The Biology and Underlying Mechanisms of Cross-Presentation of Exogenous Antigens on MHC-I Molecules.

To monitor the health of cells, the immune system tasks antigen-presenting cells with gathering antigens from other cells and bringing them to CD8 T cells in the form of peptides bound to MHC-I molecules. Most cells would be unable to perform this function because they use their MHC-I molecules to exclusively present peptides derived from the cell's own proteins. However, the immune system evolved mechanisms for dendritic cells and some other phagocytes to sample and present antigens from the extracellular milieu on MHC-I through a process called cross-presentation. How this important task is accomplished, its role in health and disease, and its potential for exploitation are the subject of this review.

An in vivo neuroimmune organoid model to study human microglia phenotypes.

Microglia are specialized brain-resident macrophages that play crucial roles in brain development, homeostasis, and disease. However, until now, the ability to model interactions between the human brain environment and microglia has been severely limited. To overcome these limitations, we developed an in vivo xenotransplantation approach that allows us to study functionally mature human microglia (hMGs) that operate within a physiologically relevant, vascularized immunocompetent human brain organoid (iHBO) model. Our data show that organoid-resident hMGs gain human-specific transcriptomic signatures that closely resemble their in vivo counterparts. In vivo two-photon imaging reveals that hMGs actively engage in surveilling the human brain environment, react to local injuries, and respond to systemic inflammatory cues. Finally, we demonstrate that the transplanted iHBOs developed here offer the unprecedented opportunity to study functional human microglia phenotypes in health and disease and provide experimental evidence for a brain-environment-induced immune response in a patient-specific model of autism with macrocephaly.

DNA hypomethylation silences anti-tumor immune genes in early prostate cancer and CTCs.

Cancer is characterized by hypomethylation-associated silencing of large chromatin domains, whose contribution to tumorigenesis is uncertain. Through high-resolution genome-wide single-cell DNA methylation sequencing, we identify 40 core domains that are uniformly hypomethylated from the earliest detectable stages of prostate malignancy through metastatic circulating tumor cells (CTCs). Nested among these repressive domains are smaller loci with preserved methylation that escape silencing and are enriched for cell proliferation genes. Transcriptionally silenced genes within the core hypomethylated domains are enriched for immune-related genes; prominent among these is a single gene cluster harboring all five CD1 genes that present lipid antigens to NKT cells and four IFI16-related interferon-inducible genes implicated in innate immunity. The re-expression of CD1 or IFI16 murine orthologs in immuno-competent mice abrogates tumorigenesis, accompanied by the activation of anti-tumor immunity. Thus, early epigenetic changes may shape tumorigenesis, targeting co-located genes within defined chromosomal loci. Hypomethylation domains are detectable in blood specimens enriched for CTCs.

Structural basis for RNA surveillance by the human nuclear exosome targeting (NEXT) complex.

RNA quality control relies on co-factors and adaptors to identify and prepare substrates for degradation by ribonucleases such as the 3' to 5' ribonucleolytic RNA exosome. Here, we determined cryogenic electron microscopy structures of human nuclear exosome targeting (NEXT) complexes bound to RNA that reveal mechanistic insights to substrate recognition and early steps that precede RNA handover to the exosome. The structures illuminate ZCCHC8 as a scaffold, mediating homodimerization while embracing the MTR4 helicase and flexibly anchoring RBM7 to the helicase core. All three subunits collaborate to bind the RNA, with RBM7 and ZCCHC8 surveying sequences upstream of the 3' end to facilitate RNA capture by MTR4. ZCCHC8 obscures MTR4 surfaces important for RNA binding and extrusion as well as MPP6-dependent recruitment and docking onto the RNA exosome core, interactions that contribute to RNA surveillance by coordinating RNA capture, translocation, and extrusion from the helicase to the exosome for decay.

Early introductions and transmission of SARS-CoV-2 variant B.1.1.7 in the United States.

The emergence and spread of SARS-CoV-2 lineage B.1.1.7, first detected in the United Kingdom, has become a global public health concern because of its increased transmissibility. Over 2,500 COVID-19 cases associated with this variant have been detected in the United States (US) since December 2020, but the extent of establishment is relatively unknown. Using travel, genomic, and diagnostic data, we highlight that the primary ports of entry for B.1.1.7 in the US were in New York, California, and Florida. Furthermore, we found evidence for many independent B.1.1.7 establishments starting in early December 2020, followed by interstate spread by the end of the month. Finally, we project that B.1.1.7 will be the dominant lineage in many states by mid- to late March. Thus, genomic surveillance for B.1.1.7 and other variants urgently needs to be enhanced to better inform the public health response.

