Infected CD4 Research Articles

The role of CD101-expressing CD4 T cells in HIV/SIV pathogenesis and persistence.

Despite the advent of effective antiretroviral therapy (ART), human immunodeficiency virus (HIV) continues to pose major challenges, with extensive pathogenesis during acute and chronic infection prior to ART initiation and continued persistence in a reservoir of infected CD4 T cells during long-term ART. CD101 has recently been characterized to play an important role in CD4 Treg potency. Using the simian immunodeficiency virus (SIV) model of HIV infection in rhesus macaques, we characterized the role and kinetics of CD101+ CD4 T cells in longitudinal SIV infection. Phenotypic analyses and single-cell RNAseq profiling revealed that CD101 marked CD4 Tregs with high immunosuppressive potential, distinct from CD101- Tregs, and these cells also were ideal target cells for HIV/SIV infection, with higher expression of CCR5 and α4β7 in the gut mucosa. Notably, during acute SIV infection, CD101+ CD4 T cells were preferentially depleted across all CD4 subsets when compared with their CD101- counterpart, with a pronounced reduction within the Treg compartment, as well as significant depletion in mucosal tissue. Depletion of CD101+ CD4 was associated with increased viral burden in plasma and gut and elevated levels of inflammatory cytokines. While restored during long-term ART, the reconstituted CD101+ CD4 T cells display a phenotypic profile with high expression of inhibitory receptors (including PD-1 and CTLA-4), immunsuppressive cytokine production, and high levels of Ki-67, consistent with potential for homeostatic proliferation. Both the depletion of CD101+ cells and phenotypic profile of these cells found in the SIV model were confirmed in people with HIV on ART. Overall, these data suggest an important role for CD101-expressing CD4 T cells at all stages of HIV/SIV infection and a potential rationale for targeting CD101 to limit HIV pathogenesis and persistence, particularly at mucosal sites.

A patient with CD4+ T cells deficiency and HIV negative with pulmonary tuberculosis, tuberculous pleuritis and meningitis tuberculosis

Background: CD4+ T cells deficiency generally occurs in human immunodeficiency virus (HIV) patients, leading to some infections such as pulmonary and extrapulmonary tuberculosis. A rare case of decreased CD4+ T cells is idiopathic CD4+ T cell lymphocytopenia, a rare and unexplained immunodeficiency syndrome with no evidence of HIV infection. In this case report, we are reporting a patient with CD4+ T cells deficiency, pulmonary tuberculosis, pleuritis tuberculosis, and meningitis with HIV test negative. Case Presentation: A 58-year-old male was referred to the Emergency Department of Dr. Soetomo General Hospital with a gradual decrease of consciousness following six days of hospitalization at a private hospital. During hospitalization, one liter of fluid was evacuated from the right lung and analyzed, revealing tuberculosis infection. History of diabetes, hypertension, stroke, hepatitis, and cardiovascular disease was denied. The patient also never had chemotherapy or radiation treatment. Based on history taking, physical examination, and laboratory results, this patient has been diagnosed new case of pulmonary tuberculosis with deficiency CD4+ T cells and altered consciousness et causes meningitis tuberculosis with pleurisy tuberculosis. After 32 days of hospitalization with anti-mycobacterium therapy, the patient was improved and was discharged. Conclusion: This case highlights the challenges of having the definitive cause of CD4+ T cells deficiency either active tuberculosis infection or idiopathic CD4+ lymphocytopenia. Therefore, serial analyses of CD4+ T cells are advised on the patient during the treatment with anti-tuberculosis drug.

Persisting Microbiota and Neuronal Imbalance Following T. gondii Infection Reliant on the Infection Route.

