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      The effect of antiretroviral treatment on selected genes in whole blood from HIV-infected adults sensitised by Mycobacterium tuberculosis

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          Abstract

          HIV-1 co-infection is a leading cause of susceptibility to tuberculosis (TB), with the risk of TB being increased at all stages of HIV-1 infection. Antiretroviral treatment (ART) is the most effective way to reduce the risk of TB in HIV-1 co-infected people. Studying protective, ART-induced, immune restoration in HIV-1 infected individuals sensitised by Mycobacterium tuberculosis ( Mtb) can thus help identify mechanisms of protection against TB. In order to understand ART-mediated prevention of TB in HIV-1 infected adults, we investigated the expression of 30 genes in whole blood from HIV-1 infected patients during the first 6 months of ART-induced immune reconstitution. The 30 selected genes were previously described to be differentially expressed between sorted Mtb specific central and effector memory CD4 T cells. HIV-1 infected persons sensitised by Mtb were recruited in Khayelitsha, South Africa, when initiating ART. RNA was extracted from whole blood at initiation and 1, 3 and 6 months of ART. qRT-PCR was used to determine gene expression and three reference ‘housekeeping’ genes were used to calculate the fold change in the expression of each gene relative to day 0 of ART. Results were assessed longitudinally. We observed a decrease in the expression of a number of genes at 6 months of ART, reflecting a decrease in immune activation. However, following correction for multiple comparisons and increasing CD4 counts, only the decrease in CD27 gene expression remained statistically significant. While not statistically significant, a number of genes also showed increased expression at various timepoints, illustrating the broad regeneration of the T cell pool in HIV-1 infected adults on ART. Our findings generate hypotheses underlying ART- induced protective immune reconstitution and may pave the way for future studies to evaluate ART mediated prevention of TB in HIV-1 infected persons.

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          Two subsets of memory T lymphocytes with distinct homing potentials and effector functions.

          Naive T lymphocytes travel to T-cell areas of secondary lymphoid organs in search of antigen presented by dendritic cells. Once activated, they proliferate vigorously, generating effector cells that can migrate to B-cell areas or to inflamed tissues. A fraction of primed T lymphocytes persists as circulating memory cells that can confer protection and give, upon secondary challenge, a qualitatively different and quantitatively enhanced response. The nature of the cells that mediate the different facets of immunological memory remains unresolved. Here we show that expression of CCR7, a chemokine receptor that controls homing to secondary lymphoid organs, divides human memory T cells into two functionally distinct subsets. CCR7- memory cells express receptors for migration to inflamed tissues and display immediate effector function. In contrast, CCR7+ memory cells express lymph-node homing receptors and lack immediate effector function, but efficiently stimulate dendritic cells and differentiate into CCR7- effector cells upon secondary stimulation. The CCR7+ and CCR7- T cells, which we have named central memory (TCM) and effector memory (TEM), differentiate in a step-wise fashion from naive T cells, persist for years after immunization and allow a division of labour in the memory response.
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            Utility of the housekeeping genes 18S rRNA, beta-actin and glyceraldehyde-3-phosphate-dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes.

            The accuracy of 18S rRNA, beta-actin mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as indicators of cell number when used for normalization in gene expression analysis of T lymphocytes at different activation stages was investigated. Quantitative real-time reverse transcriptase-polymerase chain reaction was used to determine the expression level of 18S rRNA, beta-actin mRNA, GAPDH mRNA and mRNA for six cytokines in carefully counted samples of resting human peripheral blood mononuclear cells (PBMCs), intestinal lymphocytes and PBMCs subjected to polyclonal T-cell activation. The 18S rRNA level in activated and resting PBMCs and intestinal lymphocytes was essentially the same, while the levels of beta-actin and GAPDH mRNAs fluctuated markedly upon activation. When isolated gammadeltaTCR(+), CD4(+) and CD8(+) subpopulations were studied, 18S rRNA levels remained unchanged after 21 h of activation but increased slightly after 96 h. In contrast, there was a 30-70-fold increase of GAPDH mRNA/cell in these cell populations upon activation. Cytokine analysis revealed that only normalization to 18S rRNA gave a result that satisfactorily reflected their mRNA expression levels per cell. In conclusion, 18S rRNA was the most stable housekeeping gene and hence superior for normalization in comparative analyses of mRNA expression levels in human T lymphocytes.
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              With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies

              The choice of reference genes that are stably expressed amongst treatment groups is a crucial step in real-time quantitative PCR gene expression studies. Recent guidelines have specified that a minimum of two validated reference genes should be used for normalisation. However, a quantitative review of the literature showed that the average number of reference genes used across all studies was 1.2. Thus, the vast majority of studies continue to use a single gene, with β-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being commonly selected in studies of vertebrate gene expression. Few studies (15%) tested a panel of potential reference genes for stability of expression before using them to normalise data. Amongst studies specifically testing reference gene stability, few found ACTB or GAPDH to be optimal, whereby these genes were significantly less likely to be chosen when larger panels of potential reference genes were screened. Fewer reference genes were tested for stability in non-model organisms, presumably owing to a dearth of available primers in less well characterised species. Furthermore, the experimental conditions under which real-time quantitative PCR analyses were conducted had a large influence on the choice of reference genes, whereby different studies of rat brain tissue showed different reference genes to be the most stable. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies.
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                Author and article information

                Contributors
                Role: Data curationRole: Formal analysisRole: Funding acquisitionRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: ResourcesRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: Writing – review & editing
                Role: ConceptualizationRole: ResourcesRole: Writing – review & editing
                Role: Formal analysisRole: Writing – review & editing
                Role: Project administration
                Role: ConceptualizationRole: Funding acquisitionRole: ResourcesRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: Project administrationRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                27 December 2018
                2018
                : 13
                : 12
                : e0209516
                Affiliations
                [1 ] Wellcome Centre for Infectious Diseases Research in Africa, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa
                [2 ] South African Tuberculosis Vaccine Initiative, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa
                [3 ] Department of Pathology, University of Cape Town, Cape Town, South Africa
                [4 ] Department of Medicine, University of Cape Town, Cape Town, South Africa
                [5 ] The Francis Crick Institute, London, United Kingdom
                [6 ] Department of Medicine, Imperial College London, London, United Kingdom
                Institut de Pharmacologie et de Biologie Structurale, FRANCE
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                [¤a]

                Current address: Institute of Immunity and Transplantation, University College London, Royal Free Campus, London, United Kingdom

                [¤b]

                Current address: Walter and Eliza Hall Institute of Medical Research, Parkville, Australia

                Author information
                http://orcid.org/0000-0001-6102-2375
                http://orcid.org/0000-0002-7086-2621
                http://orcid.org/0000-0002-9796-2040
                Article
                PONE-D-18-28678
                10.1371/journal.pone.0209516
                6307796
                30589870
                01269000-68c5-43fa-a1ff-4317f2de811a
                © 2018 Jhilmeet et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 2 October 2018
                : 6 December 2018
                Page count
                Figures: 5, Tables: 3, Pages: 13
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100004440, Wellcome Trust;
                Award ID: 104803
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100004440, Wellcome Trust;
                Award ID: 087754
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100004440, Wellcome Trust;
                Award ID: 203135
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100007601, Horizon 2020;
                Award ID: 643381
                Award Recipient :
                Research supported by The Wellcome Trust (104803, 087754, 203135) to RJW, The South African National Research Foundation (443386), the European Union Horizon 2020 research and innovation programme under grant agreement No 643381 to RJW and The Francis Crick Institute which receives support from UKRI (FC001218), Cancer Research UK (FC001218) and Wellcome (FC001218). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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