Recognition of microbial viability via TLR8 drives TFH cell differentiation and vaccine responses.

Live attenuated vaccines are generally highly efficacious and often superior to inactivated vaccines, yet the underlying mechanisms of this remain largely unclear. Here we identify recognition of microbial viability as a potent stimulus for follicular helper T cell (TFH cell) differentiation and vaccine responses. Antigen-presenting cells (APCs) distinguished viable bacteria from dead bacteria through Toll-like receptor 8 (TLR8)-dependent detection of bacterial RNA. In contrast to dead bacteria and other TLR ligands, live bacteria, bacterial RNA and synthetic TLR8 agonists induced a specific cytokine profile in human and porcine APCs, thereby promoting TFH cell differentiation. In domestic pigs, immunization with a live bacterial vaccine induced robust TFH cell and antibody responses, but immunization with its heat-killed counterpart did not. Finally, a hypermorphic TLR8 polymorphism was associated with protective immunity elicited by vaccination with bacillus Calmette-Guérin (BCG) in a human cohort. We have thus identified TLR8 as an important driver of TFH cell differentiation and a promising target for TFH cell-skewing vaccine adjuvants.

Single-cell RNA sequencing uncovers the nuclear decoy lincRNA PIRAT as a regulator of systemic monocyte immunity during COVID-19.

The systemic immune response to viral infection is shaped by master transcription factors, such as NF-κB, STAT1, or PU.1. Although long noncoding RNAs (lncRNAs) have been suggested as important regulators of transcription factor activity, their contributions to the systemic immunopathologies observed during SARS-CoV-2 infection have remained unknown. Here, we employed a targeted single-cell RNA sequencing approach to reveal lncRNAs differentially expressed in blood leukocytes during severe COVID-19. Our results uncover the lncRNA PIRAT (PU.1-induced regulator of alarmin transcription) as a major PU.1 feedback-regulator in monocytes, governing the production of the alarmins S100A8/A9, key drivers of COVID-19 pathogenesis. Knockout and transgene expression, combined with chromatin-occupancy profiling, characterized PIRAT as a nuclear decoy RNA, keeping PU.1 from binding to alarmin promoters and promoting its binding to pseudogenes in naïve monocytes. NF-κB-dependent PIRAT down-regulation during COVID-19 consequently releases a transcriptional brake, fueling alarmin production. Alarmin expression is additionally enhanced by the up-regulation of the lncRNA LUCAT1, which promotes NF-κB-dependent gene expression at the expense of targets of the JAK-STAT pathway. Our results suggest a major role of nuclear noncoding RNA networks in systemic antiviral responses to SARS-CoV-2 in humans.

Noncoding RNA MaIL1 is an integral component of the TLR4-TRIF pathway.

RNA has been proposed as an important scaffolding factor in the nucleus, aiding protein complex assembly in the dense intracellular milieu. Architectural contributions of RNA to cytosolic signaling pathways, however, remain largely unknown. Here, we devised a multidimensional gradient approach, which systematically locates RNA components within cellular protein networks. Among a subset of noncoding RNAs (ncRNAs) cosedimenting with the ubiquitin-proteasome system, our approach unveiled ncRNA MaIL1 as a critical structural component of the Toll-like receptor 4 (TLR4) immune signal transduction pathway. RNA affinity antisense purification-mass spectrometry (RAP-MS) revealed MaIL1 binding to optineurin (OPTN), a ubiquitin-adapter platforming TBK1 kinase. MaIL1 binding stabilized OPTN, and consequently, loss of MaIL1 blunted OPTN aggregation, TBK1-dependent IRF3 phosphorylation, and type I interferon (IFN) gene transcription downstream of TLR4. MaIL1 expression was elevated in patients with active pulmonary infection and was highly correlated with IFN levels in bronchoalveolar lavage fluid. Our study uncovers MaIL1 as an integral RNA component of the TLR4-TRIF pathway and predicts further RNAs to be required for assembly and progression of cytosolic signaling networks in mammalian cells.

Follicular Helper-like T Cells in the Lung Highlight a Novel Role of B Cells in Sarcoidosis.

Rationale: Pulmonary sarcoidosis is generally presumed to be a T-helper cell type 1- and macrophage-driven disease. However, mouse models have recently revealed that chronically inflamed lung tissue can also comprise T follicular helper (Tfh)-like cells and represents a site of active T-cell/B-cell cooperation. Objectives: To assess the role of pulmonary Tfh- and germinal center-like lymphocytes in sarcoidosis. Methods: BAL fluid, lung tissue, and peripheral blood samples from patients with sarcoidosis were analyzed by flow cytometry, immunohistology, RNA sequencing, and in vitro T-cell/B-cell cooperation assays for phenotypic and functional characterization of germinal center-like reactions in inflamed tissue. Measurements and Main Results: We identified a novel population of Tfh-like cells characterized by high expression of the B helper molecules CD40L and IL-21 in BAL of patients with sarcoidosis. Transcriptome analysis further confirmed a phenotype that was both Tfh-like and tissue resident. BAL T cells provided potent help for B cells to differentiate into antibody-producing cells. In lung tissue, we observed large peribronchial infiltrates with T and B cells in close contact, and many IgA+ plasmablasts. Most clusters were nonectopic; that is, they did not contain follicular dendritic cells. Patients with sarcoidosis also showed elevated levels of PD-1high CXCR5- CD40Lhigh ICOShigh Tfh-like cells, but not classical CXCR5+ Tfh cells, in the blood. Conclusions: Active T-cell/B-cell cooperation and local production of potentially pathogenic antibodies in the inflamed lung represents a novel pathomechanism in sarcoidosis and should be considered from both diagnostic and therapeutic perspectives.

Clonal expansion of CD4+CD8+ T cells in an adult patient with Mycoplasma pneumoniae-associated Erythema multiforme majus.

BACKGROUND: Erythema multiforme (EM) is an acute, immune-mediated mucocutaneous disease, most often preceded by herpes simplex virus (HSV) infection or reactivation. Mycoplasma pneumoniae (Mp) is considered the second major trigger of EM and is often associated with an atypical and more severe presentation of disease, characterized by prominent mucosal involvement. However, contrary to HSV-associated Erythema multiforme (HAEM), immunological mechanisms of Mp-associated EM remain unclear. CASE PRESENTATION: We present the case of a 50-year-old male patient presenting with community-acquired pneumonia (CAP) and erythema multiforme majus (EMM). Acute Mp infection was diagnosed by seroconversion, with no evidence of HSV infection as a cause of EMM. We performed immune phenotyping of blister fluid (BF) and peripheral blood (PB) T cells and detected a clonally expanded TCRVß2+ T cell population that was double positive for CD4 and CD8, and expressed the cytotoxic markers granulysin and perforin. This CD4+CD8+ population comprised up to 50.7% of BF T cells and 24.9% of PB T cells. Two years prior to the onset of disease, the frequency of PB CD4+CD8+T cells had been within normal range and it gradually returned to baseline levels with the resolution of symptoms, suggesting an involvement of this population in EMM disease pathophysiology. CONCLUSIONS: This report is the first to provide a phenotypic description of lesional T cells in Mp-associated EMM. Characterizing the local immune response might help to address pathophysiological questions and warrants further systematic research.