Immunization-induced antigen archiving enhances local memory CD8+ T cell responses following an unrelated viral infection.

Antigens from viruses or immunizations can persist or are archived in lymph node stromal cells such as lymphatic endothelial cells (LEC) and fibroblastic reticular cells (FRC). Here, we find that, during the time frame of antigen archiving, LEC apoptosis caused by a second, but unrelated, innate immune stimulus such as vaccina viral infection or CpG DNA administration resulted in cross-presentation of archived antigens and boosted memory CD8 + T cells specific to the archived antigen. In contrast to "bystander" activation associated with unrelated infections, the memory CD8 + T cells specific to the archived antigen from the immunization were significantly higher than memory CD8 + T cells of a different antigen specificity. Finally, the boosted memory CD8 + T cells resulted in increased protection against Listeria monocytogenes expressing the antigen from the immunization, but only for the duration that the antigen was archived. These findings outline an important mechanism by which lymph node stromal cell archived antigens, in addition to bystander activation, can augment memory CD8 + T cell responses during repeated inflammatory insults.

Pro-inflammatory feedback loops define immune responses to pathogenic Lentivirus infection.

BACKGROUND: The Lentivirus human immunodeficiency virus (HIV) causes chronic inflammation and AIDS in humans, with variable rates of disease progression between individuals driven by both host and viral factors. Similarly, simian lentiviruses vary in their pathogenicity based on characteristics of both the host species and the virus strain, yet the immune underpinnings that drive differential Lentivirus pathogenicity remain incompletely understood. METHODS: We profile immune responses in a unique model of differential lentiviral pathogenicity where pig-tailed macaques are infected with highly genetically similar variants of SIV that differ in virulence. We apply longitudinal single-cell transcriptomics to this cohort, along with single-cell resolution cell-cell communication techniques, to understand the immune mechanisms underlying lentiviral pathogenicity. RESULTS: Compared to a minimally pathogenic lentiviral variant, infection with a highly pathogenic variant results in a more delayed, broad, and sustained activation of inflammatory pathways, including an extensive global interferon signature. Conversely, individual cells infected with highly pathogenic Lentivirus upregulated fewer interferon-stimulated genes at a lower magnitude, indicating that highly pathogenic Lentivirus has evolved to partially escape from interferon responses. Further, we identify CXCL10 and CXCL16 as important molecular drivers of inflammatory pathways specifically in response to highly pathogenic Lentivirus infection. Immune responses to highly pathogenic Lentivirus infection are characterized by amplifying regulatory circuits of pro-inflammatory cytokines with dense longitudinal connectivity. CONCLUSIONS: Our work presents a model of lentiviral pathogenicity where failures in early viral control mechanisms lead to delayed, sustained, and amplifying pro-inflammatory circuits, which in turn drives disease progression.

Early cellular and molecular signatures correlate with severity of West Nile virus infection.

Infection with West Nile virus (WNV) drives a wide range of responses, from asymptomatic to flu-like symptoms/fever or severe cases of encephalitis and death. To identify cellular and molecular signatures distinguishing WNV severity, we employed systems profiling of peripheral blood from asymptomatic and severely ill individuals infected with WNV. We interrogated immune responses longitudinally from acute infection through convalescence employing single-cell protein and transcriptional profiling complemented with matched serum proteomics and metabolomics as well as multi-omics analysis. At the acute time point, we detected both elevation of pro-inflammatory markers in innate immune cell types and reduction of regulatory T cell activity in participants with severe infection, whereas asymptomatic donors had higher expression of genes associated with anti-inflammatory CD16+ monocytes. Therefore, we demonstrated the potential of systems immunology using multiple cell-type and cell-state-specific analyses to identify correlates of infection severity and host cellular activity contributing to an effective anti-viral response.

Vaccine-induced antigen archiving enhances local memory CD8+ T cell responses following an unrelated viral infection.

