HIV-1-infected T cell clones are shared across cerebrospinal fluid and blood during ART.

The central nervous system HIV reservoir is incompletely understood and is a major barrier to HIV cure. We profiled people with HIV (PWH) and uninfected controls through single-cell transcriptomic and T cell receptor (TCR) sequencing to understand the dynamics of HIV persistence in the CNS. In PWH on ART, we found that most participants had single cells containing HIV-1 RNA, which was found predominantly in CD4 central memory T cells, in both cerebrospinal fluid (CSF) and blood. HIV-1 RNA-containing cells were found more frequently in CSF than blood, indicating a higher burden of reservoir cells in the CNS than blood for some PWH. Most CD4 T cell clones containing infected cells were compartment specific, while some (22%) - including rare clones with members of the clone containing detectable HIV RNA in both blood and CSF - were found in both CSF and blood. These results suggest that infected T cells trafficked between tissue compartments and that maintenance and expansion of infected T cell clones contributed to the CNS reservoir in PWH on ART.

ScISOr-ATAC reveals convergent and divergent splicing and chromatin specificities between matched cell types across cortical regions, evolution, and in Alzheimer's Disease.

Multimodal measurements have become widespread in genomics, however measuring open chromatin accessibility and splicing simultaneously in frozen brain tissues remains unconquered. Hence, we devised Single-Cell-ISOform-RNA sequencing coupled with the Assay-for-Transposase-Accessible-Chromatin (ScISOr-ATAC). We utilized ScISOr-ATAC to assess whether chromatin and splicing alterations in the brain convergently affect the same cell types or divergently different ones. We applied ScISOr-ATAC to three major conditions: comparing (i) the Rhesus macaque (Macaca mulatta) prefrontal cortex (PFC) and visual cortex (VIS), (ii) cross species divergence of Rhesus macaque versus human PFC, as well as (iii) dysregulation in Alzheimer's disease in human PFC. We found that among cortical-layer biased excitatory neuron subtypes, splicing is highly brain-region specific for L3-5/L6 IT_RORB neurons, moderately specific in L2-3 IT_CUX2.RORB neurons and unspecific in L2-3 IT_CUX2 neurons. In contrast, at the chromatin level, L2-3 IT_CUX2.RORB neurons show the highest brain-region specificity compared to other subtypes. Likewise, when comparing human and macaque PFC, strong evolutionary divergence on one molecular modality does not necessarily imply strong such divergence on another molecular level in the same cell type. Finally, in Alzheimer's disease, oligodendrocytes show convergently high dysregulation in both chromatin and splicing. However, chromatin and splicing dysregulation most strongly affect distinct oligodendrocyte subtypes. Overall, these results indicate that chromatin and splicing can show convergent or divergent results depending on the performed comparison, justifying the need for their concurrent measurement to investigate complex systems. Taken together, ScISOr-ATAC allows for the characterization of single-cell splicing and chromatin patterns and the comparison of sample groups in frozen brain samples.

Retroelement-Age Clocks: Epigenetic Age Captured by Human Endogenous Retrovirus and LINE-1 DNA methylation states.

Human endogenous retroviruses (HERVs), the remnants of ancient viral infections embedded within the human genome, and long interspersed nuclear elements 1 (LINE-1), a class of autonomous retrotransposons, are silenced by host epigenetic mechanisms including DNA methylation. The resurrection of particular retroelements has been linked to biological aging. Whether the DNA methylation states of locus specific HERVs and LINEs can be used as a biomarker of chronological age in humans remains unclear. We show that highly predictive epigenetic clocks of chronological age can be constructed from retroelement DNA methylation states in the immune system, across human tissues, and pan-mammalian species. We found retroelement epigenetic clocks were reversed during transient epigenetic reprogramming, accelerated in people living with HIV-1, responsive to antiretroviral therapy, and accurate in estimating long-term culture ages of human brain organoids. Our findings support the hypothesis of epigenetic dysregulation of retroelements as a potential contributor to the biological hallmarks of aging.

The latency-reversing agent HODHBt synergizes with IL-15 to enhance cytotoxic function of HIV-specific T cells.

