The effect of age on the magnitude and longevity of Th1-directed CD4 T-cell responses to SARS-CoV-2.

Age is associated with changes in the immune system which increase the risk for severe COVID-19. Here, we investigate SARS-CoV-2-reactive CD4 T cells from individuals recovered from SARS-CoV-2 infection with mild COVID-19 symptoms after 3, 6 and 9 months using incubation with SARS-CoV-2 S1, S2 and N-peptide pools, followed by flow cytometry for a Th1-activation profile or proliferation analyses. We found that SARS-CoV-2-reactive CD4 T cells are decreasing on average after 9 months but highly polyfunctional CD4 T cells can peak after 6-month recovery. We show that individuals older than 60 years of age have significantly more SARS-CoV-2-reactive T cells in their blood after 3 months of recovery compared to younger individuals and that the percentage of SARS-CoV-2-reactive Th1-directed CD4 T cells in the blood of mild-COVID-19-recovered individuals correlates with age. Finally, we show that individuals over the age of 40 have significantly increased the amounts of highly polyfunctional SARS-CoV-2-S-peptide-reactive CD4 T cells, compared to SARS-CoV-2 naïve individuals, than those under the age of 40. These findings suggest that in individuals recovered from mild COVID-19, increased age is associated with significantly more highly polyfunctional SARS-CoV-2-reactive CD4 T cells with a Th1-profile and that these responses persist over time.

Cytomegalovirus inhibition of extrinsic apoptosis determines fitness and resistance to cytotoxic CD8 T cells.

Viral immune evasion is currently understood to focus on deflecting CD8 T cell recognition of infected cells by disrupting antigen presentation pathways. We evaluated viral interference with the ultimate step in cytotoxic T cell function, the death of infected cells. The viral inhibitor of caspase-8 activation (vICA) conserved in human cytomegalovirus (HCMV) and murine CMV (MCMV) prevents the activation of caspase-8 and proapoptotic signaling. We demonstrate the key role of vICA from either virus, in deflecting antigen-specific CD8 T cell-killing of infected cells. vICA-deficient mutants, lacking either UL36 or M36, exhibit greater susceptibility to CD8 T cell control than mutants lacking the set of immunoevasins known to disrupt antigen presentation via MHC class I. This difference is evident during infection in the natural mouse host infected with MCMV, in settings where virus-specific CD8 T cells are adoptively transferred. Finally, we identify the molecular mechanism through which vICA acts, demonstrating the central contribution of caspase-8 signaling at a point of convergence of death receptor-induced apoptosis and perforin/granzyme-dependent cytotoxicity.

A Novel Triple-Fluorescent HCMV Strain Reveals Gene Expression Dynamics and Anti-Herpesviral Drug Mechanisms.

Human Cytomegalovirus (HCMV) infection may result in severe outcomes in immunocompromised individuals such as AIDS patients, transplant recipients, and neonates. To date, no vaccines are available and there are only few drugs for anti-HCMV therapy. Adverse effects and the continuous emergence of drug-resistance strains require the identification of new drug candidates in the near future. Identification and characterization of such compounds and biological factors requires sensitive and reliable detection techniques of HCMV infection, gene expression and spread. In this work, we present and validate a novel concept for multi-reporter herpesviruses, identified through iterative testing of minimally invasive mutations. We integrated up to three fluorescence reporter genes into replication-competent HCMV strains, generating reporter HCMVs that allow the visualization of replication cycle stages of HCMV, namely the immediate early (IE), early (E), and late (L) phase. Fluorescent proteins with clearly distinguishable emission spectra were linked by 2A peptides to essential viral genes, allowing bicistronic expression of the viral and the fluorescent protein without major effects on viral fitness. By using this triple color reporter HCMV, we monitored gene expression dynamics of the IE, E, and L genes by measuring the fluorescent signal of the viral gene-associated fluorophores within infected cell populations and at high temporal resolution. We demonstrate distinct inhibitory profiles of foscarnet, fomivirsen, phosphonoacetic acid, ganciclovir, and letermovir reflecting their mode-of-action. In conclusion, our data argues that this experimental approach allows the identification and characterization of new drug candidates in a single step.

Chromatin-Remodeling Factor SPOC1 Acts as a Cellular Restriction Factor against Human Cytomegalovirus by Repressing the Major Immediate Early Promoter.