Viral and vaccine antigens persist or are archived in lymph node stromal cells (LNSC) such as lymphatic endothelial cells (LEC) and fibroblastic reticular cells (FRC). Here, we find that, during the time frame of antigen archiving, LEC apoptosis caused by a second, but unrelated, innate immune stimulus such as vaccina viral infection or CpG DNA administration boosted memory CD8+ T cells specific to the archived antigen. In contrast to "bystander" activation associated with unrelated infections, the memory CD8+ T cells specific to the vaccine archived antigen were significantly higher than memory CD8+ T cells of a different antigen specificity. Finally, the boosted memory CD8+ T cells resulted in increased protection against Listeria monocytogenes expressing the vaccine antigen, but only for the duration that the vaccine antigen was archived. These findings outline a novel mechanism by which LNSC archived antigens, in addition to bystander activation, can augment memory CD8+ T cell responses during repeated inflammatory insults.

Single-cell RNA sequencing of liver fine-needle aspirates captures immune diversity in the blood and liver in chronic hepatitis B patients.

BACKGROUND AND AIMS: HBV infection is restricted to the liver, where it drives exhaustion of virus-specific T and B cells and pathogenesis through dysregulation of intrahepatic immunity. Our understanding of liver-specific events related to viral control and liver damage has relied almost solely on animal models, and we lack useable peripheral biomarkers to quantify intrahepatic immune activation beyond cytokine measurement. Our objective was to overcome the practical obstacles of liver sampling using fine-needle aspiration and develop an optimized workflow to comprehensively compare the blood and liver compartments within patients with chronic hepatitis B using single-cell RNA sequencing. APPROACH AND RESULTS: We developed a workflow that enabled multi-site international studies and centralized single-cell RNA sequencing. Blood and liver fine-needle aspirations were collected, and cellular and molecular captures were compared between the Seq-Well S 3 picowell-based and the 10× Chromium reverse-emulsion droplet-based single-cell RNA sequencing technologies. Both technologies captured the cellular diversity of the liver, but Seq-Well S 3 effectively captured neutrophils, which were absent in the 10× dataset. CD8 T cells and neutrophils displayed distinct transcriptional profiles between blood and liver. In addition, liver fine-needle aspirations captured a heterogeneous liver macrophage population. Comparison between untreated patients with chronic hepatitis B and patients treated with nucleoside analogs showed that myeloid cells were highly sensitive to environmental changes while lymphocytes displayed minimal differences. CONCLUSIONS: The ability to electively sample and intensively profile the immune landscape of the liver, and generate high-resolution data, will enable multi-site clinical studies to identify biomarkers for intrahepatic immune activity in HBV and beyond.

Single-cell profiling of environmental enteropathy reveals signatures of epithelial remodeling and immune activation.

Environmental enteropathy (EE) is a subclinical condition of the small intestine that is highly prevalent in low- and middle-income countries. It is thought to be a key contributing factor to childhood malnutrition, growth stunting, and diminished oral vaccine responses. Although EE has been shown to be the by-product of a recurrent enteric infection, its full pathophysiology remains unclear. Here, we mapped the cellular and molecular correlates of EE by performing high-throughput, single-cell RNA-sequencing on 33 small intestinal biopsies from 11 adults with EE in Lusaka, Zambia (eight HIV-negative and three HIV-positive), six adults without EE in Boston, United States, and two adults in Durban, South Africa, which we complemented with published data from three additional individuals from the same clinical site. We analyzed previously defined bulk-transcriptomic signatures of reduced villus height and decreased microbial translocation in EE and showed that these signatures may be driven by an increased abundance of surface mucosal cells-a gastric-like subset previously implicated in epithelial repair in the gastrointestinal tract. In addition, we determined cell subsets whose fractional abundances associate with EE severity, small intestinal region, and HIV infection. Furthermore, by comparing duodenal EE samples with those from three control cohorts, we identified dysregulated WNT and MAPK signaling in the EE epithelium and increased proinflammatory cytokine gene expression in a T cell subset highly expressing a transcriptional signature of tissue-resident memory cells in the EE cohort. Together, our work elucidates epithelial and immune correlates of EE and nominates cellular and molecular targets for intervention.

