Doherty Institute - Research Publications
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ItemMemory CD4+ T cells that co-express PD1 and CTLA4 have reduced response to activating stimuli facilitating HIV latency(CELL PRESS, 2022-10-18)Programmed cell death 1 (PD1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4) suppress CD4+ T cell activation and may promote latent HIV infection. By performing leukapheresis (n = 21) and lymph node biopsies (n = 8) in people with HIV on antiretroviral therapy (ART) and sorting memory CD4+ T cells into subsets based on PD1/CTLA4 expression, we investigate the role of PD1 and CTLA 4 in HIV persistence. We show that double-positive (PD1+CTLA4+) cells in blood contain more HIV DNA compared with double-negative (PD1-CTLA4-) cells but still have a lower proportion of cells producing multiply spliced HIV RNA after stimulation as well as reduced upregulation of T cell activation and proliferation markers. Transcriptomics analyses identify differential expression of key genes regulating T cell activation and proliferation with MAF, KLRB1, and TIGIT being upregulated in double-positive compared with double-negative cells, whereas FOS is downregulated. We conclude that, in addition to being enriched for HIV DNA, double-positive cells are characterized by negative signaling and a reduced capacity to respond to stimulation, favoring HIV latency.
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ItemHuman Immunodeficiency Virus (HIV)-Infected CCR6+ Rectal CD4+ T Cells and HIV Persistence On Antiretroviral Therapy(OXFORD UNIV PRESS INC, 2020-03-01)BACKGROUND: Identifying where human immunodeficiency virus (HIV) persists in people living with HIV and receiving antiretroviral therapy is critical to develop cure strategies. We assessed the relationship of HIV persistence to expression of chemokine receptors and their chemokines in blood (n = 48) and in rectal (n = 20) and lymph node (LN; n = 8) tissue collected from people living with HIV who were receiving suppressive antiretroviral therapy. METHODS: Cell-associated integrated HIV DNA, unspliced HIV RNA, and chemokine messenger RNA were quantified by quantitative polymerase chain reaction. Chemokine receptor expression on CD4+ T cells was determined using flow cytometry. RESULTS: Integrated HIV DNA levels in CD4+ T cells, CCR6+CXCR3+ memory CD4+ T-cell frequency, and CCL20 expression (ligand for CCR6) were highest in rectal tissue, where HIV-infected CCR6+ T cells accounted for nearly all infected cells (median, 89.7%). Conversely in LN tissue, CCR6+ T cells were infrequent, and there was a statistically significant association of cell-associated HIV DNA and RNA with CCL19, CCL21, and CXCL13 chemokines. CONCLUSIONS: HIV-infected CCR6+ CD4+ T cells accounted for the majority of infected cells in rectal tissue. The different relationships between HIV persistence and T-cell subsets and chemokines in rectal and LN tissue suggest that different tissue-specific strategies may be required to eliminate HIV persistence and that assessment of biomarkers for HIV persistence may not be generalizable between blood and other tissues.
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ItemHIV integration sites in latently infected cell lines: evidence of ongoing replication(BIOMED CENTRAL LTD, 2017-01-13)BACKGROUND: Assessing the location and frequency of HIV integration sites in latently infected cells can potentially inform our understanding of how HIV persists during combination antiretroviral therapy. We developed a novel high throughput sequencing method to evaluate HIV integration sites in latently infected cell lines to determine whether there was virus replication or clonal expansion in these cell lines observed as multiple integration events at the same position. RESULTS: We modified a previously reported method using random DNA shearing and PCR to allow for high throughput robotic processing to identify the site and frequency of HIV integration in latently infected cell lines. Latently infected cell lines infected with intact virus demonstrated multiple distinct HIV integration sites (28 different sites in U1, 110 in ACH-2 and 117 in J1.1 per 150,000 cells). In contrast, cell lines infected with replication-incompetent viruses (J-Lat cells) demonstrated single integration sites. Following in vitro passaging of the ACH-2 cell line, we observed a significant increase in the frequency of unique HIV integration sites and there were multiple mutations and large deletions in the proviral DNA. When the ACH-2 cell line was cultured with the integrase inhibitor raltegravir, there was a significant decrease in the number of unique HIV integration sites and a transient increase in the frequency of 2-LTR circles consistent with virus replication in these cells. CONCLUSION: Cell lines latently infected with intact HIV demonstrated multiple unique HIV integration sites indicating that these cell lines are not clonal and in the ACH-2 cell line there was evidence of low level virus replication. These findings have implications for the use of latently infected cell lines as models of HIV latency and for the use of these cells as standards.
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ItemUnderstanding Factors That Modulate the Establishment of HIV Latency in Resting CD4+T-Cells In Vitro(PUBLIC LIBRARY SCIENCE, 2016-07-06)Developing robust in vitro models of HIV latency is needed to better understand how latency is established, maintained and reversed. In this study, we examined the effects of donor variability, HIV titre and co-receptor usage on establishing HIV latency in vitro using two models of HIV latency. Using the CCL19 model of HIV latency, we found that in up to 50% of donors, CCL19 enhanced latent infection of resting CD4+ T-cells by CXCR4-tropic HIV in the presence of low dose IL-2. Increasing the infectious titre of CXCR4-tropic HIV increased both productive and latent infection of resting CD4+ T-cells. In a different model where myeloid dendritic cells (mDC) were co-cultured with resting CD4+ T-cells, we observed a higher frequency of latently infected cells in vitro than CCL19-treated or unstimulated CD4+ T-cells in the presence of low dose IL-2. In the DC-T-cell model, latency was established with both CCR5- and CXCR4-tropic virus but higher titres of CCR5-tropic virus was required in most donors. The establishment of latency in vitro through direct infection of resting CD4+ T-cells is significantly enhanced by CCL19 and mDC, but the efficiency is dependent on virus titre, co-receptor usage and there is significant donor variability.
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ItemPolymorphisms in the CD14 and TLR4 genes independently predict CD4+ T-cell recovery in HIV-infected individuals on antiretroviral therapy(LIPPINCOTT WILLIAMS & WILKINS, 2016-09-10)BACKGROUND: Chronic HIV infection leads to marked depletion of CD4 T cells in the gastrointestinal tract and increased microbial translocation measured by an increase in circulating lipopolysaccharide (LPS) levels. Here, we hypothesized that single-nucleotide polymorphisms (SNPs) in genes encoding the Toll-like receptor 4 (TLR4) and CD14, the principal receptors for LPS, were associated with CD4 T-cell recovery postantiretroviral therapy (ART). METHODS: Prospective study of predominantly white HIV-infected participants receiving suppressive ART for at least 12 months. We analysed the CD14 SNPs C-260T and the TLR4 SNPs A+896G, C+1196T. We also determined the levels of LPS and soluble CD14 in plasma samples collected pre-ART and post-ART initiation. CD4 T-cell recovery was assessed by linear mixed models. RESULTS: Following ART, individuals with a TT genotype compared with a CT or CC genotype for CD14 C-260T SNP showed higher levels of soluble CD14 (P = 0.008 and 0.003, respectively). The CC genotype for the CD14 C-260T SNP, compared with CT or TT, and the TLR4 SNP (AC/GT), compared with the homozygous genotype (AA/CC), were both independently associated with enhanced long-term CD4 T-cell recovery (>3 months; P < 0.001). CONCLUSION: Polymorphisms in CD14 and TLR4 are independently associated with long-term CD4 T-cell recovery in HIV-infected individuals post-ART.