Toxoplasma gondii is a highly successful parasite capable of infecting all warm-blooded animals. The natural way of infection in intermediate hosts is the oral ingestion of parasite-contaminated water or food. In murine experimental models, oral infection (p.o.) of mice with T. gondii is applied to investigate mucosal and peripheral immune cell dynamics, whereas intraperitoneal infection (i.p.) is frequently used to study peripheral inflammation as well as immune cell – neuronal interaction in the central nervous system (CNS). However, the two infection routes have not yet been systematically compared along the course of infection. Here, C57BL/6 mice were infected p.o. or i.p. with a low dose of T. gondii cysts, and the acute and chronic stages of infection were compared. A more severe course of infection was detected following i.p. challenge, characterized by an increased weight loss and marked expression of proinflammatory cytokines particularly in the CNS during the chronic stage. The elevated proinflammatory cytokine expression in the ileum was more prominent after p.o. challenge that continued following the acute phase in both i.p. or p.o. infected mice. This resulted in sustained microbial dysbiosis, especially after p.o. challenge, highlighted by increased abundance of pathobionts from the phyla proteobacteria and a reduction of beneficial commensal species. Further, we revealed that in the CNS of i.p. infected mice CD4 and CD8 T cells displayed higher IFNγ production in the chronic stage. This corresponded with an increased expression of C1q and CD68 in the CNS and reduced expression of genes involved in neuronal signal transmission. Neuroinflammation-associated synaptic alterations, especially PSD-95, VGLUT, and EAAT2 expression, were more pronounced in the cortex upon i.p. infection highlighting the profound interplay between peripheral inflammation and CNS homeostasis.

Id3 expression identifies CD4+ memory Th1 cells

Memory CD4+ T cells play a pivotal role in mediating long-term protective immunity, positioning them as an important target in vaccine development. However, multiple functionally distinct helper CD4+ T-cell subsets can arise in response to a single invading pathogen, complicating the identification of rare populations of memory precursor cells during the effector phase of infection and memory CD4+ T cells following pathogen clearance and the contraction phase of infection. Furthermore, current literature remains unclear regarding whether a single CD4+ memory T-cell lineage gives rise to secondary CD4+ T helper subsets or if there are unique memory precursor cells within each helper lineage. A majority of T follicular helper (Tfh) cells, which have established memory potential, express Id3, an inhibitor of E protein transcription factors, following acute viral infection. We show that expression of Id3 definitively identified a subset of cells within both the CD4+ Tfh and T helper 1 (Th1) lineages at memory time points that exhibited memory potential, with the capacity for significant re-expansion in response to secondary infection. Notably, we demonstrate that a subset of Th1 cells that survive into the memory phase were marked by Id3 expression and possessed the potential for enhanced expansion and generation of both Th1 and Tfh secondary effector cell populations in a secondary response to pathogen. Additionally, these cells exhibited enrichment of key molecules associated with memory potential when compared with Id3lo Th1 cells. Therefore, we propose that Id3 expression serves as an important marker to indicate multipotent potential in memory CD4+ T cells.

Calcitriol decreases HIV-1 transfer in vitro from monocyte-derived dendritic cells to CD4 + T cells, and downregulates the expression of DC-SIGN and SIGLEC-1.

Dendritic cells (DCs) promote HIV-1 transmission by acting as Trojan horses, capturing viral particles, facilitating the infection of CD4+ T-cells. Vitamin D (VitD) has shown to decrease T cell activation, reducing susceptibility to HIV-1 infection of CD4+ T-cells in vitro; however, if VitD decreases viral transfer from DCs to CD4+ T-cells is unknown. In this study, we co-cultured HIV-1-pulsed immature and LPS mature monocytes-derived DCs (iDCs and LmDCs, respectively), differentiated in presence or absence of calcitriol (VitD active form), with PHA-activated autologous CD4+ T-cells from 16 healthy donors. In co-cultures of iDCs and LmDCs treated with calcitriol, there was a significant decrease in frequency of infected CD4+ T-cells, evaluated by flow cytometry. However, p24 levels evaluated by ELISA were not significantly reduced in culture supernatants. Moreover, calcitriol-treated iDCs exhibited decreased expression of genes involved in HIV-1 transfer compared to the control. Both, calcitriol-treated iDCs and LmDCs exhibit a similar gene expression profile, probably related to a transcriptional balance achieved after long treatment with calcitriol. Since calcitriol-differentiated DCs express on their surface a lower amount of DC-SIGN and SIGLEC-1 molecules, widely associated with HIV-1 transfer, suggesting that this mechanism contributes to a lower transfer of viral particles by the DCs.

