subdominant epitope-specific CD8+ T cells. Flow-cytometric measurements of protein expression and anti-viral functionality during chronic infection confirmed these phenotypic and functional differences. Expression analyses of exhaustion-associated genes indicated that LAG-3 and CTLA-4 were more highly expressed in the dominant epitope-specific cells during acute SIV infection. Interestingly, only LAG-3 expression remained high during chronic infection in dominant epitope-specific cells. We also explored the binding interaction between peptide:MHC (pMHC) complexes and their cognate TCRs to determine their role in the establishment of immunodominance hierarchies. We found that epitope dominance was associated with higher TCR:pMHC affinity. These studies demonstrate that significant functional differences exist between dominant and subdominant epitope-specific CD8+ T cells within MHC-restricted immunodominance hierarchies and suggest that TCR:pMHC affinity may play an important role in determining the frequency and functionality of these cell populations. These findings advance our understanding of the regulation of T cell immunodominance and will aid HIV vaccine design.

SUBMITTER: Osuna CE

PROVIDER: S-EPMC3990730 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Publications

TCR affinity associated with functional differences between dominant and subdominant SIV epitope-specific CD8+ T cells in Mamu-A*01+ rhesus monkeys.

Osuna Christa E CE Gonzalez Ana Maria AM Chang Hsun-Hsien HH Hung Amy Shi AS Ehlinger Elizabeth E Anasti Kara K Alam S Munir SM Letvin Norman L NL

PLoS pathogens 20140417 4

Many of the factors that contribute to CD8+ T cell immunodominance hierarchies during viral infection are known. However, the functional differences that exist between dominant and subdominant epitope-specific CD8+ T cells remain poorly understood. In this study, we characterized the phenotypic and functional differences between dominant and subdominant simian immunodeficiency virus (SIV) epitope-specific CD8+ T cells restricted by the major histocompatibility complex (MHC) class I allele Mamu-A ...[more]

PMID: 24743648

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Project description:CD8+ T cells that bind different regions of proteins derived from a virus are often present at different frequencies. It is thought that those virus-specific CD8+ T cells that are present at the highest frequency are predominantly responsible for control of viral infections. While the number of virus-specific CD8+ T cells is undoubtedly important, the functionality of these cells may also play a role in their anti-viral capacity. It was not known if virus-specific CD8+ T cells present at high frequencies are more functionally effective against viral infection than those present at low frequencies. In this study, we characterized the functional differences between the SIV-specific cells present at high frequencies to those present at low frequencies in the blood of rhesus monkeys infected with simian immunodeficiency virus (SIV). We found that the high-and low-frequency SIV-specific cells had different functional capacities both during acute and chronic SIV infection. We also found that the strength with which a cell interacts with the proteins derived from the virus may contribute to the functional differences between the high- and low-frequency cells. These findings further our understanding of anti-viral responses and may inform HIV vaccine design. Peripheral blood was collected on days 14, 21, 28, 35, 42, 56, 70, and and 210 after inoculation with SIVmac251. Plasma viral RNA from these samples was measured using an ultra-sensitive branched DNA amplification assay (Siemens Diagnostics, Berkeley, CA). PBMC were stained with p11C and p54AS tetramers and CD3 and CD8 antibodies at 4°C. Tetramer-positive single CD3+CD8+ lymphocytes were sorted to greather than 95% purity into RNAprotect (Qiagen) at 4°C. RNA was isolated using a Trizol (Invitrogen) extraction protocol. Briefly, 0.8 ml of Trizol were added to the cell pellet and incubated for 5 minutes at room temperature and 0.16 ml of chloroform were added, shaken vigorously by hand for 15 seconds, incubated at room temperature for 2-3 minutes and centrifuged at 13,000 rpm for 15 minutes at 4ºC. The colorless upper aqueous phase was carefully collected and transferred to a new tube containing 2 ?l of linear acrylamide for mixing. An equal volume of isopropyl alcohol was then added and mixed. The mixture was incubated at room temperature for 10 minutes and centrifuged at 13,000 rpm for 10 minutes at 4ºC. The supernatant was collected and the RNA was washed with 1 ml of 70% ethanol and centrifuged at 10,500 rpm for 5 minutes at 4ºC. The supernatant was completely removed and the RNA pellet was allowed to air-dry. The RNA was then resuspended in RNase-free water and stored at -80ºC. RNA integrity was tested using an Agilent Bioanalyzer. RNA was then amplified using the TargetAmp 2-Round Biotin-aRNA Amplification Kit 3.0 (Epicentre Biotechnologies) according the manufacturer’s instructions. Amplified biotinylated antisense-RNA (aRNA) was resuspended in RNase-free water and stored at -80ºC. Nanodrop ND-1000 was used to determine the biotinylatedaRNA concentration and an Agilent Bioanalyzer was used to determine its integrity.Amplified aRNA was hybridized to Illumina Human HT-12 Expression BeadChips according to the manufacturer instructions and was stained with Streptavidin Cy3 for detection (Illumina, San Diego, CA, USA).

Dataset Information

TCR affinity associated with functional differences between dominant and subdominant SIV epitope-specific CD8+ T cells in Mamu-A*01+ rhesus monkeys.

Publications

TCR affinity associated with functional differences between dominant and subdominant SIV epitope-specific CD8+ T cells in Mamu-A*01+ rhesus monkeys.

Similar Datasets

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