Role of TCR:peptide/MHC Class I Affinity in Directing Different Maturation Phenotypes of Dominant and Subdominant Epitope-Specific CD8+ T Cells in Mamu-A*01+ Rhesus Monkeys
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ABSTRACT:
ORGANISM(S): Homo sapiens Macaca mulatta
PROVIDER: GSE54001 | GEO | 2014/04/24
SECONDARY ACCESSION(S): PRJNA234329
REPOSITORIES: GEO
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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).
2014-04-24 | E-GEOD-54001 | biostudies-arrayexpress
Project description:The goal of an effective AIDS vaccine is to generate immunity that will prevent HIV-1 acquisition. Despite limited progress towards this goal, renewed optimism has followed the recent success of the RV144 vaccine trial in Thailand. However, the lack of complete protection in this trial suggests that breakthroughs, where infection occurs despite adequate vaccination, will be a reality for many vaccine candidates. We previously reported that neutralizing antibodies elicited by DNA prime/rAd5 boost vaccination with SIVmac239 Gag/Pol and Env provided protection against pathogenic SIVsmE660 acquisition after repeated mucosal challenge. Here, we report that SIV-specific CD8+ T cells elicited by that vaccine lowered both peak and set-point viral loads in macaques that became infected despite vaccination. These SIV-specific CD8+ T cells showed strong virus inhibitory activity (VIA) and displayed an effector memory (EM) phenotype. VIA correlated with high levels of CD107a mobilization and perforin expression in SIV-specific CD8+ T cells. Remarkably, both the frequency and the number of Gag CM9-specific public clonotypes were strongly correlated with VIA mediated by EM CD8+ T cells. The ability to elicit such virus-specific EM CD8+ T cells might contribute substantially to an efficacious HIV/AIDS vaccine, even after breakthrough infection. Gag CM9-specific EM CD8+ T cells (CD28 low CD95 high tetramer+) from SIV-negative macaques at 12 wks post-DNA/rAd5 immunization were sorted by flow cytometry for microarray studies. RNA samples from strong VIA animals with (n=3) or without (n=6) CM9 peptide stimulation, along with CM9 peptide stimulated samples from weak VIA animals (n=2) were prepared using the Illumina beads station assay and hybridized to the Illumina HumanHT-12 version 4 Expression BeadChip.
2012-06-14 | E-GEOD-35628 | biostudies-arrayexpress
Project description:Some viruses have established an equilibrium with their host. African green monkeys (AGM) display persistent high viral replication in blood and intestine during Simian immunodeficiency virus (SIV) infection but resolve systemic inflammation after acute infection and lack intestinal immune or tissue damage during chronic infection. We show that NKG2 a/c + CD8 + T cells increase in blood and intestine of AGM in response to SIVagm infection in contrast to SIVmac infection in macaques, the latter modeling HIV infection. NKG2 a/c + CD8 + T cells were not expanded in lymph nodes and CXCR5 + NKG2 a/c + CD8 + T cell frequencies further decreased after SIV infection. Genome-wide transcriptome analysis of NKG2 a/c + CD8 + T cells from AGM revealed the expression of NK cell receptors, and of molecules with cytotoxic effector, gut homing, immunoregulatory and gut barrier function, including CD73. NKG2 a/c + CD8 + T cells correlated negatively with IL-23 in the intestine during SIVmac infection. The data suggest a potential regulatory role of NKG2 a/c + CD8 + T cells in intestinal inflammation during SIV/HIV infections.
2021-03-11 | E-MTAB-8664 | biostudies-arrayexpress
Project description:MHC-I-restricted, virus-specific cytotoxic CD8+ T cells control HIV/SIV replication via the recognition and killing of productively infected CD4+ T cells. Several studies in SIV-infected macaques suggest that CD8+ T cells may also decrease virus production by suppressing viral transcription. RNA sequencing is used to identify candidate cellular pathways that are involved in the virus-silencing mediated by these CD8+ T cells.