Wnt/β-Catenin Protects Lymphocytes from HIV-Mediated Apoptosis via Induction of Bcl-xL.

HIV infection mediates the apoptosis of lymphocytes, the mechanism of which is multifaceted. Here, we evaluated the role of Wnt/β-catenin signaling in HIV-associated T cell apoptosis, as Wnt/β-catenin regulates the transcriptional activity of genes impacting apoptosis. We specifically investigated the role of the Wnt/β-catenin pathway in the HIV-associated apoptosis of CD4+ T cells and CD4dimCD8bright T cells, a population that is infected by HIV. We found that the induction of β-catenin, via a 6-bromoindirubin-3-oxime (BIO), significantly rescued HIV-infected CD4+ and CD4dimCD8bright T cells from apoptosis by >40–50%. Further, a small-molecule inhibitor of the Wnt/β-catenin pathway (PNU-74654) reversed BIO-mediated protection from HIV-associated apoptosis. BIO also induced Bcl-xL, an anti-apoptotic protein, and a target gene of Wnt/β-catenin, in CD4+ and CD4dimCD8bright T cells by approximately 3-fold. Inhibiting Bcl-xL by WEHI-539 abrogated β-catenin-mediated apoptotic protection in infected CD4+ and CD4dimCD8bright T cells. Collectively, these findings demonstrate that engaging Wnt/β-catenin signaling in HIV-infected T cells protects them from HIV-associated apoptosis by inducing Bcl-xL.

Endocytosed HIV-1 Envelope Glycoprotein Traffics to Rab14+ Late Endosomes and Lysosomes to Regulate Surface Levels in T-Cell Lines.

ABSTRACTProduction of infectious HIV-1 particles requires incorporation of the viral envelope glycoprotein (Env) at the plasma membrane (PM) of infected CD4+ T cells. Env trafficking to the PM exposes viral epitopes that can be exploited by the host immune system; however, HIV-1 can evade this response by endocytosis of excess Env from the PM. The fate of Env after internalization remains unclear, with evidence suggesting several different vesicular trafficking steps may be involved, including recycling pathways. To date, there have been very few studies documenting the trafficking pathways of native Env in infected T cells. Furthermore, it remains unclear whether there are T-cell-specific endosomal pathways regulating the fate of endocytic Env. Here, we use a pulse-labeling approach with a monovalent anti-Env Fab probe to characterize the trafficking of internalized Env within infected CD4+ T-cell lines, together with CRISPR/Cas9-mediated endogenous protein tagging, to assess the role of host cell Rab GTPases in Env trafficking. We show that endocytosed Env traffics to Rab14+ compartments that possess hallmarks of late endosomes and lysosomes. We also demonstrate that Env can recycle back to the PM, although we find that recycling does not occur at high rates when compared to the model recycling protein transferrin. These results help to resolve open questions about the fate and relevance of endocytosed Env in HIV-infected cells and suggest a novel role for Rab14 in a cell-type-specific late-endosomal/lysosomal trafficking pathway in T cells.IMPORTANCE HIV-1 envelope glycoprotein (Env) evades immune neutralization through many mechanisms. One immune evasion strategy may result from the internalization of excess surface-exposed Env to prevent antibody-dependent cellular cytotoxicity or neutralization. Characterization of the fate of endocytosed Env is critical to understand which vesicular pathways could be targeted to promote display of Env epitopes to the immune system. In this study, we characterize the endocytic fate of native Env, expressed from infected human T-cell lines. We demonstrate that Env is rapidly trafficked to a late-endosome/lysosome-like compartment and can be recycled to the cell surface for incorporation into virus assembly sites. This study implicates a novel intracellular compartment, marked by host-cell Rab14 GTPases, for the sequestration of Env. Therapeutic approaches aimed at mobilizing this intracellular pool of Env could lead to stronger immune control of HIV-1 infection via antibody-dependent cell-mediated cytotoxicity.

