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Regulatory T cells (T reg cells) can activate multiple suppressive mechanisms in vitro after activation via the T cell antigen receptor, resulting in antigen-independent suppression. However, it remains unclear whether similar pathways operate in vivo. Here we found that antigen-specific T reg cells activated by dendritic cells (DCs) pulsed with two antigens suppressed conventional naive T cells (T naive cells) specific for both cognate antigens and non-cognate antigens in vitro but suppressed only T naive cells specific for cognate antigen in vivo. Antigen-specific T reg cells formed strong interactions with DCs, resulting in selective inhibition of the binding of T naive cells to cognate antigen yet allowing bystander T naive cell access. Strong binding resulted in the removal of the complex of cognate peptide and major histocompatibility complex class II (pMHCII) from the DC surface, reducing the capacity of DCs to present antigen. The enhanced binding of T reg cells to DCs, coupled with their capacity to deplete pMHCII, represents a novel pathway for T reg cell–mediated suppression and may be a mechanism by which T reg cells maintain immune homeostasis. Regulatory T cells suppress target cells through diverse mechanisms. Shevach and colleagues demonstrate that regulatory T cells in vivo strip complexes of cognate peptide and major histocompatibility complex class II from dendritic cells and thereby help to maintain immune homeostasis.

SignificanceSingle-cell flow cytometry and genomic methods are rapidly increasing our knowledge of the diversity of cell types in metazoan tissues. However, suitably robust methods for placing these cells in a spatial context that reveals how their localization and putative interactions contribute to tissue physiology and pathology are still lacking. Here we provide a readily accessible pipeline (IBEX) for highly multiplex immunofluorescent imaging that enables a fine-grained analysis of cells in their tissue context. Additionally, we describe extensions of the IBEX workflow to handle hard-to-image tissue preparations and a method to facilitate direct integration of the imaging data with flow cytometry and sequencing technologies. The diverse composition of mammalian tissues poses challenges for understanding the cell–cell interactions required for organ homeostasis and how spatial relationships are perturbed during disease. Existing methods such as single-cell genomics, lacking a spatial context, and traditional immunofluorescence, capturing only two to six molecular features, cannot resolve these issues. Imaging technologies have been developed to address these problems, but each possesses limitations that constrain widespread use. Here we report a method that overcomes major impediments to highly multiplex tissue imaging. “Iterative bleaching extends multiplexity” (IBEX) uses an iterative staining and chemical bleaching method to enable high-resolution imaging of >65 parameters in the same tissue section without physical degradation. IBEX can be employed with various types of conventional microscopes and permits use of both commercially available and user-generated antibodies in an “open” system to allow easy adjustment of staining panels based on ongoing marker discovery efforts. We show how IBEX can also be used with amplified staining methods for imaging strongly fixed tissues with limited epitope retention and with oligonucleotide-based staining, allowing potential cross-referencing between flow cytometry, cellular indexing of transcriptomes and epitopes by sequencing, and IBEX analysis of the same tissue. To facilitate data processing, we provide an open-source platform for automated registration of iterative images. IBEX thus represents a technology that can be rapidly integrated into most current laboratory workflows to achieve high-content imaging to reveal the complex cellular landscape of diverse organs and tissues.

B cells are activated by two temporally distinct signals, the first provided by the binding of antigen to the B cell antigen receptor (BCR), and the second provided by helper T cells. Here we found that B cells responded to antigen by rapidly increasing their metabolic activity, including both oxidative phosphorylation and glycolysis. In the absence of a second signal, B cells progressively lost mitochondrial function and glycolytic capacity, which led to apoptosis. Mitochondrial dysfunction was a result of the gradual accumulation of intracellular calcium through calcium response–activated calcium channels that, for approximately 9 h after the binding of B cell antigens, was preventable by either helper T cells or signaling via the receptor TLR9. Thus, BCR signaling seems to activate a metabolic program that imposes a limited time frame during which B cells either receive a second signal and survive or are eliminated. B cells need at least two signals to terminally differentiate into antibody-secreting cells. Pierce and colleagues show that persistent exposure to antigen in the absence of T cell help or ‘pathogen pattern motifs’ leads to B cell death via a calcium-dependent ‘metabolic timer’.

