Ongoing production of tissue-resident macrophages from hematopoietic stem cells in healthy adult macaques.

ABSTRACT: Macrophages orchestrate tissue immunity from the initiation and resolution of antimicrobial immune responses to the repair of damaged tissue. Murine studies demonstrate that tissue-resident macrophages are a heterogenous mixture of yolk sac-derived cells that populate the tissue before birth, and bone marrow-derived replacements recruited in adult tissues at steady-state and in increased numbers in response to tissue damage or infection. How this translates to species that are constantly under immunologic challenge, such as humans, is unknown. To understand the ontogeny and longevity of tissue-resident macrophages in nonhuman primates (NHPs), we use a model of autologous hematopoietic stem progenitor cell (HSPC) transplantation with HSPCs genetically modified to be marked with clonal barcodes, allowing for subsequent analysis of clonal ontogeny. We study the contribution of HSPCs to tissue macrophages, their clonotypic profiles relative to leukocyte subsets in the peripheral blood, and their transcriptomic and epigenetic landscapes. We find that HSPCs contribute to tissue-resident macrophage populations in all anatomic sites studied. Macrophage clonotypic profiles are dynamic and overlap significantly with the clonal hierarchy of contemporaneous peripheral blood monocytes. Epigenetic and transcriptomic landscapes of HSPC-derived macrophages are similar to tissue macrophages isolated from NHPs that did not undergo transplantation. We also use in vivo bromodeoxyuridine infusions to monitor tissue macrophage turnover in NHPs that did not undergo transplantation and find evidence for macrophage turnover at steady state. These data demonstrate that the life span of most tissue-resident macrophages is limited and can be replenished continuously from HSPCs.

Distinct transcriptomic and epigenomic modalities underpin human memory T cell subsets and their activation potential.

Human memory T cells (MTC) are poised to rapidly respond to antigen re-exposure. Here, we derived the transcriptional and epigenetic programs of resting and ex vivo activated, circulating CD4+ and CD8+ MTC subsets. A progressive gradient of gene expression from naïve to TCM to TEM is observed, which is accompanied by corresponding changes in chromatin accessibility. Transcriptional changes suggest adaptations of metabolism that are reflected in altered metabolic capacity. Other differences involve regulatory modalities comprised of discrete accessible chromatin patterns, transcription factor binding motif enrichment, and evidence of epigenetic priming. Basic-helix-loop-helix factor motifs for AHR and HIF1A distinguish subsets and predict transcription networks to sense environmental changes. Following stimulation, primed accessible chromatin correlate with an augmentation of MTC gene expression as well as effector transcription factor gene expression. These results identify coordinated epigenetic remodeling, metabolic, and transcriptional changes that enable MTC subsets to ultimately respond to antigen re-encounters more efficiently.

Prolonged experimental CD4+ T-cell depletion does not cause disease progression in SIV-infected African green monkeys.

CD4+ T-cell depletion is a hallmark of HIV infection, leading to impairment of cellular immunity and opportunistic infections, but its contribution to SIV/HIV-associated gut dysfunction is unknown. Chronically SIV-infected African Green Monkeys (AGMs) partially recover mucosal CD4+ T-cells, maintain gut integrity and do not progress to AIDS. Here we assess the impact of prolonged, antibody-mediated CD4 + T-cell depletion on gut integrity and natural history of SIV infection in AGMs. All circulating CD4+ T-cells and >90% of mucosal CD4+ T-cells are depleted. Plasma viral loads and cell-associated viral RNA in tissues are lower in CD4+-cell-depleted animals. CD4+-cell-depleted AGMs maintain gut integrity, control immune activation and do not progress to AIDS. We thus conclude that CD4+ T-cell depletion is not a determinant of SIV-related gut dysfunction, when gastrointestinal tract epithelial damage and inflammation are absent, suggesting that disease progression and resistance to AIDS are independent of CD4+ T-cell restoration in SIVagm-infected AGMs.

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 α4ß7 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.

Epigenetic Reprogramming Leads to Downregulation of CD4 and Functional Changes in African Green Monkey Memory CD4+ T Cells.