The cellular protein SPOC1 (survival time-associated PHD [plant homeodomain] finger protein in ovarian cancer 1) acts as a regulator of chromatin structure and the DNA damage response. It binds H3K4me2/3-containing chromatin and promotes DNA condensation by recruiting corepressors such as KAP-1 and H3K9 methyltransferases. Previous studies identified SPOC1 as a restriction factor against human adenovirus (HAdV) infection that is antagonized by E1B-55K/E4-orf6-dependent proteasomal degradation. Here, we demonstrate that, in contrast to HAdV-infected cells, SPOC1 is transiently upregulated during the early phase of human cytomegalovirus (HCMV) replication. We show that the expression of immediate early protein 1 (IE1) is sufficient and necessary to induce SPOC1. Additionally, we discovered that during later stages of infection, SPOC1 is downregulated in a glycogen synthase kinase 3ß (GSK-3ß)-dependent manner. We provide evidence that SPOC1 overexpression severely impairs HCMV replication by repressing the initiation of viral immediate early (IE) gene expression. Consistently, we observed that SPOC1-depleted primary human fibroblasts displayed an augmented initiation of viral IE gene expression. This occurs in a multiplicity of infection (MOI)-dependent manner, a defining hallmark of intrinsic immunity. Interestingly, repression requires the presence of high SPOC1 levels at the start of infection, while later upregulation had no negative impact, suggesting distinct temporal roles of SPOC1 during the HCMV replicative cycle. Mechanistically, we observed a highly specific association of SPOC1 with the major immediate early promoter (MIEP), strongly suggesting that SPOC1 inhibits HCMV replication by MIEP binding and the subsequent recruitment of heterochromatin-building factors. Thus, our data add SPOC1 as a novel factor to the endowment of a host cell to restrict cytomegalovirus infections.IMPORTANCE Accumulating evidence indicates that during millennia of coevolution, host cells have developed a sophisticated compilation of cellular factors to restrict cytomegalovirus infections. Defining this equipment is important to understand cellular barriers against viral infection and to develop strategies to utilize these factors for antiviral approaches. So far, constituents of PML nuclear bodies and interferon gamma-inducible protein 16 (IFI16) were known to mediate intrinsic immunity against HCMV. In this study, we identify the chromatin modulator SPOC1 as a novel restriction factor against HCMV. We show that preexisting high SPOC1 protein levels mediate a silencing of HCMV gene expression via a specific association with an important viral cis-regulatory element, the major immediate early promoter. Since SPOC1 expression varies between cell types, this factor may play an important role in tissue-specific defense against HCMV.

Myeloid Dendritic Cells Repress Human Cytomegalovirus Gene Expression and Spread by Releasing Interferon-Unrelated Soluble Antiviral Factors.

Cytomegalovirus (CMV) is a betaherpesvirus that latently infects most adult humans worldwide and is a major cause of morbidity and mortality in immunocompromised hosts. Latent human CMV (HCMV) is believed to reside in precursors of myeloid-lineage leukocytes and monocytes, which give rise to macrophages and dendritic cells (DC). We report here that human monocyte-derived DC (mo-DC) suppress HCMV infection in coculture with infected fibroblast target cells in a manner dependent on the effector-to-target ratio. Intriguingly, optimal activation of mo-DC was achieved under coculture conditions and not by direct infection with HCMV, implying that mo-DC may recognize unique molecular patterns on, or within, infected fibroblasts. We show that HCMV is controlled by secreted factors that act by priming defenses in target cells rather than by direct viral neutralization, but we excluded a role for interferons (IFNs) in this control. The expression of lytic viral genes in infected cells and the progression of infection were significantly slowed, but this effect was reversible, indicating that the control of infection depended on the transient induction of antiviral effector molecules in target cells. Using immediate early or late-phase reporter HCMVs, we show that soluble factors secreted in the cocultures suppress HCMV replication at both stages of the infection and that their antiviral effects are robust and comparable in numerous batches of mo-DC as well as in primary fibroblasts and stromal cells.IMPORTANCE Human cytomegalovirus is a widespread opportunistic pathogen that can cause severe disease and complications in vulnerable individuals. This includes newborn children, HIV AIDS patients, and transplant recipients. Although the majority of healthy humans carry this virus throughout their lives without symptoms, it is not exactly clear which tissues in the body are the main reservoirs of latent virus infection or how the delicate balance between the virus and the immune system is maintained over an individual's lifetime. Here, for the first time, we provide evidence for a novel mechanism of direct virus control by a subset of human innate immune cells called dendritic cells, which are regarded as a major site of virus latency and reactivation. Our findings may have important implications in HCMV disease prevention as well as in development of novel therapeutic approaches.

UL36 Rescues Apoptosis Inhibition and In vivo Replication of a Chimeric MCMV Lacking the M36 Gene.