Genomic Epidemiology of SARS-CoV-2 in Guangdong Province, China.

Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 infection and was first reported in central China in December 2019. Extensive molecular surveillance in Guangdong, China's most populous province, during early 2020 resulted in 1,388 reported RNA-positive cases from 1.6 million tests. In order to understand the molecular epidemiology and genetic diversity of SARS-CoV-2 in China, we generated 53 genomes from infected individuals in Guangdong using a combination of metagenomic sequencing and tiling amplicon approaches. Combined epidemiological and phylogenetic analyses indicate multiple independent introductions to Guangdong, although phylogenetic clustering is uncertain because of low virus genetic variation early in the pandemic. Our results illustrate how the timing, size, and duration of putative local transmission chains were constrained by national travel restrictions and by the province's large-scale intensive surveillance and intervention measures. Despite these successes, COVID-19 surveillance in Guangdong is still required, because the number of cases imported from other countries has increased.

Travel Surveillance and Genomics Uncover a Hidden Zika Outbreak during the Waning Epidemic.

The Zika epidemic in the Americas has challenged surveillance and control. As the epidemic appears to be waning, it is unclear whether transmission is still ongoing, which is exacerbated by discrepancies in reporting. To uncover locations with lingering outbreaks, we investigated travel-associated Zika cases to identify transmission not captured by reporting. We uncovered an unreported outbreak in Cuba during 2017, a year after peak transmission in neighboring islands. By sequencing Zika virus, we show that the establishment of the virus was delayed by a year and that the ensuing outbreak was sparked by long-lived lineages of Zika virus from other Caribbean islands. Our data suggest that, although mosquito control in Cuba may initially have been effective at mitigating Zika virus transmission, such measures need to be maintained to be effective. Our study highlights how Zika virus may still be "silently" spreading and provides a framework for understanding outbreak dynamics. VIDEO ABSTRACT.

A Heterochromatin-Specific RNA Export Pathway Facilitates piRNA Production.

PIWI-interacting RNAs (piRNAs) guide transposon silencing in animals. The 22-30 nt piRNAs are processed in the cytoplasm from long non-coding RNAs that often lack RNA processing hallmarks of export-competent transcripts. By studying how these transcripts achieve nuclear export, we uncover an RNA export pathway specific for piRNA precursors in the Drosophila germline. This pathway requires Nxf3-Nxt1, a variant of the hetero-dimeric mRNA export receptor Nxf1-Nxt1. Nxf3 interacts with UAP56, a nuclear RNA helicase essential for mRNA export, and CG13741/Bootlegger, which recruits Nxf3-Nxt1 and UAP56 to heterochromatic piRNA source loci. Upon RNA cargo binding, Nxf3 achieves nuclear export via the exportin Crm1 and accumulates together with Bootlegger in peri-nuclear nuage, suggesting that after export, Nxf3-Bootlegger delivers precursor transcripts to the piRNA processing sites. These findings indicate that the piRNA pathway bypasses nuclear RNA surveillance systems to export unprocessed transcripts to the cytoplasm, a strategy also exploited by retroviruses.

The RNA Exosome Syncs IAV-RNAPII Transcription to Promote Viral Ribogenesis and Infectivity.

The nuclear RNA exosome is an essential multi-subunit complex that controls RNA homeostasis. Congenital mutations in RNA exosome genes are associated with neurodegenerative diseases. Little is known about the role of the RNA exosome in the cellular response to pathogens. Here, using NGS and human and mouse genetics, we show that influenza A virus (IAV) ribogenesis and growth are suppressed by impaired RNA exosome activity. Mechanistically, the nuclear RNA exosome coordinates the initial steps of viral transcription with RNAPII at host promoters. The viral polymerase complex co-opts the nuclear RNA exosome complex and cellular RNAs en route to 3' end degradation. Exosome deficiency uncouples chromatin targeting of the viral polymerase complex and the formation of cellular:viral RNA hybrids, which are essential RNA intermediates that license transcription of antisense genomic viral RNAs. Our results suggest that evolutionary arms races have shaped the cellular RNA quality control machinery.