A chimeric thermostable M2e and H3 stalk-based universal influenza A virus vaccine

We developed a new chimeric M2e and H3 hemagglutinin (HA) stalk protein vaccine (M2e-H3 stalk) by genetic engineering of modified H3 stalk domain conjugated with conserved M2e epitopes to overcome the drawbacks of low efficacy by monomeric domain-based universal vaccines. M2e-H3 stalk protein expressed and purified from Escherichia coli was thermostable, displaying native-like antigenic epitopes recognized by antisera of different HA subtype proteins and influenza A virus infections. Adjuvanted M2e-H3 stalk vaccination induced M2e and stalk-specific IgG antibodies recognizing viral antigens on virus particles and on the infected cell surface, CD4+ and CD8+ T-cell responses, and antibody-dependent cytotoxic cell surrogate activity in mice. M2e-H3 stalk was found to confer protection against heterologous and heterosubtypic cross-group subtype viruses (H1N1, H5N1, H9N2, H3N2, H7N9) at similar levels in adult and aged mice. These results provide evidence that M2e-H3 stalk chimeric proteins can be developed as a universal influenza A virus vaccine candidate for young and aged populations.

Factors associated with early biological aging in older people with HIV

ABSTRACT Advances and availability of antiretroviral treatment has enabled a longer life expectancy for HIV population. However, with its chronicity, premature aging challenges the management of people living with HIV. This study, conducted between 2018–2020, aimed to identify an association between risk factors and premature aging, using the biological age estimated by artificial intelligence (AI) based on deep learning (Aging 3.0). This was a cross-sectional, analytical study, involving older people living with HIV (OPLHIV), 66.1% of whom were men. Premature aging was identified in 67.8%. The presence of cannabis and diabetes were significant (p = 0.045 and p = 0.042, respectively). For current and nadir CD4 + cell counts, participants were divided into groups comparing biological age (BA) and chronological age (CA). Just one group presented no premature aging, whereas the group with premature aging was subdivided into BA > CA up to 4 years and BA > CA in 5 or more years. In conclusion, premature aging was present in most of the OPLHIV. The use of cannabis was self-reported in those with higher BAs and those with a lower BA presented a higher prevalence of diabetes. Factors directly linked to HIV infection, lower current and nadir CD4 + counts were associated with premature aging.

Stochastic HIV model coupled with pharmacokinetics and drug adherence may explain intermittent viral blips

We develop a stochastic HIV model by integrating drug adherence into a pharmacokinetic model and by coupling a pharmacodynamic model with a viral dynamic model. Numerical simulations show that the proposed model can generate viral blips, which have been observed in HIV patients receiving suppressive antiretroviral therapy. We calculate the probability density function of infected CD4+ T cells by developing the generalized density evolution equation. We fit the model to the clinical data of four HIV patients exhibiting viral blips. The results demonstrate that poor drug adherence can be a reason explaining the occurrence of viral blips in treated HIV patients. We also find that viral dynamics are sensitive to drug-adherence parameters, which have a significant impact on the frequency and amplitude of viral blips. The modeling and methods can be applied to the study of long-term therapy of other chronic diseases in which drug adherence might also be an issue.

Stability analysis of within-host SARS-CoV-2/HIV coinfection model.

The world has been suffering from the coronavirus disease 2019 (COVID‐19) since late 2019. COVID‐19 is caused by a virus called the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). The human immunodeficiency virus (HIV) coinfection with SARS‐CoV‐2 has been reported in many patients around the world. This has raised the alarm for the importance of understanding the dynamics of coinfection and its impact on the lives of patients. As in other pandemics, mathematical modeling is one of the important tools that can help medical and experimental studies of COVID‐19. In this paper, we develop a within‐host SARS‐CoV‐2/HIV coinfection model. The model consists of six ordinary differential equations. It depicts the interactions between uninfected epithelial cells, infected epithelial cells, free SARS‐CoV‐2 particles, uninfected CD4+ T cells, infected CD4+ T cells, and free HIV particles. We confirm that the solutions of the developed model are biologically acceptable by proving their nonnegativity and boundedness. We compute all possible steady states and derive their positivity conditions. We choose suitable Lyapunov functions to prove the global asymptotic stability of all steady states. We run some numerical simulations to enhance the global stability results. Based on our model, weak CD4+ T cell immune response or low CD4+ T cell counts in SARS‐CoV‐2/HIV coinfected patient increase the concentrations of infected epithelial cells and SARS‐CoV‐2 viral load. This causes the coinfected patient to suffer from severe SARS‐CoV‐2 infection. This result agrees with many studies which showed that HIV patients are at greater risk of suffering from severe COVID‐19 when infected. More studies are needed to understand the nature of SARS‐CoV‐2/HIV coinfection and the role of different immune responses during infection.