Tissue-resident macrophages are critical for tissue homeostasis and repair. We previously showed that dermis-resident macrophages produce CCL24 which mediates their interaction with IL-4 + eosinophils, required to maintain their M2-like properties in the T H 1 environment of the Leishmania major infected skin. Here, we show that thymic stromal lymphopoietin (TSLP) and IL-5 + type 2 innate lymphoid cells are also required to maintain dermis-resident macrophages and promote infection. Single cell RNA sequencing reveals the dermis-resident macrophages as the sole source of TSLP and CCL24. Generation of Ccl24-cre mice permits specific labeling of dermis-resident macrophages and interstitial macrophages from other organs. Selective ablation of TSLP in dermis-resident macrophages reduces the numbers of IL-5 + type 2 innate lymphoid cells, eosinophils and dermis-resident macrophages, and ameliorates infection. Our findings demonstrate that dermis-resident macrophages are self-maintained as a replicative niche for L. major by orchestrating localized type 2 circuitries with type 2 innate lymphoid cells and eosinophils. Tissue resident macrophages are intricately involved in homeostatic processes but also in tissue repair. Here Lee and colleagues show dermis-resident macrophages are a source of thymic stromal lymphopoietin and CCL24, which act on type 2 innate lymphoid cells and eosinophils respectively, to maintain their M2 properties and promote non-healing cutaneous leishmaniasis.

Delineating the complex network of interactions between antigen-specific T cells and antigen presenting cells (APCs) is crucial for effective precision therapies against cancer, chronic infections, and autoimmunity. However, the existing arsenal for examining antigen-specific T cell interactions is restricted to a select few antigen-T cell receptor pairs, with limited in situ utility. This lack of versatility is largely due to the disruptive effects of reagents on the immune synapse, which hinder real-time monitoring of antigen-specific interactions. To address this limitation, we have developed a novel and versatile immune monitoring strategy by adding a short cysteine-rich tag to antigenic peptides that emits fluorescence upon binding to thiol-reactive biarsenical hairpin compounds. Our findings demonstrate the specificity and durability of the novel antigen-targeting probes during dynamic immune monitoring in vitro and in vivo. This strategy opens new avenues for biological validation of T-cell receptors with newly identified epitopes by revealing the behavior of previously unrecognized antigen-receptor pairs, expanding our understanding of T cell responses.

Hepatitis B virus (HBV)-associated acute liver failure (ALF) is a dramatic clinical syndrome leading to death or liver transplantation in 80% of cases. Due to the extremely rapid clinical course, the difficulties in obtaining liver specimens, and the lack of an animal model, the pathogenesis of ALF remains largely unknown. Here, we performed a comprehensive genetic and functional characterization of the virus and the host in liver tissue from HBV-associated ALF and compared the results with those of classic acute hepatitis B in chimpanzees. In contrast with acute hepatitis B, HBV strains detected in ALF livers displayed highly mutated HBV core antigen (HBcAg), associated with increased HBcAg expression ex vivo, which was independent of viral replication levels. Combined gene and miRNA expression profiling revealed a dominant B cell disease signature, with extensive intrahepatic production of IgM and IgG in germline configuration exclusively targeting HBcAg with subnanomolar affinities, and complement deposition. Thus, HBV ALF appears to be an anomalous T cell-independent, HBV core-driven B cell disease, which results from the rare and unfortunate encounter between a host with an unusual B cell response and an infecting virus with a highly mutated core antigen.

Reassortment of influenza viral RNA (vRNA) segments in co-infected cells can lead to the emergence of viruses with pandemic potential. Replication of influenza vRNA occurs in the nucleus of infected cells, while progeny virions bud from the plasma membrane. However, the intracellular mechanics of vRNA assembly into progeny virions is not well understood. Here we used recent advances in microscopy to explore vRNA assembly and transport during a productive infection. We visualized four distinct vRNA segments within a single cell using fluorescent in situ hybridization (FISH) and observed that foci containing more than one vRNA segment were found at the external nuclear periphery, suggesting that vRNA segments are not exported to the cytoplasm individually. Although many cytoplasmic foci contain multiple vRNA segments, not all vRNA species are present in every focus, indicating that assembly of all eight vRNA segments does not occur prior to export from the nucleus. To extend the observations made in fixed cells, we used a virus that encodes GFP fused to the viral polymerase acidic (PA) protein (WSN PA-GFP) to explore the dynamics of vRNA assembly in live cells during a productive infection. Since WSN PA-GFP colocalizes with viral nucleoprotein and influenza vRNA segments, we used it as a surrogate for visualizing vRNA transport in 3D and at high speed by inverted selective-plane illumination microscopy. We observed cytoplasmic PA-GFP foci colocalizing and traveling together en route to the plasma membrane. Our data strongly support a model in which vRNA segments are exported from the nucleus as complexes that assemble en route to the plasma membrane through dynamic colocalization events in the cytoplasm.