Endothelial cells give a boost to senescence surveillance.

Senescence is a specialized form of cell cycle arrest induced in response to damage and stress. In certain settings, senescent cells can promote their own removal by recruitment of the immune system, a process that is thought to decline in efficiency with age. In this issue of Genes & Development, Yin et al. (pp. 533-549) discover a surprising cross-talk where senescent cells instruct endothelial cells to help organize the clearance of the senescent population. This uncovers yet another layer of complexity in senescent cell biology, with implications for cancer treatment and aging.

Senescence-induced endothelial phenotypes underpin immune-mediated senescence surveillance.

Senescence is a stress-responsive tumor suppressor mechanism associated with expression of the senescence-associated secretory phenotype (SASP). Through the SASP, senescent cells trigger their own immune-mediated elimination, which if evaded leads to tumorigenesis. Senescent parenchymal cells are separated from circulating immunocytes by the endothelium, which is targeted by microenvironmental signaling. Here we show that SASP induces endothelial cell NF-κB activity and that SASP-induced endothelial expression of the canonical NF-κB component Rela underpins senescence surveillance. Using human liver sinusoidal endothelial cells (LSECs), we show that SASP-induced endothelial NF-κB activity regulates a conserved transcriptional program supporting immunocyte recruitment. Furthermore, oncogenic hepatocyte senescence drives murine LSEC NF-κB activity in vivo. Critically, we show two distinct endothelial pathways in senescence surveillance. First, endothelial-specific loss of Rela prevents development of Stat1-expressing CD4+ T lymphocytes. Second, the SASP up-regulates ICOSLG on LSECs, with the ICOS-ICOSLG axis contributing to senescence cell clearance. Our results show that the endothelium is a nonautonomous SASP target and an organizing center for immune-mediated senescence surveillance.

Endocrine Autoimmune Disease as a Fragility of Immune Surveillance against Hypersecreting Mutants.

Some endocrine organs are frequent targets of autoimmune attack. Here, we addressed the origin of autoimmune disease from the viewpoint of feedback control. Endocrine tissues maintain mass through feedback loops that balance cell proliferation and removal according to hormone-driven regulatory signals. We hypothesized the existence of a dedicated mechanism that detects and removes mutant cells that missense the signal and therefore hyperproliferate and hypersecrete with potential to disrupt organismal homeostasis. In this mechanism, hypersecreting cells are preferentially eliminated by autoreactive T cells at the cost of a fragility to autoimmune disease. The "autoimmune surveillance of hypersecreting mutants" (ASHM) hypothesis predicts the presence of autoreactive T cells in healthy individuals and the nature of self-antigens as peptides from hormone secretion pathway. It explains why some tissues get prevalent autoimmune disease, whereas others do not and instead show prevalent mutant-expansion disease (e.g., hyperparathyroidism). The ASHM hypothesis is testable, and we discuss experimental follow-up.

Targeting KDM4A epigenetically activates tumor-cell-intrinsic immunity by inducing DNA replication stress.

Developing strategies to activate tumor-cell-intrinsic immune response is critical for improving tumor immunotherapy by exploiting tumor vulnerability. KDM4A, as a histone H3 lysine 9 trimethylation (H3K9me3) demethylase, has been found to play a critical role in squamous cell carcinoma (SCC) growth and metastasis. Here we report that KDM4A inhibition promoted heterochromatin compaction and induced DNA replication stress, which elicited antitumor immunity in SCC. Mechanistically, KDM4A inhibition promoted the formation of liquid-like HP1γ puncta on heterochromatin and stall DNA replication, which activated tumor-cell-intrinsic cGAS-STING signaling through replication-stress-induced cytosolic DNA accumulation. Moreover, KDM4A inhibition collaborated with PD1 blockade to inhibit SCC growth and metastasis by recruiting and activating CD8+ T cells. In vivo lineage tracing demonstrated that KDM4A inhibition plus PD1 blockade efficiently eliminated cancer stem cells. Altogether, our results demonstrate that targeting KDM4A can activate anti-tumor immunity and enable PD1 blockade immunotherapy by aggravating replication stress in SCC cells.

Cancer statistics for the US Hispanic/Latino population, 2021.

