Project description:We assessed correlates of protection from disease progression in a rare subset of HIV-infected individuals, viremic non-progressors (VNPs). These individuals have high viral load for several years, but in contrast to the majority of infected individuals, they do not progress to AIDS. Here we found this lack of progression was associated with selective preservation of two essential subsets of memory CD4+ T cells, central memory (TCM) and stem-cell memory (TSCM) cells. Compared to HIV-infected putative progressors, VNPs had higher proliferation of these indispensable subsets of memory cells, which was associated with the number of TCM. In addition, the long-lived CD4+ TCM and TSCM cells in VNPs had decreased HIV infection compared to the less critical effector memory CD4+ T cells, which indicates a possible mechanism by which VNPs maintain their CD4+ T cell pool after several years of infection, and remain free from AIDS progression. 6 HIV-infected patients fitting the clinical criteria of Viremic Non-Progressors were identified. VNPs were defined as having confirmed HIV-1 infection for at least 9 years with sustained plasma HIV RNA levels >10,000 copies/ml and maintenance of peripheral blood CD4+ T cell counts >500 cells/mm3 and a CD4% (of all lymphocytes) >15%. As controls, 7 HIV-infected Putative Progressors were identified. PPs were defined as having plasma HIV RNA levels >10,000 copies/mL, CD4+ T cell counts >400 cells/mm3 and having been initially infected with HIV 2-24 months prior to the index visit. The estimated date of initial HIV infection was calculated according to published algorithms that incorporate âde-tunedâ anti-HIV-1 antibody ELISA results or by a documented sero-conversion window of <6 months. All participants were required to be antiretroviral therapy (ART)-naÃ¯ve. RNA from 6 VNP and 7 PPs was purified from PAXgene whole blood tubes and hybridized to Affymetrix U133 Plus 2.0 arrays. During data analysis, one VNP patient (PID 4015) was determined to be an outlier and removed from further analysis. Thus, 5 VNPs and 7 PPs are represented in this Series.

Project description:BACKGROUND: Combination antiretroviral therapy (cART) is able to control HIV-1 viral replication, however long-lived latent infection in resting memory CD4+ T-cells persist. The mechanisms for establishment and maintenance of latent infection in resting memory CD4+ T-cells remain unclear. Previously we have shown that HIV-1 infection of resting CD4+ T-cells co-cultured with CD11c+ myeloid dendritic cells (mDC) produced a population of non-proliferating T-cells with latent infection. Here we asked whether different antigen presenting cells (APC), including subpopulations of DC and monocytes, were able to induce post-integration latent infection in resting CD4+ T-cells, and examined potential cell interactions that may be involved using RNA-seq. RESULTS: mDC (CD1c+), SLAN+ DC and CD14+ monocytes were most efficient in stimulating proliferation of CD4+ T-cells during syngeneic culture and in generating post-integration latent infection in non-proliferating CD4+ T-cells following HIV-1 infection of APC-T-cell co-cultures. In comparison, plasmacytoid DC (pDC) and B-cells did not induce latent infection in APC-T-cell co-cultures. We compared the RNA expression profiles of APC subpopulations that could and could not induce latency in non-proliferating CD4+ T-cells. Gene expression analysis, comparing the mDC, SLAN+ DC and CD14+ monocyte subpopulations to pDC identified 53 upregulated genes that encode proteins expressed on the plasma membrane that could signal to CD4+ T-cells via cell-cell interactions (32 genes), immune checkpoints (IC) (5 genes), T-cell activation (9 genes), regulation of apoptosis (5 genes), antigen presentation (1 gene) and through unknown ligands (1 gene). CONCLUSIONS: APC subpopulations from the myeloid lineage, specifically mDC subpopulations and CD14+ monocytes, were able to efficiently induce post-integration HIV-1 latency in non-proliferating CD4+ T-cells in vitro. Inhibition of key pathways involved in mDC-T-cell interactions and HIV-1 latency may provide novel targets to eliminate HIV latency. mRNA profiles of sorted, pure antigen presenting cells including, CD1c+ myleoid dendirtic cells (mDC), SLAN+ mDC, CD14+ monocytes and plasmacytoid DC (pDC), were generated using next generation sequencing in triplicate, using Illumina Illumina Hiseq 2000.