Antiretroviral therapy duration and immunometabolic state determine efficacy of ex vivo dendritic cell-based treatment restoring functional HIV-specific CD8+ T cells in people living with HIV

SummaryBackgroundDysfunction of CD8+ T cells in people living with HIV-1 (PLWH) receiving anti-retroviral therapy (ART) has restricted the efficacy of dendritic cell (DC)-based immunotherapies against HIV-1. Heterogeneous immune exhaustion and metabolic states of CD8+ T cells might differentially associate with dysfunction. However, specific parameters associated to functional restoration of CD8+ T cells after DC treatment have not been investigated.MethodsWe studied association of restoration of functional HIV-1-specific CD8+ T cell responses after stimulation with Gag-adjuvant-primed DC with ART duration, exhaustion, metabolic and memory cell subsets profiles.FindingsHIV-1-specific CD8+ T cell responses from a larger proportion of PLWH on long-term ART (more than 10 years; LT-ARTp) improved polyfunctionality and capacity to eliminate autologous p24+ infected CD4+ T cells in vitro. In contrast, functional improvement of CD8+ T cells from PLWH on short-term ART (less than a decade; ST-ARTp) after DC treatment was limited. This was associated with lower frequencies of central memory CD8+ T cells, increased co-expression of PD1 and TIGIT and reduced mitochondrial respiration and glycolysis induction upon TCR activation. In contrast, CD8+ T cells from LT-ARTp showed increased frequencies of TIM3+ PD1− cells and preserved induction of glycolysis. Treatment of dysfunctional CD8+ T cells from ST-ARTp with combined anti-PD1 and anti-TIGIT antibodies plus a glycolysis promoting drug restored their ability to eliminate infected CD4+ T cells.InterpretationTogether, our study identifies specific immunometabolic parameters for different PLWH subgroups potentially useful for future personalized DC-based HIV-1 vaccines.FundingNIH (R21AI140930), MINECO/FEDER RETOS (RTI2018-097485-A-I00) and CIBERINF grants.

Activation of α7 nicotinic acetylcholine receptor promotes HIV-1 transcription.

Alpha7 nicotinic acetylcholine receptor (α7 nAChR), a hub of the cholinergic anti-inflammatory pathway (CAP), is required for the treatment of inflammatory diseases. HIV-1 infection can upregulate the expression of α7 nAChR in T lymphocytes and affect the role of CAP. However, whether α7 nAChR regulates HIV-1 infection in CD4+ T cells is unclear. In this study, we first found that activation of α7 nAChR by GTS-21 (an α7 nAChR agonist) can promote the transcription of HIV-1 proviral DNA. Then, through transcriptome sequencing analysis, we found that p38 MAPK signaling was enriched in GTS-21 treated HIV-latent T cells. Mechanistically, activation of α7 nAChR could increase reactive oxygen species (ROS), reduce DUSP1 and DUSP6, and consequently enhance the phosphorylation of p38 MAPK. By co-immunoprecipitation and liquid chromatography tandem mass spectrometry, we found that p-p38 MAPK interacted with Lamin B1 (LMNB1). Activation of α7 nAChR increased the binding between p-p38 MAPK and LMNB1. We confirmed that knockdown of MAPK14 significantly downregulated NFATC4, a key activator of HIV-1 transcription. Taken together, activation of the α7 nAChR could trigger ROS/p-p38 MAPK/LMNB1/NFATC4 signaling pathway enhancing HIV-1 transcription. We have revealed an unrecognized mechanism of α7 nAChR-mediated neuroimmune regulation of HIV infection.

