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The hallmark of HIV/AIDS is a gradual depletion of CD4 T cells. Despite effective control by antiretroviral therapy (ART), a significant subgroup of people living with HIV (PLHIV) fails to achieve complete immune reconstitution, deemed as immune non-responders (INRs). The mechanisms underlying incomplete CD4 T cell recovery in PLHIV remain unclear. In this study, CD4 T cells from PLHIV were phenotyped and functionally characterized, focusing on their mitochondrial functions. The results show that while total CD4 T cells are diminished, cycling cells are expanded in PLHIV, especially in INRs. HIV-INR CD4 T cells are more activated, displaying exhausted and senescent phenotypes with compromised mitochondrial functions. Transcriptional profiling and flow cytometry analysis showed remarkable repression of mitochondrial transcription factor A (mtTFA) in CD4 T cells from PLHIV, leading to abnormal mitochondrial and T cell homeostasis. These results demonstrate a sequential cellular paradigm of T cell over-activation, proliferation, exhaustion, senescence, apoptosis, and depletion, which correlates with compromised mitochondrial functions. Therefore, reconstituting the mtTFA pathway may provide an adjunctive immunological approach to revitalizing CD4 T cells in ART-treated PLHIV, especially in INRs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhao, Schank, Wang, Li, Nguyen, Dang, Cao, Khanal, Nguyen, Thakuri, Ogbu, Lu, Wu, Morrison, Gazzar, Liu, Zhang, Ning, Moorman and Yao.)

T cells are critical for the control of viral infections and T cell responses are regulated by a dynamic network of non-coding RNAs, including microRNAs (miR) and long non-coding RNAs (lncRNA). Here we show that an activation-induced decline of lncRNA growth arrest-specific transcript 5 (GAS5) activates DNA damage response (DDR), and regulates cellular functions and apoptosis in CD4 T cells derived from people living with HIV (PLHIV) via upregulation of miR-21. Notably, GAS5-miR21-mediated DDR and T cell dysfunction are observed in PLHIV on antiretroviral therapy (ART), who often exhibit immune activation due to low-grade inflammation despite robust virologic control. We found that GAS5 negatively regulates miR-21 expression, which in turn controls critical signaling pathways involved in DNA damage and cellular response. The sustained stimulation of T cells decreased GAS5, increased miR-21 and, as a result, caused dysfunction and apoptosis in CD4 T cells. Importantly, this inflammation-driven T cell over-activation and aberrant apoptosis in ART-controlled PLHIV and healthy subjects (HS) could be reversed by antagonizing the GAS5-miR-21 axis. Also, mutation of the miR-21 binding site on exon 4 of GAS5 gene to generate a GAS5 mutant abolished its ability to regulate miR-21 expression as well as T cell activation and apoptosis markers compared to the wild-type GAS5 transcript. Our data suggest that GAS5 regulates TCR-mediated activation and apoptosis in CD4 T cells during HIV infection through miR-21-mediated signaling. However, GAS5 effects on T cell exhaustion during HIV infection may be mediated by a mechanism beyond the GAS5-miR-21-mediated signaling. These results indicate that targeting the GAS5-miR-21 axis may improve activity and longevity of CD4 T cells in ART-treated PLHIV. This approach may also be useful for targeting other infectious or inflammatory diseases associated with T cell over-activation, exhaustion, and premature immune aging., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Nguyen, Nguyen, Zhao, Schank, Dang, Cao, Khanal, Chand Thakuri, Lu, Zhang, Li, Morrison, Wu, El Gazzar, Ning, Wang, Moorman and Yao.)

CD4 T cell death or survival following initial HIV infection is crucial for the development of viral reservoirs and latent infection, making its evaluation critical in devising strategies for HIV cure. Here we infected primary CD4 T cells with a wild-type HIV-1 and investigated the death and survival mechanisms in productively infected and bystander cells during early HIV infection. We found that HIV-infected cells exhibited increased programmed cell death, such as apoptosis, pyroptosis, and ferroptosis, than uninfected cells. However, productively infected (p24 + ) cells and bystander (p24 - ) cells displayed different patterns of cell death due to differential expression of pro-/anti-apoptotic proteins and signaling molecules. Cell death was triggered by an aberrant DNA damage response (DDR), as evidenced by increases in γH2AX levels, which inversely correlated with telomere length and telomerase levels during HIV infection. Mechanistically, HIV-infected cells exhibited a gradual shortening of telomeres following infection. Notably, p24 + cells had longer telomeres compared to p24 - cells, and telomere length positively correlated with the telomerase, pAKT, and pATM expressions in HIV-infected CD4 T cells. Importantly, blockade of viral entry attenuated the HIV-induced inhibition of telomerase, pAKT, and pATM as well as the associated telomere erosion and cell death. Moreover, ATM inhibition promoted survival of HIV-infected CD4 T cells, especially p24 + cells, and rescued telomerase and AKT activities by inhibiting cell activation, HIV infection, and DDR. These results indicate that productively infected and bystander CD4 T cells employ different mechanisms for their survival and death, suggesting a possible pro-survival, pro-reservoir mechanism during early HIV infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cao, Khanal, Wang, Li, Zhao, Nguyen, Nguyen, Dang, Schank, Thakuri, Zhang, Lu, Wu, Morrison, El Gazzar, Ning, Moorman and Yao.)