HIV-1 Nucleic Acids Identify Rectal HIV Exposures in Self-Collected Rectal Swabs, Whereas Y-Chromosome Single Tandem Repeat Mixtures Are Not Reliable Biomarkers of Condomless Receptive Anal Intercourse.

BACKGROUND: To focus interventions, biomarkers of HIV-1 exposure could help in identifying subpopulations at highest risk of acquisition. We assessed whether Y-chromosome single tandem repeat (YSTR) mixtures obtained from rectal swabs could serve as a biomarker of condomless receptive anal intercourse (CRAI) among men who have sex with men and transgender women and evaluated the feasibility of detecting HIV-1 virions to assess exposures. METHODS: Twenty-nine sexually active HIV-seronegative men who have sex with men and one transgender woman from New York City answered on-site and mobile app sexual behavior questionnaires. They were randomized to collecting self-administered rectal swabs every morning or after receptive anal intercourse (RAI). YSTR profiles were assessed from blood sample and swabs; HIV-1 exposure was measured by conducting quantitative polymerase chain reaction in swabs. RESULTS: After 2 months, the daily mobile survey had 135%-201% more instances of anal sex acts and 170%-193% more RAI than on-site surveys. Daily mobile reporting had 11%-35% less CRAI events than those reported on-site (Pdaily = 0.001; Pper-sex = 0.047). The daily swabbing arm reported less RAI (P < 0.001) and CRAI (P < 0.038) and had 2.95 lower odds of detecting YSTR mixtures (P = 0.021) than the per-sex-event arm. Surprisingly, YSTR detection was not significantly modified by report of bowel movements and lubricant, enema, or condom use. No participant became HIV-1 infected, yet HIV-1 total nucleic acids were detected in 6 independent episodes of CRAI in 2 participants taking pre-exposure prophylaxis. CONCLUSIONS: YSTR mixtures demonstrated 80% specificity but only 30% sensitivity as a biomarker of CRAI in self-collected rectal swabs. However, detection of HIV-1 exposures in self-collected swabs may help in identifying those needing further HIV risk reduction strategies.

Single-cell immunophenotyping of the skin lesion erythema migrans identifies IgM memory B cells.

The skin lesion erythema migrans (EM) is an initial sign of the Ixodes tick-transmitted Borreliella spirochetal infection known as Lyme disease. T cells and innate immune cells have previously been shown to predominate the EM lesion and promote the reaction. Despite the established importance of B cells and antibodies in preventing infection, the role of B cells in the skin immune response to Borreliella is unknown. Here, we used single-cell RNA-Seq in conjunction with B cell receptor (BCR) sequencing to immunophenotype EM lesions and their associated B cells and BCR repertoires. We found that B cells were more abundant in EM in comparison with autologous uninvolved skin; many were clonally expanded and had circulating relatives. EM-associated B cells upregulated the expression of MHC class II genes and exhibited preferential IgM isotype usage. A subset also exhibited low levels of somatic hypermutation despite a gene expression profile consistent with memory B cells. Our study demonstrates that single-cell gene expression with paired BCR sequencing can be used to interrogate the sparse B cell populations in human skin and reveals that B cells in the skin infection site in early Lyme disease expressed a phenotype consistent with local antigen presentation and antibody production.

Leukocyte dynamics after intracerebral hemorrhage in a living patient reveal rapid adaptations to tissue milieu.