Project description:HIV-1 infection of resting memory CD4+ T cells forms a barrier to curing HIV-1. Identification of immune biomarkers that correlate with HIV-1 reservoir size could aid with assessing efficacy of HIV-1 eradication strategies, especially in pediatric infections where blood sampling is limited. In adults, the immune exhaustion marker PD-1 on central memory CD4+ T cells (Tcm) correlates with HIV-1 reservoir size. Immune correlates of HIV-1 reservoir size are less defined in perinatal infection. Using multi-parameter flow cytometry, we examined immune activation (CD69, CD25, HLA-DR), and exhaustion (PD-1, TIGIT, LAG-3 and TIM-3) markers on CD4+ T cell subsets (naïve (Tn), central memory (Tcm), and a combination (Ttem) of transitional (Ttm) and effector memory (Tem) in 10 children living with HIV-1 (median age 15.9 years; median duration of virologic suppression 7.0 years), in whom HIV-1 reservoir size was determined with both the Intact Proviral HIV-1 DNA assay (IPDA) and the Tat/Rev limiting dilution assay (TILDA). Total HIV-1 DNA in CD4+ T cells was also measured. Correlations between immune activation, and exhaustion markers on T cell subsets with the various markers of proviral reservoir size, and baseline CD4+ T cell transcriptomes were examined. The median total HIV-1 DNA concentration was 211.9 copies per million CD4+ T cells, with a median intact proviral load in individuals with HIV-1 subtype B of 8.0 copies per million CD4+ T cells. Levels of HLA-DR and TIGIT on Ttem were strongly correlated with total HIV-1 DNA (r=0.758, p=0.015) and (r=0.721, p=0.023), respectively, but not with intact proviral load or inducible reservoir size. HIV-1 DNA load was also positively correlated with transcriptional clusters associated with HLA-DR. In contrast, PD-1 expression on Tcm was inversely correlated with both total HIV-1 DNA (r=-0.67, p=0.039) and HLA-DR in Ttem (r=-0.89, p=0.060). Gene expression profiles for HLA-DR and PD-1 were also inversely correlated. In conclusion, with virologically suppressed perinatal HIV-1 infection, HLA-DR and TIGIT on Ttem CD4+ T cells correlate with total HIV-1 DNA, and may serve as immune biomarkers for transcriptionally active proviruses, including defectives persisting on ART.

Project description:BACKGROUND: Combination antiretroviral therapy (cART) is able to control HIV-1 viral replication, however long-lived latent infection in resting memory CD4+ T-cells persist. The mechanisms for establishment and maintenance of latent infection in resting memory CD4+ T-cells remain unclear. Previously we have shown that HIV-1 infection of resting CD4+ T-cells co-cultured with CD11c+ myeloid dendritic cells (mDC) produced a population of non-proliferating T-cells with latent infection. Here we asked whether different antigen presenting cells (APC), including subpopulations of DC and monocytes, were able to induce post-integration latent infection in resting CD4+ T-cells, and examined potential cell interactions that may be involved using RNA-seq. RESULTS: mDC (CD1c+), SLAN+ DC and CD14+ monocytes were most efficient in stimulating proliferation of CD4+ T-cells during syngeneic culture and in generating post-integration latent infection in non-proliferating CD4+ T-cells following HIV-1 infection of APC-T-cell co-cultures. In comparison, plasmacytoid DC (pDC) and B-cells did not induce latent infection in APC-T-cell co-cultures. We compared the RNA expression profiles of APC subpopulations that could and could not induce latency in non-proliferating CD4+ T-cells. Gene expression analysis, comparing the mDC, SLAN+ DC and CD14+ monocyte subpopulations to pDC identified 53 upregulated genes that encode proteins expressed on the plasma membrane that could signal to CD4+ T-cells via cell-cell interactions (32 genes), immune checkpoints (IC) (5 genes), T-cell activation (9 genes), regulation of apoptosis (5 genes), antigen presentation (1 gene) and through unknown ligands (1 gene). CONCLUSIONS: APC subpopulations from the myeloid lineage, specifically mDC subpopulations and CD14+ monocytes, were able to efficiently induce post-integration HIV-1 latency in non-proliferating CD4+ T-cells in vitro. Inhibition of key pathways involved in mDC-T-cell interactions and HIV-1 latency may provide novel targets to eliminate HIV latency.