Apoptosis is an important defense mechanism mounted by the immune system to control virus replication. Hence, cytomegaloviruses (CMV) evolved and acquired numerous anti-apoptotic genes. The product of the human CMV (HCMV) UL36 gene, pUL36 (also known as vICA), binds to pro-caspase-8, thus inhibiting death-receptor apoptosis and enabling viral replication in differentiated THP-1 cells. In vivo studies of the function of HCMV genes are severely limited due to the strict host specificity of cytomegaloviruses, but CMV orthologues that co-evolved with other species allow the experimental study of CMV biology in vivo. The mouse CMV (MCMV) homolog of the UL36 gene is called M36, and its protein product (pM36) is a functional homolog of vICA that binds to murine caspase-8 and inhibits its activation. M36-deficient MCMV is severely growth impaired in macrophages and in vivo. Here we show that pUL36 binds to the murine pro-caspase-8, and that UL36 expression inhibits death-receptor apoptosis in murine cells and can replace M36 to allow MCMV growth in vitro and in vivo. We generated a chimeric MCMV expressing the UL36 ORF sequence instead of the M36 one. The newly generated MCMVUL36 inhibited apoptosis in macrophage lines RAW 264.7, J774A.1, and IC-21 and its growth was rescued to wild type levels. Similarly, growth was rescued in vivo in the liver and spleen, but only partially in the salivary glands of BALB/c and C57BL/6 mice. In conclusion, we determined that an immune-evasive HCMV gene is conserved enough to functionally replace its MCMV counterpart and thus allow its study in an in vivo setting. As UL36 and M36 proteins engage the same molecular host target, our newly developed model can facilitate studies of anti-viral compounds targeting pUL36 in vivo.

Cyclic dinucleotides modulate induced type I IFN responses in innate immune cells by degradation of STING.

The cyclic dinucleotides, GMP-AMP (cGAMP) and c-di-AMP [bis-(3',5')-cyclic dimeric AMP], are potent type I IFN inducers via STING-TBK1-IRF3 cascade. They are promising adjuvants that promote antigen-specific humoral and cellular immune responses in different preclinical models; however, an optimal outcome of vaccination depends on a balanced immune activation. Here, we characterize the process of IFN-ß induction by c-di-AMP and cGAMP in an in vitro model on the basis of primary mouse dendritic cells. Results obtained show decreased IFN-ß production upon prolonged cell stimulation. We demonstrate that this effect depends on c-di-AMP/cGAMP-mediated down-regulation of stimulator of IFN gene (STING) protein levels. These results were confirmed by using human peripheral blood mononuclear cell-derived dendritic cells. Studies performed to explore the potential mechanism of STING modulation suggested proteolytic degradation to be a contributing factor to the observed decrease in cellular STING levels. Our work contributes to the elucidation of the molecular mode of action of vaccine constituents, which, in turn, is a prerequisite for the rational design of vaccines with predictable efficacy and safety profiles-Rueckert, C., Rand, U., Roy, U., Kasmapour, B., Strowig, T., Guzmán, C. A. Cyclic dinucleotides modulate induced type I IFN responses in innate immune cells by degradation of STING.

Experimental selection of long-term intracellular mycobacteria.

Some intracellular bacteria are known to cause long-term infections that last decades without compromising the viability of the host. Although of critical importance, the adaptations that intracellular bacteria undergo during this long process of residence in a host cell environment remain obscure. Here, we report a novel experimental approach to study the adaptations of mycobacteria imposed by a long-term intracellular lifestyle. Selected Mycobacterium bovis BCG through continuous culture in macrophages underwent an adaptation process leading to impaired phenolic glycolipids (PGL) synthesis, improved usage of glucose as a carbon source and accumulation of neutral lipids. These changes correlated with increased survival of mycobacteria in macrophages and mice during re-infection and also with the specific expression of stress- and survival-related genes. Our findings identify bacterial traits implicated in the establishment of long-term cellular infections and represent a tool for understanding the physiological states and the environment that bacteria face living in fluctuating intracellular environments.

Study of phagolysosome biogenesis in live macrophages.

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosomal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

Spatial distribution of phagolysosomes is independent of the regulation of lysosome position by Rab34.