Intracerebral hemorrhage (ICH) is a devastating form of stroke with a high mortality rate and few treatment options. Discovery of therapeutic interventions has been slow given the challenges associated with studying acute injury in the human brain. Inflammation induced by exposure of brain tissue to blood appears to be a major part of brain tissue injury. Here, we longitudinally profiled blood and cerebral hematoma effluent from a patient enrolled in the Minimally Invasive Surgery with Thrombolysis in Intracerebral Hemorrhage Evacuation trial, offering a rare window into the local and systemic immune responses to acute brain injury. Using single-cell RNA-Seq (scRNA-Seq), this is the first report to our knowledge that characterized the local cellular response during ICH in the brain of a living patient at single-cell resolution. Our analysis revealed shifts in the activation states of myeloid and T cells in the brain over time, suggesting that leukocyte responses are dynamically reshaped by the hematoma microenvironment. Interestingly, the patient had an asymptomatic rebleed that our transcriptional data indicated occurred prior to detection by CT scan. This case highlights the rapid immune dynamics in the brain after ICH and suggests that sensitive methods such as scRNA-Seq would enable greater understanding of complex intracerebral events.

Inflammasomes within Hyperactive Murine Dendritic Cells Stimulate Long-Lived T Cell-Mediated Anti-tumor Immunity.

Central to anti-tumor immunity are dendritic cells (DCs), which stimulate long-lived protective T cell responses. Recent studies have demonstrated that DCs can achieve a state of hyperactivation, which is associated with inflammasome activities within living cells. Herein, we report that hyperactive DCs have an enhanced ability to migrate to draining lymph nodes and stimulate potent cytotoxic T lymphocyte (CTL) responses. This enhanced migratory activity is dependent on the chemokine receptor CCR7 and is associated with a unique transcriptional program that is not observed in conventionally activated or pyroptotic DCs. We show that hyperactivating stimuli are uniquely capable of inducing durable CTL-mediated anti-tumor immunity against tumors that are sensitive or resistant to PD-1 inhibition. These protective responses are intrinsic to the cDC1 subset of DCs, depend on the inflammasome-dependent cytokine IL-1ß, and enable tumor lysates to serve as immunogens. If these activities are verified in humans, hyperactive DCs may impact immunotherapy.

Functional compensation precedes recovery of tissue mass following acute liver injury.

The liver plays a central role in metabolism, protein synthesis and detoxification. It possesses unique regenerative capacity upon injury. While many factors regulating cellular proliferation during liver repair have been identified, the mechanisms by which the injured liver maintains vital functions prior to tissue recovery are unknown. Here, we identify a new phase of functional compensation following acute liver injury that occurs prior to cellular proliferation. By coupling single-cell RNA-seq with in situ transcriptional analyses in two independent murine liver injury models, we discover adaptive reprogramming to ensure expression of both injury response and core liver function genes dependent on macrophage-derived WNT/ß-catenin signaling. Interestingly, transcriptional compensation is most prominent in non-proliferating cells, clearly delineating two temporally distinct phases of liver recovery. Overall, our work describes a mechanism by which the liver maintains essential physiological functions prior to cellular reconstitution and characterizes macrophage-derived WNT signals required for this compensation.

Selection for tandem stop codons in ciliate species with reassigned stop codons.

The failure of mRNA translation machinery to recognize a stop codon as a termination signal and subsequent translation of the 3' untranslated region (UTR) is referred to as stop codon readthrough, the frequency of which is related to the length, composition, and structure of mRNA sequences downstream of end-of-gene stop codons. Secondary in-frame stop codons within a few positions downstream of the primary stop codons, so-called tandem stop codons (TSCs), serve as backup termination signals, which limit the effects of readthrough: polypeptide product degradation, mislocalization, and aggregation. In this study, ciliate species with UAA and UAG stop codons reassigned to code for glutamine are found to possess statistical excesses of TSCs at the beginning of their 3' UTRs. The overrepresentation of TSCs in these species is greater than that observed in standard code organisms. Though the overall numbers of TSCs are lower in most species with alternative stop codons because they use fewer than three unique stop codons, the relatively great overrepresentation of TSCs in alternative-code ciliate species suggests that there exist stronger selective pressures to maintain TSCs in these organisms compared to standard code organisms.