Development of an HIV reporter virus that identifies latently infected CD4+ T cells

SummaryThere is no cure for HIV infection, as the virus establishes a latent reservoir, which escapes highly active antiretroviral treatments. One major obstacle is the difficulty identifying cells that harbor latent proviruses. We devised a single-round viral vector that carries a series of versatile reporter molecules that are expressed in an LTR-dependent or LTR-independent manner and make it possible to accurately distinguish productive from latent infection. Using primary human CD4+ T cells, we show that transcriptionally silent proviruses are found in more than 50% of infected cells. The latently infected cells harbor proviruses but lack evidence for multiple spliced transcripts. LTR-silent integrations occurred to variable degrees in all CD4+ T subsets examined, with CD4+ TEM and CD4+ TREG displaying the highest frequency of latent infections. This viral vector permits the interrogation of HIV latency at single-cell resolution, revealing mechanisms of latency establishment and allowing the characterization of effective latency-reversing agents.

Dynamics of an HTLV-I infection model with delayed CTLs immune response

This paper deals with a four dimensional mathematical model for Human T-cell leukemia virus type-I (HTLV-I) infection that includes delayed CD8+ cytotoxic T-cells (CTLs) immune response. The proposed system has three biologically feasible steady states, namely disease-free steady state, CTL-inactive steady state and an interior steady state. Our theoretical analysis demonstrates that local and global stability analysis are established by the two critical parameters R0 and R1, basic reproduction numbers due to viral infection and due to CTLs immune response, respectively. The disease-free steady state E0 is globally stable if R0≤1, and the HTLV-I infections are eliminated. The asymptotic-carrier steady state E1 is globally stable if R1≤1<R0, which indicates HTLV-I infection is chronic without persistence of CTLs immune response. The interior steady state E2 is globally asymptotic stable if R1>1, which implies that the HTLV-I infection is choric in persistence of CTLs immune response. Due to immune response delay, our proposed model undergoes a destabilization of the interior steady state leading to Hopf bifurcation and periodic oscillations. We estimate the length of time delay that preserve the stability of period-1 limit cycle. We also derived the direction and stability of Hopf bifurcation around the interior steady state by center manifold theory and normal form method. To determine the robustness of the model, we performed normalized forward sensitivity analysis with reference to R0 and R1. Our proposed model undergoes Hopf bifurcation with respect to the production rate of uninfected CD4+T cells h, removal rate of virus-specific CTLs d4, spontaneous infected CD4+T cell activation d2 and transmissibility coefficient β. Implications of our numerical illustrations to the pathogenesis of HTLV-I infection and the development of HTLV-I related HAM/TSP are explored.

HIV-1 cell-to-cell spread overcomes the virus entry block of non-macrophage-tropic strains in macrophages

Macrophages (MΦ) are increasingly recognized as HIV-1 target cells involved in the pathogenesis and persistence of infection. Paradoxically, in vitro infection assays suggest that virus isolates are mostly T-cell-tropic and rarely MΦ-tropic. The latter are assumed to emerge under CD4+ T-cell paucity in tissues such as the brain or at late stage when the CD4 T-cell count declines. However, assays to qualify HIV-1 tropism use cell-free viral particles and may not fully reflect the conditions of in vivo MΦ infection through cell-to-cell viral transfer. Here, we investigated the capacity of viruses expressing primary envelope glycoproteins (Envs) with CCR5 and/or CXCR4 usage from different stages of infection, including transmitted/founder Envs, to infect MΦ by a cell-free mode and through cell-to-cell transfer from infected CD4+ T cells. The results show that most viruses were unable to enter MΦ as cell-free particles, in agreement with the current view that non-M-tropic viruses inefficiently use CD4 and/or CCR5 or CXCR4 entry receptors on MΦ. In contrast, all viruses could be effectively cell-to-cell transferred to MΦ from infected CD4+ T cells. We further showed that viral transfer proceeded through Env-dependent cell-cell fusion of infected T cells with MΦ targets, leading to the formation of productively infected multinucleated giant cells. Compared to cell-free infection, infected T-cell/MΦ contacts showed enhanced interactions of R5 M- and non-M-tropic Envs with CD4 and CCR5, resulting in a reduced dependence on receptor expression levels on MΦ for viral entry. Altogether, our results show that virus cell-to-cell transfer overcomes the entry block of isolates initially defined as non-macrophage-tropic, indicating that HIV-1 has a more prevalent tropism for MΦ than initially suggested. This sheds light into the role of this route of virus cell-to-cell transfer to MΦ in CD4+ T cell rich tissues for HIV-1 transmission, dissemination and formation of tissue viral reservoirs.