Objective: Myeloid-derived suppressor cells (MDSCs) contribute to HIV progression by impairing antiviral immunity; however, the mechanisms responsible for MDSC development during HIV infection are incompletely understood. HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) is a long noncoding RNA (lncRNA) that plays a pivotal role in regulating myeloid cell development via targeting HOXA1. The role of HOTAIRM1--HOXA1 in the differentiation and functions of MDSCs during HIV infection remains unclear., Methods: In this study, we measured MDSC induction and suppressive functions by flow cytometry, RT-PCR, and co-culture experiments using CD33 myeloid cells derived from people living with HIV (PLHIV) on antiretroviral therapy (ART). We also manipulated the HOTAIRM1--HOXA1 axis in myeloid cells using knockdown and overexpression approaches., Results: We demonstrate that HOTAIRM1 and HOXA1 expressions are reciprocally upregulated and are responsible for increased levels of immunosuppressive molecules, such as arginase 1 (Arg1), inducible nitric oxide synthase (iNOS), signal transducer and activator of transcription 3 (STAT3), and reactive oxygen species (ROS), in CD33 myeloid cells derived from PLHIV on ART. We found that overexpression of HOTAIRM1 or HOXA1 in CD33 cells isolated from healthy individuals promoted the differentiation and suppressive functions of MDSCs, whereas silencing of HOTAIRM1 or HOXA1 expression in MDSCs derived from PLHIV significantly inhibited the frequency of MDSCs and expressions of the immunosuppressive molecules and reduced their immunosuppressive effects on T cells., Conclusion: These results indicate that the HOTAIRM1--HOXA1 axis enhances differentiation and suppressive functions of MDSCs and could be a potential therapeutic target for immunomodulation during latent HIV infection.

CD4 T-cell depletion is a hallmark of HIV/AIDS, but the underlying mechanism is still unclear. We have recently shown that ataxia-telangiectasia-mutated (ATM) deficiency in CD4 T cells accelerates DNA damage, telomere erosion, and cell apoptosis in HIV-infected individuals on antiretroviral therapy (ART). Whether these alterations in ART-treated HIV subjects occur in vitro in HIV-infected CD4 T cells remains unknown. In this study, we employed a cellular model of HIV infection to characterize the mechanisms underlying CD4 T-cell destruction by analyzing the telomeric DNA damage response (DDR) and cellular apoptosis in highly permissive SupT1 cells, followed by the validation of our observations in primary CD4 T cells with active or drug-suppressed HIV infection. Specifically, we established an in vitro HIV T-cell culture system with viral replication and raltegravir (RAL; an integrase inhibitor) suppression, mimicking active and ART-controlled HIV infection in vivo We demonstrated that HIV-induced, telomeric DDR plays a pivotal role in triggering telomere erosion, premature T-cell aging, and CD4 T-cell apoptosis or depletion via dysregulation of the PI3K/ATM pathways. This in vitro model provides a new tool to investigate HIV pathogenesis, and our results shed new light on the molecular mechanisms of telomeric DDR and CD4 T-cell homeostasis during HIV infection. IMPORTANCE The hallmark of HIV infection is a gradual depletion of CD4 T cells, with a progressive decline of host immunity. How CD4 T cells are depleted in individuals with active and virus-suppressed HIV infection remains unclear. In this study, we employed a cellular model of HIV infection to characterize the mechanisms underlying CD4 T-cell destruction by analyzing the chromosome end (telomere) DNA damage response (DDR) and cellular apoptosis in a T-cell line (highly permissive SupT1 cells), as well as in primary CD4 T cells with active or drug-suppressed HIV infection. We demonstrated that HIV-induced telomeric DDR plays a critical role in inducing telomere loss, premature cell aging, and CD4 T-cell apoptosis or depletion via dysregulation of the PI3K/ATM pathways. This study sheds new light on the molecular mechanisms of telomeric DDR and its role in CD4 T-cell homeostasis during HIV infection., (Copyright © 2020 American Society for Microbiology.)

HIV infection leads to a phenomenon of inflammaging, in which chronic inflammation induces an immune aged phenotype, even in individuals on combined antiretroviral therapy (cART) with undetectable viremia. In this study, we investigated T cell homeostasis and telomeric DNA damage and repair machineries in cART-controlled HIV patients at risk for inflammaging. We found a significant depletion of CD4 T cells, which was inversely correlated with the cell apoptosis in virus-suppressed HIV subjects compared to age-matched healthy subjects (HS). In addition, HIV CD4 T cells were prone to DNA damage that extended to chromosome ends-telomeres, leading to accelerated telomere erosion-a hallmark of cell senescence. Mechanistically, the DNA double-strand break (DSB) sensors MRE11, RAD50, and NBS1 (MRN complex) remained intact, but both expression and activity of the DNA damage checkpoint kinase ataxia-telangiectasia mutated (ATM) and its downstream checkpoint kinase 2 (CHK2) were significantly suppressed in HIV CD4 T cells. Consistently, ATM/CHK2 activation, DNA repair, and cellular functions were also impaired in healthy CD4 T cells following ATM knockdown or exposure to the ATM inhibitor KU60019 in vitro , recapitulating the biological effects observed in HIV-derived CD4 T cells in vivo . Importantly, ectopic expression of ATM was essential and sufficient to reduce the DNA damage, apoptosis, and cellular dysfunction in HIV-derived CD4 T cells. These results demonstrate that failure of DSB repair due to ATM deficiency leads to increased DNA damage and renders CD4 T cells prone to senescence and apoptotic death, contributing to CD4 T cell depletion or dysfunction in cART-controlled, latent HIV infection., (Copyright © 2019 Zhao, Nguyen, Nguyen, Dang, Cao, Khanal, Schank, Thakuri, Ogbu, Morrison, Wu, Li, Zou, El Gazzar, Ning, Wang, Moorman and Yao.)