IL-15 is under clinical investigation toward the goal of curing HIV infection because of its abilities to reverse HIV latency and enhance immune effector function. However, increased potency through combination with other agents may be needed. 3-Hydroxy-1,2,3-benzotriazin-4(3H)-one (HODHBt) enhances IL-15-mediated latency reversal and NK cell function by increasing STAT5 activation. We hypothesized that HODHBt would also synergize with IL-15, via STAT5, to directly enhance HIV-specific cytotoxic T cell responses. We showed that ex vivo IL-15 + HODHBt treatment markedly enhanced HIV-specific granzyme B-releasing T cell responses in PBMCs from antiretroviral therapy-suppressed (ART-suppressed) donors. We also observed upregulation of antigen processing and presentation in CD4+ T cells and increased surface MHC-I. In ex vivo PBMCs, IL-15 + HODHBt was sufficient to reduce intact proviruses in 1 of 3 ART-suppressed donors. Our findings reveal the potential for second-generation IL-15 studies incorporating HODHBt-like therapeutics. Iterative studies layering on additional latency reversal or other agents are needed to achieve consistent ex vivo reservoir reductions.

Distinct mucosal and systemic immunological characteristics in transgender women potentially relating to HIV acquisition.

Transgender women (TGW) are disproportionally affected by HIV infection, with a global estimated prevalence of 19.9%, often attributed to behavioral risk factors, with less known about biological factors. We evaluated potential biological risk factors for HIV acquisition in TGW at the sites of viral entry by assessing immune parameters of the neovaginal surface and gut mucosa. The neovagina in TGW, compared with the vagina in cisgender women (CW), shows distinct cell composition and may pose a more inflammatory environment, evidenced by increased CD4+ T cell activation and higher levels of soluble markers of inflammation (C-reactive protein, soluble CD30). Increased inflammation may be driven by microbiome composition, as shown by a greater abundance of Prevotella and a higher Shannon Diversity Index. In addition, we have observed higher frequency of CD4+CCR5+ target cells and decreased DNA methylation of the CCR5 gene in the gut mucosa of TGW compared with CW and men who have sex with men, which was inversely correlated with testosterone levels. The rectal microbiome composition in TGW appears to favor a proinflammatory milieu as well as mucosal barrier disruption. Thus, it is possible that increased inflammation and higher frequencies of CCR5-expressing target cells at sites of mucosal viral entry may contribute to increased risk of HIV acquisition in TGW, with further validation in larger studies warranted.

Disruption of the CCL5/RANTES-CCR5 Pathway Restores Immune Homeostasis and Reduces Plasma Viral Load in Critical COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is now pandemic with nearly three million cases reported to date. Although the majority of COVID-19 patients experience only mild or moderate symptoms, a subset will progress to severe disease with pneumonia and acute respiratory distress syndrome (ARDS) requiring mechanical ventilation. Emerging results indicate a dysregulated immune response characterized by runaway inflammation, including cytokine release syndrome (CRS), as the major driver of pathology in severe COVID-19. With no treatments currently approved for COVID-19, therapeutics to prevent or treat the excessive inflammation in severe disease caused by SARS-CoV-2 infection are urgently needed. Here, in 10 terminally-ill, critical COVID-19 patients we report profound elevation of plasma IL-6 and CCL5 (RANTES), decreased CD8+ T cell levels, and SARS-CoV-2 plasma viremia. Following compassionate care treatment with the CCR5 blocking antibody leronlimab, we observed complete CCR5 receptor occupancy on macrophage and T cells, rapid reduction of plasma IL-6, restoration of the CD4/CD8 ratio, and a significant decrease in SARS-CoV-2 plasma viremia. Consistent with reduction of plasma IL-6, single-cell RNA-sequencing revealed declines in transcriptomic myeloid cell clusters expressing IL-6 and interferon-related genes. These results demonstrate a novel approach to resolving unchecked inflammation, restoring immunologic deficiencies, and reducing SARS-CoV-2 plasma viral load via disruption of the CCL5-CCR5 axis, and support randomized clinical trials to assess clinical efficacy of leronlimab-mediated inhibition of CCR5 for COVID-19.