Human papillomaviruses are small DNA viruses that cause chronic infection of cutaneous and mucosal epithelium. In some cases, persistent infection with HPV can result in cancer, and 5% of human cancers are the result of HPV infection. The life cycle of human papillomavirus (HPV) depends on keratinocyte differentiation as the virus modulates and takes advantage of cellular pathways to replicate its genome and assemble viral particles in differentiated cells. Viral genomes are amplified in nuclear replication foci in differentiated keratinocytes, and DNA repair factors from the DNA damage response signaling pathway are recruited to replicate viral DNA. The HPV genome is associated with cellular histones at all stages of the infectious cycle, and here, we show that the histone variant macroH2A1 is bound to the HPV genome and enriched in viral replication foci in differentiated cells. macroH2A1 isoforms play important roles in cellular transcriptional repression, double-strand break repair, and replication stress. The viral E8^E2 protein also binds to the HPV genome and inhibits viral replication and gene expression by recruiting NCoR/SMRT complexes. We show here that E8^E2 and SMRT also localize within replication foci, though independently from macroH2A1. Conversely, transcription complexes containing RNA polymerase II and Brd4 are located on the surface of the foci. Foci generated with an HPV16 E8^E2 mutant genome are not enriched for SMRT or macroH2A1 but contain transcriptional complexes throughout the foci. We propose that both the cellular macroH2A1 protein and viral E8^E2 protein help to spatially separate replication and transcription activities within viral replication foci. IMPORTANCE Human papillomaviruses are small DNA viruses that cause chronic infection of cutaneous and mucosal epithelium. In some cases, persistent infection with HPV can result in cancer, and 5% of human cancers are the result of HPV infection. In differentiated cells, HPV amplifies viral DNA in nuclear replication factories and transcribes late mRNAs to produce capsid proteins. However, very little is known about the spatial organization of these activities in the nucleus. Here, we show that repressive viral and cellular factors localize within the foci to suppress viral transcription, while active transcription takes place on the surface. The cellular histone variant macroH2A1 is important for this spatial organization.

In murine models of visceral leishmaniasis (VL), the parasitization of resident Kupffer cells (resKCs) drives early Leishmania infantum growth in the liver, leading to granuloma formation and subsequent parasite control. Using the chronic VL model, we demonstrate that polyclonal resKCs redistributed to form granulomas outside the sinusoids, creating an open sinusoidal niche that was gradually repopulated by monocyte-derived KCs (moKCs) acquiring a tissue specific, homeostatic profile. Early-stage granulomas predominantly consisted of CLEC4F + KCs. In contrast, late-stage granulomas led to remodeling of the sinusoidal network and contained monocyte-derived macrophages (momacs) along with KCs that downregulated CLEC4F, with both populations expressing iNOS and pro-inflammatory chemokines. During late-stage infection, parasites were largely confined to CLEC4F - KCs. Reduced monocyte recruitment and increased resKCs proliferation in infected Ccr2 −/− mice impaired parasite control. These findings show that the ontogenic heterogeneity of granuloma macrophages is closely linked to granuloma maturation and the development of hepatic immunity in VL. Here, Pessenda et al show that during visceral leishmaniasis, Kupffer cells relocate outside of the liver sinusoids to form granulomas which contain CLEC4F − KCs and monocyte-derived macrophages essential for effective parasite control.

Human Th17/type 17 cells express the chemokine receptor CCR6, but the functions of CCR6 and other chemokine receptors in human type 17 Th cell extravasation have not been fully delineated. Here we show that human peripheral blood CD4 + CCR6 + T cells co-expressing CCR2 have a pathogenic Th17 signature, can produce inflammatory cytokines without T cell receptor activation, and show enhanced expression of pathogenicity-associated and activation-associated genes in the cerebrospinal fluid of patients with multiple sclerosis as compared to controls. In flow chambers with activated endothelial cell (EC) monolayers, CD4 + CCR6 + CCR2 + T cells are efficient at transendothelial migration (TEM). Ligands for CCR5, CCR6 and CXCR3 localize to EC surfaces and mediate only arrest, whereas CCR2 ligands fail to bind well to ECs and mediate only TEM. Conversely, expressing a chimeric CCR2 ligand engineered to bind glycosaminoglycans on ECs results in CCR2-mediated arrest but blocks TEM induction. Our results from human pathogenic-like type 17 cells thus suggest that T cell migration arrest requires chemokine bound to EC surfaces, whereas TEM requires a transendothelial chemokine gradient. Human pathogenic-like Th17 cells express the chemokine receptor CCR6, but how CCR6, together with other chemokine receptors, modulate Th17 functions is still unclear. Here the authors show that CCR2 is coexpressed with CCR6 on these cells, and specifically mediates transendothelial migration, while CCR6 and other chemokine receptors induce T cell arrest on activated endothelial cells.