Rapamycin limits CD4+ T cell proliferation in simian immunodeficiency virus-infected rhesus macaques on antiretroviral therapy.

Proliferation of latently infected CD4+ T cells with replication-competent proviruses is an important mechanism contributing to HIV persistence during antiretroviral therapy (ART). One approach to targeting this latent cell expansion is to inhibit mTOR, a regulatory kinase involved with cell growth, metabolism, and proliferation. Here, we determined the effects of chronic mTOR inhibition with rapamycin with or without T cell activation in SIV-infected rhesus macaques (RMs) on ART. Rapamycin perturbed the expression of multiple genes and signaling pathways important for cellular proliferation and substantially decreased the frequency of proliferating CD4+ memory T cells (TM cells) in blood and tissues. However, levels of cell-associated SIV DNA and SIV RNA were not markedly different between rapamycin-treated RMs and controls during ART. T cell activation with an anti-CD3LALA antibody induced increases in SIV RNA in plasma of RMs on rapamycin, consistent with SIV production. However, upon ART cessation, both rapamycin and CD3LALA–treated and control-treated RMs rebounded in less than 12 days, with no difference in the time to viral rebound or post-ART viral load set points. These results indicate that, while rapamycin can decrease the proliferation of CD4+ TM cells, chronic mTOR inhibition alone or in combination with T cell activation was not sufficient to disrupt the stability of the SIV reservoir.

Single-Cell Profiling of Latently SIV-Infected CD4+ T Cells Directly Ex Vivo to Reveal Host Factors Supporting Reservoir Persistence.

ABSTRACTHIV-1 cure strategies aiming to eliminate persistent infected cell reservoirs are hampered by a poor understanding of cells harboring viral DNA in vivo. We describe a novel method to identify, enumerate, and characterize in detail individual cells infected in vivo using a combination of single-cell multiplexed assays for integrated proviral DNA, quantitative viral and host gene expression, and quantitative surface protein expression without any in vitro manipulation. Latently infected CD4+ T cells, defined as harboring integrated provirus in the absence of spliced viral mRNA, were identified from macaque lymph nodes during acute, chronic, and combination antiretroviral therapy (cART)-suppressed simian immunodeficiency virus (SIV) infection. Latently infected CD4+ T cells were most abundant during acute SIV (~8% of memory CD4+ T cells) and persisted in chronic and cART-suppressed infection. Productively infected cells actively transcribing viral mRNA, by contrast, were much more labile and declined substantially between acute and chronic or cART-suppressed infection. Expression of most surface proteins and host genes was similar between latently infected cells and uninfected cells. Elevated FLIP mRNA and surface CD3 expression among latently infected cells suggest increased survival potential and capacity to respond to T cell receptor stimulation. These findings point to a large pool of latently infected CD4+ T cells established very early in acute infection and upregulated host factors that may facilitate their persistence in vivo, both of which pose potential challenges to eliminating HIV-1 reservoirs.IMPORTANCE Effective combination antiretroviral therapy controls HIV-1 infection but fails to eliminate latent viral reservoirs that give rise to viremia upon treatment interruption. Strategies to eradicate latently infected cells require a better understanding of their biology and distinguishing features to promote their elimination. Tools for studying these cells from patients are currently limited. Here, we developed a single-cell method to identify cells latently infected in vivo and to characterize these cells for expression of surface proteins and host genes without in vitro manipulation, capturing their in vivo state from SIV-infected macaques. Host factors involved in cell survival and proliferation were upregulated in latently infected cells, which were abundant in the earliest stages of acute infection. These studies provide insight into the basic biology of latently infected cells as well as potential mechanisms underlying the persistence of HIV-1/SIV reservoirs to inform development of novel HIV-1 cure strategies.