2020-08-23 | GSE130368 | GEO
Project description:HIV cure efforts are increasingly focused on harnessing CD8 T cell functions; however, a deeper understanding of CD8 T cells promoting HIV control is necessary to properly inform therapeutic approaches. Here, we identified a novel TOX-expressing CD8 T cell population associated with control of SIV infection in lymphoid tissue of rhesus macaques defined as an antigen-responsive TCF1+ CD39+ subset expressing high levels of TOX and inhibitory receptors but lower levels of canonical cytolytic molecules such as granzyme B, granzyme A, and perforin. Transcriptional analysis of SIV-specific CD8 T cells, as well as proteomic analysis of purified CD8 T cell subsets, revealed these TCF1+ CD39+ cells as an intermediate effector population retaining stem-like features while maintaining a lineage relationship with terminal effector cells. TCF1+ CD39+ CD8 T cells expressed higher levels of CXCR5 than terminally differentiated cells, were found at higher frequency in follicular micro-environments, and were preferentially located in the proximity of SIV-RNA+ cells both in lymph node T cell zone and B cell follicles. Importantly, their frequency was strongly associated with reduced plasma viremia and lower reservoir size. Finally, we confirmed the presence of a highly similar TOX-enriched TCF1+ CD39+ cell population in lymph node biopsies from ART-naïve and ART-treated people living with HIV. Collectively, these data identify a unique population of lymphoid CD8 T cells possessing both stem-like and effector properties that contribute to limiting HIV/SIV persistence.
2024-06-11 | PXD050498 | Pride
Project description:The goal of an effective AIDS vaccine is to generate immunity that will prevent HIV-1 acquisition. Despite limited progress towards this goal, renewed optimism has followed the recent success of the RV144 vaccine trial in Thailand. However, the lack of complete protection in this trial suggests that breakthroughs, where infection occurs despite adequate vaccination, will be a reality for many vaccine candidates. We previously reported that neutralizing antibodies elicited by DNA prime/rAd5 boost vaccination with SIVmac239 Gag/Pol and Env provided protection against pathogenic SIVsmE660 acquisition after repeated mucosal challenge. Here, we report that SIV-specific CD8+ T cells elicited by that vaccine lowered both peak and set-point viral loads in macaques that became infected despite vaccination. These SIV-specific CD8+ T cells showed strong virus inhibitory activity (VIA) and displayed an effector memory (EM) phenotype. VIA correlated with high levels of CD107a mobilization and perforin expression in SIV-specific CD8+ T cells. Remarkably, both the frequency and the number of Gag CM9-specific public clonotypes were strongly correlated with VIA mediated by EM CD8+ T cells. The ability to elicit such virus-specific EM CD8+ T cells might contribute substantially to an efficacious HIV/AIDS vaccine, even after breakthrough infection.
2012-06-15 | GSE35628 | GEO
Project description:CD8+ T cells contribute to the antiviral response in simian immunodeficiency virus (SIV) infection in nonhuman primates subsequent to recognition of viral epitopes via the T cell receptor (TCR). In previous studies, we examined the TCR repertoire of CD8+ T cells specific for a single SIV immunodominant epitope, Gag-CM9 (CTPYDINQM), throughout SIV infection. We identified tissue specific TCR sequences and TCRs shared by multiple anatomical sites. We now sought to evaluate if the tissue localization or TCR sequence of a CM9 specific CD8+ T cell corresponds with a transcriptional phenotype. CM9 specific CD8+ T cells were sorted from blood, lymph nodes, spleen, and liver from SIV infected rhesus macaques. The cells were processed through a single cell sequencing protocol, creating a TCR amplified library and an RNA gene expression library for each cell. Gene Set Enrichment Analysis (GSEA) revealed no distinct transcriptional profiles for CM9 specific CD8+ T cells between different anatomical sites and between cells with shared or tissue specific TCRs. Similarly, no clear transcriptional profiles were associated with public clonotypes. CM9 specific CD8+ T cells from posttreatment controllers did exhibit enrichment of select pathways compared to non-controllers. These data suggest that, while antigen specific CD8+ T cells from non-controllers exhibit largely similar transcriptional profiles, altered transcription in antigen specific CD8+ T cells from post-treatment controllers may associate with viral control.