Project description:Persistence of memory CD4+ T cells in ECs was coupled with the inactivation of FOXO3a transcriptional activities, which we have previously identified as a critical regulator of TCM survival. Indeed, expression levels of transcriptional targets of FOXO3a, endowed with pro-apoptotic and anti-proliferative functions, were lower in TCM and TEM from ECs as compared to ST individuals. Silencing the transcriptionally active form of FOXO3a by siRNA rescued TCM and TEM of STs from Fas-mediated apoptosis. Moreover the expression of FOXO3a dominant negative form (FOXO3a Nt) rescued the long-term survival of TCM from STs as these cells persisted as long as those derived from ECs. Overall, these studies indicate that FOXO3a activation is an important mediator of the shortened survival and heighteined turnover of TCM and TEM in chronic HIV infection. Targeting this pathway may provide a strategy to preserve memory T cell numbers in HIV infection. Keywords: comparative gene profile, cell-type comparison Isolation of CD4+T cell sub-populations. Peripheral blood mononuclear cells (PBMCs) from healthy adult individuals were isolated by Ficoll-HyPaque (Pharmacia) density gradient. We first enriched for CD4+ T cells using negative immunomagnetic beads selection (Automacs, Myltenii), cells were then labeled with anti-CD4-APCcy7, anti-CD45RA-ECD, anti-CD27-FITC and anti-CCR7-PEcy7 and sorted into Naive cells described as CD4+, CD45RA+, CD27+ and CCR7+, Central Memory cells (TCM) described as CD4+, CD45RA-, CD27+ and CCR7+ cells and Effector Memory cells (TEM) described as CD4+, CD45RA-, CD27- and CCR7- cells. Sorting was performed using a BDAria (BD Pharmingen). Purity of the TCM and TEM sub-populations was ranging from 96 to 99%. All procedures were done at 4C to avoid any changes in cell phenotype or gene expression. Sample RNA was extracted using an RNA extraction kit (Quiagen), then amplified using the MessageAmp RNA kit (Ambion) as per the manufacturer’s instructions. The amplified RNA (aRNA) was then verified for quality and quantity using the Agilent Bioanalyser and measuring the OD. Universal human RNA (Stratagene) was also prepared in the same way. Sample probes were prepared by direct labelling with 3 µg of the aRNA Cy-5 (R values) fluorescent dye while the universal RNA probes were prepared by direct labelling of universal aRNA with Cy-3 (G values). All patient samples were hybridized against amplified universal RNA at 37 ºC for 18h on a custom human Immune array. Detailed information on the labeling and hybridization procedures can be obtained at the Microarray Centre web page (University Health Network).

Project description:Wodarz2007 - HIV/CD4 T-cell interaction A deterministic model illustrating how CD4 T-cells can influence HIV infection. This model is described in the article: Infection dynamics in HIV-specific CD4 T cells: does a CD4 T cell boost benefit the host or the virus? Wodarz D, Hamer DH. Math Biosci 2007 Sep; 209(1): 14-29 Abstract: Recent experimental data have shown that HIV-specific CD4 T cells provide a very important target for HIV replication. We use mathematical models to explore the effect of specific CD4 T cell infection on the dynamics of virus spread and immune responses. Infected CD4 T cells can provide antigen for their own stimulation. We show that such autocatalytic cell division can significantly enhance virus spread, and can also provide an additional reservoir for virus persistence during anti-viral drug therapy. In addition, the initial number of HIV-specific CD4 T cells is an important determinant of acute infection dynamics. A high initial number of HIV-specific CD4 T cells can lead to a sudden and fast drop of the population of HIV-specific CD4 T cells which results quickly in their extinction. On the other hand, a low initial number of HIV-specific CD4 T cells can lead to a prolonged persistence of HIV-specific CD4 T cell help at higher levels. The model suggests that boosting the population of HIV-specific CD4 T cells can increase the amount of virus-induced immune impairment, lead to less efficient anti-viral effector responses, and thus speed up disease progression, especially if effector responses such as CTL have not been sufficiently boosted at the same time. This model is hosted on BioModels Database and identified by: BIOMD0000000663. To cite BioModels Database, please use: Chelliah V et al. BioModels: ten-year anniversary. Nucl. Acids Res. 2015, 43(Database issue):D542-8. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

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Expression data from Human CD4 T cells subpopulations of HIV+ patients and HIV- controls

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