Within a cell, the regulation of organelle positioning is considered to be critical in spatio-temporal responses. The position of late endocytic organelles (named here lysosomes for simplicity) is tightly controlled and has a functional impact on processes like endocytosis, phagocytosis and autophagocytosis. The cytoplasmic distribution profile of lysosomes can be easily determined in cells where the cytoplasm/nuclear ratio in a cross-section area is high. However, determining lysosomal position in cells with lower cytoplasm/nuclear ratio, such as macrophages is more challenging. Here, we describe a method that can be efficiently and accurately used to determine the position of organelles in macrophages using confocal microscopy in two-dimensional (2D) images. Using this approach in macrophages, we confirmed previous observations in epithelial cells that both changes in cytoplasmic pH and the levels of active Rab34 induced a re-distribution of lysosomes to the cell centre or periphery. Noteworthy is that this Rab34-dependent re-distribution of lysosomes did not significantly affect the spatial distribution profile of phagolysosomes in the cytoplasm. We conclude that although Rab34 regulates both lysosomal positioning and lysosome to phagosome fusion, the latter effect is not due to the regulation of the cytoplasmic accessibility of lysosomes to phagosomes by Rab34.

Size-dependent mechanism of cargo sorting during lysosome-phagosome fusion is controlled by Rab34.

Phagosome maturation is an essential part of the innate and adaptive immune response. Although it is well established that several Ras-related proteins in brain (Rab) proteins become associated to phagosomes, little is known about how these phagosomal Rab proteins influence phagosome maturation. Here, we show a specific role for Rab34 and mammalian uncoordinated 13-2 (Munc13-2) in phagolysosome biogenesis and cargo delivery. Rab34 knockdown impaired the fusion of phagosomes with late endosomes/lysosomes and high levels of active Rab34 promoted this process. We demonstrate that Rab34 enhances phagosome maturation independently of Rab7 and coordinates phagolysosome biogenesis through size-selective transfer of late endosomal/lysosomal cargo into phagosomes. More importantly, we show that Rab34 mediates phagosome maturation through the recruitment of the protein Munc13-2. Finally, we report that the alternative maturation pathway controlled by Rab34 is critical for mycobacterial killing because Rab34 silencing resulted in mycobacterial survival, and Rab34 expression led to mycobacterial killing. Altogether, our studies uncover Rab34/Munc13-2 as a critical part of an alternative Rab7-independent phagosome maturation machinery and lysosome-mediated killing of mycobacteria.

Internalization, phagolysosomal biogenesis and killing of mycobacteria in enucleated epithelial cells.

Bacterial and parasitic intracellular pathogens or their secreted products have been shown to induce host cell transcriptional responses, which may benefit the host, favour the microorganism or be unrelated to the infection. In most instances, however, it is not known if the host cell nucleus is proximately required for the development of an intracellular infection. This information can be obtained by the infection of artificially enucleated host cells (cytoplasts). This model, although rather extensively used in studies of viral infection, has only been applied to few bacterial pathogens, which do not include Mycobacterium spp. Here, we investigate the internalization, phagosome biogenesis and survival of M. smegmatis in enucleated type II alveolar epithelial cells. Cytoplasts were infected with M. smegmatis, but the percentage of infection was significantly lower than that of nucleated cells. Scanning electron microscopy indicated that in both cells and cytoplasts, bacteria were internalized by a phagocytosis-like mechanism. Interestingly, phagosome fusion with lysosomes and mycobacterial killing were both more efficient in enucleated than in nucleated cells, a finding that may be correlated with the increased number of autophagic vesicles developed in cytoplasts. We provide evidence that although quantitative changes were observed, the full development of the infection, as well as mycobacterial killing did not require the presence of the host cell nucleus.

Golgi-to-phagosome transport of acid sphingomyelinase and prosaposin is mediated by sortilin.

Sortilin, also known as neurotensin receptor 3 (NTR3), is a transmembrane protein with a dual function. It acts as a receptor for neuromediators and growth factors at the plasma membrane, but it has also been implicated in binding and transport of some lysosomal proteins. However, the role of sortilin during phagosome maturation has not been investigated before. Here, we show that in macrophages, sortilin is mainly localized in the Golgi and transported to latex-bead phagosomes (LBPs). Using live-cell imaging and electron microscopy, we found that sortilin is delivered to LBPs in a manner that depends on its cytoplasmic tail. We also show that sortilin participates in the direct delivery of acid sphingomyelinase (ASM) and prosaposin (PS) to the phagosome, bypassing fusion with lysosomal compartments. Further analysis confirmed that ASM and PS are targeted to the phagosome by sortilin in a Brefeldin-A-sensitive pathway. Analysis of primary macrophages isolated from Sort1(-/-) mice indicated that the delivery of ASM and PS, but not pro-cathepsin D, to LBPs was severely impaired. We propose a pathway mediated by sortilin by which selected lysosomal proteins are transported to the phagosome along a Golgi-dependent route during the maturation of phagosomes.