2024-06-23 | GSE270230 | GEO
Project description:CD8+ T-cells inhibit virus replication in SIV-infected rhesus macaques (RM). However, it is unclear to what extent the viral suppression mediated by CD8+ T-cells reflects direct killing of infected cells as opposed to indirect, non-cytolytic mechanisms. In this study, we used functional genomics to investigate potential mechanisms of in vivo viral suppression mediated by CD8+ lymphocytes. Eight chronically SIVmac239-infected RMs underwent CD8+ lymphocyte depletion, and RNA from whole blood was obtained prior to depletion, at the nadir of CD8+ lymphocytes (5 days post-depletion), and during the repopulation phase (11 days post-depletion). Principal components analysis demonstrated that overall gene expression during the nadir of CD8+ T-cells was highly divergent from other intervals. Conversely, the genomic signature of samples from the CD8+ cell rebound phase was similar to that of pre-depletion samples. During CD8+ lymphocyte depletion we detected a strongly significant decrease in the expression of the genes encoding CD8α and CD8β chains, consistent with the near complete CD8+ T-cell depletion measured by flow cytometry. Of note, we observed significant down-regulation of the expression of genes encoding for factors that can suppress SIV replication, including the CCR5-binding chemokine CCL5/Rantes, several retroviral restriction factors (TRIM10, TRIM15, APOBEC3G/H) and defensins. Reduced expression of various genes related to T cell activation and proliferation was also observed. Collectively, these data indicate that depletion of CD8+ lymphocytes in SIV-infected RMs is associated with the establishment of a pattern of gene expression that may result in increased intrinsic permissivity to virus replication. A total of 60 RNA samples were hybridized on to Rhesus Affymetrix 3' Expression arrays. The study was composed of 8 replicate rhesus macaques subjected to SIVmac239 infection and followed over infection, during subsequent treatment with monocloncal antibody OKT8F to deplete CD8+ T lymphocytes, antiretroviral therapy to suppress virus. Samples were taken at various time points during acute and chronic infection, after CD8+ cell depletion, after CD8+ cell reconstition, and during ART suppression of virus.
2013-02-08 | E-GEOD-37834 | biostudies-arrayexpress
Project description:In contrast to pathogenic HIV and SIV infection of humans and macaques, SIV infection of sooty mangabeys (SMs) is typically non-pathogenic despite high virus replication. A key feature of primary SIV infection of SMs is a strong type I interferon (IFN-I) response, characterized by massive up-regulation of interferon-stimulated genes (ISG), followed by rapid resolution during the acute-to-chronic phase transition and establishment of an immune quiescent state that persists throughout the chronic infection. Based on these observations we hypothesized that low levels of IFN-I signaling may be instrumental in preventing chronic immune activation and disease progression in SIV-infected SMs. We used microarrays to characterize gene expression changes induced by IFNalpha treatment. To directly assess the effects of an experimentally-induced augmentation of IFN-I signaling in chronically SIV-infected SMs, we administered recombinant rhesus macaque IFNalpha2-IgFc (rmIFNα2) to eight naturally SIV-infected SMs weekly for 16 weeks and longitudinally monitored viral load, lymphocyte counts, immune activation, SIV-specific CD8+ T-cell responses, and gene expression profile. Administration of rmIFNα2 was bioactive in vivo with gene expression profiling revealing a strong upregulation of numerous ISGs in the blood of treated animals.
2012-07-09 | E-GEOD-35460 | biostudies-arrayexpress
Project description:Live-attenuated SIV vaccines (LAVs) remain the most efficacious of all vaccines in nonhuman primate models of HIV/AIDS, yet the basis of their robust protection remains poorly understood. Here, we demonstrate that the degree of LAV-mediated protection against intravenous (IV) wildtype (wt) SIVmac239 challenge strongly correlates with the magnitude and function of SIV-specific, effector-differentiated T cells in lymph node (LN), but not with such T cell responses in blood or with other cellular, humoral and innate immune parameters. Maintenance of protective T cell responses was associated with persistent LAV replication in LN, which occurred almost exclusively in follicular helper T cells. Thus, effective LAVs maintain lymphoid tissue-based, effector-differentiated, SIV-specific T cells that intercept and suppress early wt SIV amplification and, if present in sufficient frequencies, can completely control and perhaps clear infection -- an observation that provides rationale for development of safe, persistent vectors that can elicit and maintain such responses. There are two protection outcome groups, complete protect (CP) and non-protect (NP) animals. Memory T cells (CD4+ or CD8+) were sorted for three time points, seven days pre-challenge, four days post-challenge and fourteen days post-challenge.
2012-08-08 | E-GEOD-39968 | biostudies-arrayexpress