Journal: Frontiers in Immunology

Article Title: ATM Deficiency Accelerates DNA Damage, Telomere Erosion, and Premature T Cell Aging in HIV-Infected Individuals on Antiretroviral Therapy

doi: 10.3389/fimmu.2019.02531

Figure Lengend Snippet: ROS generation and telomeric DNA damage in CD4 T cells from HIV patients vs. HS. (A) MFI of DCFDA levels in CD4 T cells isolated from PBMCs of 10 HIV patients and 10 HS, and cultured in vitro without stimulation for 1–4 days, then analyzed by flow cytometry. (B) Flow cytometry analysis for the percentage (%) and MFI of CellROX Green as well as Av levels in CD4 T cells derived from HIV or HS and cultured ex vivo for 4 days. Representative pseudocolor plots and summary data of the MFI as well as % of the two gated cell populations (AV − ROX high and Av + ROX low ) from 3 subjects in each group are shown. (C) MFI of CellROX Green, cell apoptotic rate, as well as the correlation of ROS production and apoptotic rate in CD8 + T cells in 6 HIV and 6 HS. (D) Confocal microscopy analysis of 53BP1 and TRF1 co-localization in the nuclei of CD4 T cells isolated from 8 HIV patients vs. 8 HS. Approximately 100 cells were counted per subject. Summary data of dysfunctional telomere-induced foci (TIF) number per nuclei and the percentage of cells with ≥3 TIFs in the two groups are shown. (E) Representative plots and summary data of the percentage of γH2AX expression in purified CD4 T cells from 16 HIV patients and 16 HS, as well as the MFI in total, naïve and memory CD4 T cells in PBMCs from 8 HIV patient vs. 8 HS.

Article Snippet: Levels of reactive oxygen species (ROS) in CD4 T cells were measured using the DCFDA-based Cellular ROS Detection Kit (Abcam, Cambridge, MA) or CellROX Green ROS Detection kit (ThermoFisher Scientific, Waltham, MA) according to manufacturer's protocol.

Techniques: Isolation, Cell Culture, In Vitro, Flow Cytometry, Derivative Assay, Ex Vivo, Confocal Microscopy, Expressing, Purification

Journal: Frontiers in Immunology

Article Title: ATM Deficiency Accelerates DNA Damage, Telomere Erosion, and Premature T Cell Aging in HIV-Infected Individuals on Antiretroviral Therapy

doi: 10.3389/fimmu.2019.02531

Figure Lengend Snippet: A working model depicting the role of HIV-induced ATM deficiency in T cell cycle arrest, telomeric DNA damage, cell senescence, and apoptosis during chronic viral infection. HIV infection triggers the DNA damage response (DDR) during the early phase of infection via activation of the MRN-ATM-CHK2/P53 signaling pathways in CD4 T cells, promoting cell cycle arrest (through the p21/p27-regulated CDK/Cyclins) and allowing DNA damage repair. If the infection persists and causes irreparable DNA damage, the cell will commit suicide and undergo apoptosis. Only a very small population of HIV reservoirs harbor provirus. However, the persistent, low grade of antigenic and inflammatory or ROS stimulation in the setting of latent HIV-1 infection in individuals on cART drives the majority of uninfected, bystander T cell exhaustion (PD-1) and senescence (telomere attrition) by inducing ATM (PI3K-like kinase) activation/exhaustion and deficiency (low protein expression and phosphorylation). This leads to inflammaging and diminished DNA damage repair, which results in aberrant DNA damage (γH2AX, 53BP1 accumulation), cell apoptosis (caspase-3-dependent cleavage of PARP-1), and thus, constant CD4 T cell depletion. Excessive CD4 T cell loss necessitates homeostatic proliferation and imposes replicative stress on reactive, memory CD4 T cells as well as unprimed, naïve CD4 T cells, leading to accelerated, premature T cell aging or senescence. This working model represents a novel molecular mechanism underlying the T cell dysfunction and immune (vaccine) non-responsiveness seen in the setting of latent HIV infection.

Article Snippet: Levels of reactive oxygen species (ROS) in CD4 T cells were measured using the DCFDA-based Cellular ROS Detection Kit (Abcam, Cambridge, MA) or CellROX Green ROS Detection kit (ThermoFisher Scientific, Waltham, MA) according to manufacturer's protocol.

Techniques: Infection, Activation Assay, Expressing