The Hispanic/Latino population is the second largest racial/ethnic group in the continental United States and Hawaii, accounting for 18% (60.6 million) of the total population. An additional 3 million Hispanic Americans live in Puerto Rico. Every 3 years, the American Cancer Society reports on cancer occurrence, risk factors, and screening for Hispanic individuals in the United States using the most recent population-based data. An estimated 176,600 new cancer cases and 46,500 cancer deaths will occur among Hispanic individuals in the continental United States and Hawaii in 2021. Compared to non-Hispanic Whites (NHWs), Hispanic men and women had 25%-30% lower incidence (2014-2018) and mortality (2015-2019) rates for all cancers combined and lower rates for the most common cancers, although this gap is diminishing. For example, the colorectal cancer (CRC) incidence rate ratio for Hispanic compared with NHW individuals narrowed from 0.75 (95% CI, 0.73-0.78) in 1995 to 0.91 (95% CI, 0.89-0.93) in 2018, reflecting delayed declines in CRC rates among Hispanic individuals in part because of slower uptake of screening. In contrast, Hispanic individuals have higher rates of infection-related cancers, including approximately two-fold higher incidence of liver and stomach cancer. Cervical cancer incidence is 32% higher among Hispanic women in the continental US and Hawaii and 78% higher among women in Puerto Rico compared to NHW women, yet is largely preventable through screening. Less access to care may be similarly reflected in the low prevalence of localized-stage breast cancer among Hispanic women, 59% versus 67% among NHW women. Evidence-based strategies for decreasing the cancer burden among the Hispanic population include the use of culturally appropriate lay health advisors and patient navigators and targeted, community-based intervention programs to facilitate access to screening and promote healthy behaviors. In addition, the impact of the COVID-19 pandemic on cancer trends and disparities in the Hispanic population should be closely monitored.

Mammalian RNA Decay Pathways Are Highly Specialized and Widely Linked to Translation.

RNA decay is crucial for mRNA turnover and surveillance and misregulated in many diseases. This complex system is challenging to study, particularly in mammals, where it remains unclear whether decay pathways perform specialized versus redundant roles. Cytoplasmic pathways and links to translation are particularly enigmatic. By directly profiling decay factor targets and normal versus aberrant translation in mouse embryonic stem cells (mESCs), we uncovered extensive decay pathway specialization and crosstalk with translation. XRN1 (5'-3') mediates cytoplasmic bulk mRNA turnover whereas SKIV2L (3'-5') is universally recruited by ribosomes, tackling aberrant translation and sometimes modulating mRNA abundance. Further exploring translation surveillance revealed AVEN and FOCAD as SKIV2L interactors. AVEN prevents ribosome stalls at structured regions, which otherwise require SKIV2L for clearance. This pathway is crucial for histone translation, upstream open reading frame (uORF) regulation, and counteracting ribosome arrest on small ORFs. In summary, we uncovered key targets, components, and functions of mammalian RNA decay pathways and extensive coupling to translation.

Extraction of mRNA from Stalled Ribosomes by the Ski Complex.

The evolutionarily conserved Ski2-Ski3-Ski8 (Ski) complex containing the 3'→5' RNA helicase Ski2 binds to 80S ribosomes near the mRNA entrance and facilitates 3'→5' exosomal degradation of mRNA during ribosome-associated mRNA surveillance pathways. Here, we assayed Ski's activity using an in vitro reconstituted translation system and report that this complex efficiently extracts mRNA from 80S ribosomes in the 3'→5' direction in a nucleotide-by-nucleotide manner. The process is ATP dependent and can occur on pre- and post-translocation ribosomal complexes. The Ski complex can engage productively with mRNA and extract it from 80S complexes containing as few as 19 (but not 13) 3'-terminal mRNA nucleotides starting from the P site. The mRNA-extracting activity of the Ski complex suggests that its role in mRNA quality control pathways is not limited to acceleration of exosomal degradation and could include clearance of stalled ribosomes from mRNA, poising mRNA for degradation and rendering stalled ribosomes recyclable by Pelota/Hbs1/ABCE1.

The G3BP1-Family-USP10 Deubiquitinase Complex Rescues Ubiquitinated 40S Subunits of Ribosomes Stalled in Translation from Lysosomal Degradation.