African green monkeys (AGMs), Chlorocebus pygerythrus, are a natural host for a lentivirus related to HIV, SIV. SIV-infected AGMs rarely progress to AIDS despite robust viral replication. Though multiple mechanisms are involved, a primary component is the animals' ability to downregulate CD4 expression on mature CD4+ Th cells, rendering these cells resistant to infection by SIV. These CD8αα+ T cells retain functional characteristics of CD4+ Th cells while simultaneously acquiring abilities of cytotoxic CD8αß+ T cells. To determine mechanisms underlying functional differences between T cell subsets in AGMs, chromatin accessibility in purified populations was determined by assay for transposase-accessible chromatin sequencing. Differences in chromatin accessibility alone were sufficient to cluster cells by subtype, and accessibility at the CD4 locus reflected changes in CD4 expression. DNA methylation at the CD4 locus also correlated with inaccessible chromatin. By associating accessible regions with nearby genes, gene expression was found to correlate with accessibility changes. T cell and immune system activation pathways were identified when comparing regions that changed accessibility from CD4+ T cells to CD8αα+ T cells. Different transcription factor binding sites are revealed as chromatin accessibility changes, and these differences may elicit downstream changes in differentiation. This comprehensive description of the epigenetic landscape of AGM T cells identified genes and pathways that could have translational value in therapeutic approaches recapitulating the protective effects CD4 downregulation.

Butyrate administration is not sufficient to improve immune reconstitution in antiretroviral-treated SIV-infected macaques.

Defective gastrointestinal barrier function and, in turn, microbial translocation have been identified as significant contributors to persistent inflammation in antiretroviral (ARV)-treated people living with HIV. Metabolic supplementation of short-chain fatty acids (SCFAs), generally produced by the commensal microbiome, may improve these outcomes. Butyrate is a SCFA that is essential for the development and maintenance of intestinal immunity and has a known role in supporting epithelial integrity. Herein we assessed whether supplementation with the dietary supplement sodium butyrate would improve immune reconstitution and reduce inflammation in ARV-treated, simian immunodeficiency virus (SIV)-infected rhesus macaques. We demonstrate that butyrate supplementation does not significantly improve immune reconstitution, with no differences observed in systemic CD4+ T-cell frequencies, T-cell functionality or immune activation, microbial translocation, or transcriptional regulation. Our findings demonstrate that oral administration of sodium butyrate is insufficient to reduce persistent inflammation and microbial translocation in ARV-treated, SIV-infected macaques, suggesting that this therapeutic may not reduce co-morbidities and co-mortalities in treated people living with HIV.

A super enhancer controls expression and chromatin architecture within the MHC class II locus.

Super enhancers (SEs) play critical roles in cell type-specific gene regulation. The mechanisms by which such elements work are largely unknown. Two SEs termed DR/DQ-SE and XL9-SE are situated within the human MHC class II locus between the HLA-DRB1 and HLA-DQA1 genes and are highly enriched for disease-causing SNPs. To test the function of these elements, we used CRISPR/Cas9 to generate a series of mutants that deleted the SE. Deletion of DR/DQ-SE resulted in reduced expression of HLA-DRB1 and HLA-DQA1 genes. The SEs were found to interact with each other and the promoters of HLA-DRB1 and HLA-DQA1. DR/DQ-SE also interacted with neighboring CTCF binding sites. Importantly, deletion of DR/DQ-SE reduced the local chromatin interactions, implying that it functions as the organizer for the local three-dimensional architecture. These data provide direct mechanisms by which an MHC-II SE contributes to expression of the locus and suggest how variation in these SEs may contribute to human disease and altered immunity.

Dynamic Modulation of Expression of Lentiviral Restriction Factors in Primary CD4+ T Cells following Simian Immunodeficiency Virus Infection.

Although multiple restriction factors have been shown to inhibit HIV/SIV replication, little is known about their expression in vivo Expression of 45 confirmed and putative HIV/SIV restriction factors was analyzed in CD4+ T cells from peripheral blood and the jejunum in rhesus macaques, revealing distinct expression patterns in naive and memory subsets. In both peripheral blood and the jejunum, memory CD4+ T cells expressed higher levels of multiple restriction factors compared to naive cells. However, relative to their expression in peripheral blood CD4+ T cells, jejunal CCR5+ CD4+ T cells exhibited significantly lower expression of multiple restriction factors, including APOBEC3G, MX2, and TRIM25, which may contribute to the exquisite susceptibility of these cells to SIV infection. In vitro stimulation with anti-CD3/CD28 antibodies or type I interferon resulted in upregulation of distinct subsets of multiple restriction factors. After infection of rhesus macaques with SIVmac239, the expression of most confirmed and putative restriction factors substantially increased in all CD4+ T cell memory subsets at the peak of acute infection. Jejunal CCR5+ CD4+ T cells exhibited the highest levels of SIV RNA, corresponding to the lower restriction factor expression in this subset relative to peripheral blood prior to infection. These results illustrate the dynamic modulation of confirmed and putative restriction factor expression by memory differentiation, stimulation, tissue microenvironment and SIV infection and suggest that differential expression of restriction factors may play a key role in modulating the susceptibility of different populations of CD4+ T cells to lentiviral infection.IMPORTANCE Restriction factors are genes that have evolved to provide intrinsic defense against viruses. HIV and simian immunodeficiency virus (SIV) target CD4+ T cells. The baseline level of expression in vivo and degree to which expression of restriction factors is modulated by conditions such as CD4+ T cell differentiation, stimulation, tissue location, or SIV infection are currently poorly understood. We measured the expression of 45 confirmed and putative restriction factors in primary CD4+ T cells from rhesus macaques under various conditions, finding dynamic changes in each state. Most dramatically, in acute SIV infection, the expression of almost all target genes analyzed increased. These are the first measurements of many of these confirmed and putative restriction factors in primary cells or during the early events after SIV infection and suggest that the level of expression of restriction factors may contribute to the differential susceptibility of CD4+ T cells to SIV infection.