Identification and characterization of a novel enhancer in the HTLV-1 proviral genome

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes adult T-cell leukemia/lymphoma (ATL), a cancer of infected CD4+ T-cells. There is both sense and antisense transcription from the integrated provirus. Sense transcription tends to be suppressed, but antisense transcription is constitutively active. Various efforts have been made to elucidate the regulatory mechanism of HTLV-1 provirus for several decades; however, it remains unknown how HTLV-1 antisense transcription is maintained. Here, using proviral DNA-capture sequencing, we found a previously unidentified viral enhancer in the middle of the HTLV-1 provirus. The transcription factors, SRF and ELK-1, play a pivotal role in the activity of this enhancer. Aberrant transcription of genes in the proximity of integration sites was observed in freshly isolated ATL cells. This finding resolves certain long-standing questions concerning HTLV-1 persistence and pathogenesis. We anticipate that the DNA-capture-seq approach can be applied to analyze the regulatory mechanisms of other oncogenic viruses integrated into the host cellular genome.

Crotonate sensitizes IAPi in the disruption of latent HIV by modulating the ncNF‐κB signaling pathway at the step of p100 cleavage into p52

BackgroundAntiretroviral therapy (ART) effectively suppresses HIV‐1 (HIV) replication to an undetectable level in the periphery; however, the treatment is life‐long and HIV reservoirs cannot be eliminated by ART alone in people living with HIV (PLWH). The major obstacle that impedes HIV eradication in ART‐treated PLWH is the latent viral reservoir harbors in the resting CD4+ T cells. Recently, the activation of non‐canonical (nc) NF‐κB pathway via IAP inhibitor (IAPi) or the SMAC mimetic (SMACm) compounds has raised as an attractive mechanism to disrupt HIV from latency. IAPi/SMACm targets IAPs to activate NF‐κB inducing kinase (NIK) followed by phosphorylation of IkappaB kinase (IKK), leading to the cleavage of p100 into p52. The latter forms a complex with RelB to stimulate transcription of its target genes after translocation into the nucleus. Previously, we discovered that crotonylation stimulates HIV latency reversal that elicited by PKC/NF‐κB agonist. We hypothesize that there may exist a functional interaction between protein crotonylation and ncNF‐κB signaling, which may enhance IAPi‐induced latency reversal to optiomally disrupt HIV latency for a cure of HIV.MethodsMultiple HIV latency models were used, including Jurkat cell derived HIV latency models, the U1 promonocytic model of latency, the primary CD4+ T cell model of HIV latency and the resting CD4+ T cells isolated from ART‐suppressed PLWH. HIV expression was determined by flow cytometry and/or real‐time PCR. Protein expression and cleavage was determined by Western blot.ResultsWe found that IAPi/SMACm can disrupt latent HIV. However, its ability to reactivate latent HIV varied among different HIV latency models, with a lowest efficacy in the primary CD4+ T cell model of latency. Interestingly, IAPi/SMACm‐induced disruption of latent HIV was greatly enhanced in the presence of crotonate in all these in vitro and ex vivo models. This was possibly mediated by the enhancement of p100 cleavage into p52 in the ncNF‐κB signaling pathway.ConclusionsWe propose that a blockage of p100 cleavage prevents an efficient activation of ncNF‐κB signaling pathway in the latently infected CD4+ T cells in PLWH, which could be relieved by targeting IAP proteins to induce p100 cleavage for the activation of ncNF‐κB signaling. Crotonylation, which was recently defined by us as a new epigenetic mark for the active HIV transcription, potentiates the reactivation of HIV from latency by further releasing the blockage at the p100 cleavage step, thereby enhancing ncNF‐κB activation to disrupt HIV latency. These data indicate that crotonylation could be exploited to develop novel strategies for HIV cure or remission in the future.