Ribosome-associated quality control (RQC) purges aberrant mRNAs and nascent polypeptides in a multi-step molecular process initiated by the E3 ligase ZNF598 through sensing of ribosomes collided at aberrant mRNAs and monoubiquitination of distinct small ribosomal subunit proteins. We show that G3BP1-family-USP10 complexes are required for deubiquitination of RPS2, RPS3, and RPS10 to rescue modified 40S subunits from programmed degradation. Knockout of USP10 or G3BP1 family proteins increased lysosomal ribosomal degradation and perturbed ribosomal subunit stoichiometry, both of which were rescued by a single K214R substitution of RPS3. While the majority of RPS2 and RPS3 monoubiquitination resulted from ZNF598-dependent sensing of ribosome collisions initiating RQC, another minor pathway contributed to their monoubiquitination. G3BP1 family proteins have long been considered RNA-binding proteins, however, our results identified 40S subunits and associated mRNAs as their predominant targets, a feature shared by stress granules to which G3BP1 family proteins localize under stress.

HemK2 functions for sufficient protein synthesis and RNA stability through eRF1 methylation during Drosophila oogenesis.

HemK2 is a highly conserved methyltransferase, but the identification of its genuine substrates has been controversial, and its biological importance in higher organisms remains unclear. We elucidate the role of HemK2 in the methylation of eukaryotic Release Factor 1 (eRF1), a process essential for female germline development in Drosophila melanogaster. Knockdown of hemK2 in the germline cells (hemK2-GLKD) induces apoptosis, accompanied by a pronounced decrease in both eRF1 methylation and protein synthesis. Overexpression of a methylation-deficient eRF1 variant recapitulates the defects observed in hemK2-GLKD, suggesting that eRF1 is a primary methylation target of HemK2. Furthermore, hemK2-GLKD leads to significant reduction mRNA levels in germline cell. These defects in oogenesis and protein synthesis can be partially restored by inhibiting the No-Go Decay pathway. In addition, hemK2 knockdown is associated with increased disome formation, suggesting that disruptions in eRF1 methylation may provoke ribosomal stalling, which subsequently activates translation-coupled mRNA surveillance mechanisms that degrade actively-translated mRNAs. We propose that HemK2-mediated methylation of eRF1 is critical for ensuring efficient protein production and mRNA stability, which are vital for the generation of high-quality eggs.

Drp1-Zip1 Interaction Regulates Mitochondrial Quality Surveillance System.

Mitophagy, a mitochondrial quality control process for eliminating dysfunctional mitochondria, can be induced by a response of dynamin-related protein 1 (Drp1) to a reduction in mitochondrial membrane potential (MMP) and mitochondrial division. However, the coordination between MMP and mitochondrial division for selecting the damaged portion of the mitochondrial network is less understood. Here, we found that MMP is reduced focally at a fission site by the Drp1 recruitment, which is initiated by the interaction of Drp1 with mitochondrial zinc transporter Zip1 and Zn2+ entry through the Zip1-MCU complex. After division, healthy mitochondria restore MMP levels and participate in the fusion-fission cycle again, but mitochondria that fail to restore MMP undergo mitophagy. Thus, interfering with the interaction between Drp1 and Zip1 blocks the reduction of MMP and the subsequent mitophagic selection of damaged mitochondria. These results suggest that Drp1-dependent fission provides selective pressure for eliminating "bad sectors" in the mitochondrial network, serving as a mitochondrial quality surveillance system.

Estimation of SARS-CoV-2 fitness gains from genomic surveillance data without prior lineage classification.

The emergence of SARS-CoV-2 variants with increased fitness has had a strong impact on the epidemiology of COVID-19, with the higher effective reproduction number of the viral variants leading to new epidemic waves. Tracking such variants and their genetic signatures, using data collected through genomic surveillance, is therefore crucial for forecasting likely surges in incidence. Current methods of estimating fitness advantages of variants rely on tracking the changing proportion of a particular lineage over time, but describing successful lineages in a rapidly evolving viral population is a difficult task. We propose a method of estimating fitness gains directly from nucleotide information generated by genomic surveillance, without a priori assigning isolates to lineages from phylogenies, based solely on the abundance of single nucleotide polymorphisms (SNPs). The method is based on mapping changes in the genetic population structure over time. Changes in the abundance of SNPs associated with periods of increasing fitness allow for the unbiased discovery of new variants, thereby obviating a deliberate lineage assignment and phylogenetic inference. We conclude that the method provides a fast and reliable way to estimate fitness advantages of variants without the need for a priori assigning isolates to lineages.