Tetherin upregulation in simian immunodeficiency virus-infected macaques.

Here we show that simian immunodeficiency virus (SIV) infection of rhesus macaques results in rapid upregulation of tetherin (BST-2 or CD317) on peripheral blood lymphocytes, including the CD4(+) CCR5(+) T cell targets of virus infection, with a peak of induction that coincides with peak alpha interferon (IFN-α) levels in plasma, and that tetherin remains above baseline levels throughout chronic infection. These observations are consistent with a role for tetherin in innate immunity to immunodeficiency virus infection.

Latent Membrane Protein 1 as a molecular adjuvant for single-cycle lentiviral vaccines.

BACKGROUND: Molecular adjuvants are a promising method to enhance virus-specific immune responses and protect against HIV-1 infection. Immune activation by ligands for receptors such as CD40 can induce dendritic cell activation and maturation. Here we explore the incorporation of two CD40 mimics, Epstein Barr Virus gene LMP1 or an LMP1-CD40 chimera, into a strain of SIV that was engineered to be limited to a single cycle of infection. RESULTS: Full length LMP1 or the chimeric protein LMP1-CD40 was cloned into the nef-locus of single-cycle SIV. Human and Macaque monocyte derived macrophages and DC were infected with these viruses. Infected cells were analyzed for activation surface markers by flow cytometry. Cells were also analyzed for secretion of pro-inflammatory cytokines IL-1ß, IL-6, IL-8, IL-12p70 and TNF by cytometric bead array. CONCLUSIONS: Overall, single-cycle SIV expressing LMP1 and LMP1-CD40 produced a broad and potent T(H)1-biased immune response in human as well as rhesus macaque macrophages and DC when compared with control virus. Single-cycle SIV-LMP1 also enhanced antigen presentation by lentiviral vector vaccines, suggesting that LMP1-mediated immune activation may enhance lentiviral vector vaccines against HIV-1.

Envelope-modified single-cycle simian immunodeficiency virus selectively enhances antibody responses and partially protects against repeated, low-dose vaginal challenge.

Immunization of rhesus macaques with strains of simian immunodeficiency virus (SIV) that are limited to a single cycle of infection elicits T-cell responses to multiple viral gene products and antibodies capable of neutralizing lab-adapted SIV, but not neutralization-resistant primary isolates of SIV. In an effort to improve upon the antibody responses, we immunized rhesus macaques with three strains of single-cycle SIV (scSIV) that express envelope glycoproteins modified to lack structural features thought to interfere with the development of neutralizing antibodies. These envelope-modified strains of scSIV lacked either five potential N-linked glycosylation sites in gp120, three potential N-linked glycosylation sites in gp41, or 100 amino acids in the V1V2 region of gp120. Three doses consisting of a mixture of the three envelope-modified strains of scSIV were administered on weeks 0, 6, and 12, followed by two booster inoculations with vesicular stomatitis virus (VSV) G trans-complemented scSIV on weeks 18 and 24. Although this immunization regimen did not elicit antibodies capable of detectably neutralizing SIV(mac)239 or SIV(mac)251(UCD), neutralizing antibody titers to the envelope-modified strains were selectively enhanced. Virus-specific antibodies and T cells were observed in the vaginal mucosa. After 20 weeks of repeated, low-dose vaginal challenge with SIV(mac)251(UCD), six of eight immunized animals versus six of six naïve controls became infected. Although immunization did not significantly reduce the likelihood of acquiring immunodeficiency virus infection, statistically significant reductions in peak and set point viral loads were observed in the immunized animals relative to the naïve control animals.