Genomic deletion of Bcl6 differentially affects conventional dendritic cell subsets and compromises Tfh/Tfr/Th17 cell responses.

Conventional dendritic cells (cDC) play key roles in immune induction, but what drives their heterogeneity and functional specialization is still ill-defined. Here we show that cDC-specific deletion of the transcriptional repressor Bcl6 in mice alters the phenotype and transcriptome of cDC1 and cDC2, while their lineage identity is preserved. Bcl6-deficient cDC1 are diminished in the periphery but maintain their ability to cross-present antigen to CD8+ T cells, confirming general maintenance of this subset. Surprisingly, the absence of Bcl6 in cDC causes a complete loss of Notch2-dependent cDC2 in the spleen and intestinal lamina propria. DC-targeted Bcl6-deficient mice induced fewer T follicular helper cells despite a profound impact on T follicular regulatory cells in response to immunization and mounted diminished Th17 immunity to Citrobacter rodentium in the colon. Our findings establish Bcl6 as an essential transcription factor for subsets of cDC and add to our understanding of the transcriptional landscape underlying cDC heterogeneity.

Migratory CD103+CD11b+ cDC2s in Peyer's patches are critical for gut IgA responses following oral immunization.

Induction and regulation of specific intestinal immunoglobulin (Ig)A responses critically depend on dendritic cell (DC) subsets and the T cells they activate in the Peyer's patches (PP). We found that oral immunization with cholera toxin (CT) as an adjuvant resulted in migration-dependent changes in the composition and localization of PP DC subsets with increased numbers of cluster of differentiation (CD)103- conventional DC (cDC)2s and lysozyme-expressing DC (LysoDCs) in the subepithelial dome and of CD103+ cDC2s that expressed CD101 in the T cell zones, while oral ovalbumin (OVA) tolerization was instead associated with greater accumulation of cDC1s and peripherally induced regulatory T cells (pTregs) in this area. Decreased IgA responses were observed after CT-adjuvanted immunization in huCD207DTA mice lacking CD103+ cDC2s, while oral OVA tolerization was inefficient in cDC1-deficient Batf3-/- mice. Using OVA transgenic T cell receptor CD4 T cell adoptive transfer models, we found that co-transferred endogenous wildtype CD4 T cells can hinder the induction of OVA-specific IgA responses through secretion of interleukin-10. CT could overcome this blocking effect, apparently through a modulating effect on pTregs while promoting an expansion of follicular helper T cells. The data support a model where cDC1-induced pTreg normally suppresses PP responses for any given antigen and where CT's oral adjuvanticity effect is dependent on promoting follicular helper T cell responses through induction of CD103+ cDC2s.

IL-23 to see: Gut DCs shine bright in inductive sites.

The cytokine IL-23 plays important roles in intestinal barrier protection and integrity, but is also linked to chronic inflammation. In this issue of JEM, Ohara et al. (https://doi.org/10.1084/jem.20230923) provide clarity on the much-debated question of which cells produce IL-23.

Guidelines for DC preparation and flow cytometry analysis of mouse nonlymphoid tissues.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various nonlymphoid tissues. DC are sentinels of the immune system present in almost every mammalian organ. Since they represent a rare cell population, DC need to be extracted from organs with protocols that are specifically developed for each tissue. This article provides detailed protocols for the preparation of single-cell suspensions from various mouse nonlymphoid tissues, including skin, intestine, lung, kidney, mammary glands, oral mucosa and transplantable tumors. Furthermore, our guidelines include comprehensive protocols for multiplex flow cytometry analysis of DC subsets and feature top tricks for their proper discrimination from other myeloid cells. With this collection, we provide guidelines for in-depth analysis of DC subsets that will advance our understanding of their respective roles in healthy and diseased tissues. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all coauthors, making it an essential resource for basic and clinical DC immunologists.

A self-sustaining layer of early-life-origin B cells drives steady-state IgA responses in the adult gut.

The adult immune system consists of cells that emerged at various times during ontogeny. We aimed to define the relationship between developmental origin and composition of the adult B cell pool during unperturbed hematopoiesis. Lineage tracing stratified murine adult B cells based on the timing of output, revealing that a substantial portion originated within a restricted neonatal window. In addition to B-1a cells, early-life time-stamped B cells included clonally interrelated IgA plasma cells in the gut and bone marrow. These were actively maintained by B cell memory within gut chronic germinal centers and contained commensal microbiota reactivity. Neonatal rotavirus infection recruited recurrent IgA clones that were distinct from those arising by infection with the same antigen in adults. Finally, gut IgA plasma cells arose from the same hematopoietic progenitors as B-1a cells during ontogeny. Thus, a complex layer of neonatally imprinted B cells confer unique antibody responses later in life.

Type I Interferons Promote Germinal Centers Through B Cell Intrinsic Signaling and Dendritic Cell Dependent Th1 and Tfh Cell Lineages.

Type I interferons (IFNs) are essential for antiviral immunity, appear to represent a key component of mRNA vaccine-adjuvanticity, and correlate with severity of systemic autoimmune disease. Relevant to all, type I IFNs can enhance germinal center (GC) B cell responses but underlying signaling pathways are incompletely understood. Here, we demonstrate that a succinct type I IFN response promotes GC formation and associated IgG subclass distribution primarily through signaling in cDCs and B cells. Type I IFN signaling in cDCs, distinct from cDC1, stimulates development of separable Tfh and Th1 cell subsets. However, Th cell-derived IFN-γ induces T-bet expression and IgG2c isotype switching in B cells prior to this bifurcation and has no evident effects once GCs and bona fide Tfh cells developed. This pathway acts in synergy with early B cell-intrinsic type I IFN signaling, which reinforces T-bet expression in B cells and leads to a selective amplification of the IgG2c+ GC B cell response. Despite the strong Th1 polarizing effect of type I IFNs, the Tfh cell subset develops into IL-4 producing cells that control the overall magnitude of the GCs and promote generation of IgG1+ GC B cells. Thus, type I IFNs act on B cells and cDCs to drive GC formation and to coordinate IgG subclass distribution through divergent Th1 and Tfh cell-dependent pathways.

Rotavirus-Induced Expansion of Antigen-Specific CD8 T Cells Does Not Require Signaling via TLR3, MyD88 or the Type I Interferon Receptor.

Rotavirus (RV) infection induces strong adaptive immunity. While protection from reinfection requires humoral immunity, initial clearance of infection depends on cytotoxic CD8 T cells. Type I classical dendritic cells (cDC1) excel at CD8 T cell induction through cross-presentation and are essential for optimal cytotoxicity towards RV. Upon sensing of infection-induced innate immune signals through pattern recognition receptors (PRRs), cumulating in autocrine type I interferon (IFN) signaling, cDC1 mature and migrate to the draining lymph nodes (LNs), where they prime adaptive immune cells. To analyze which PRR pathways lead to robust cytotoxicity in the context of RV infection, we measured RV-specific CD8 T cell priming in mice deficient for Toll-like receptor 3 (TLR3), recognizing double-stranded RNA, or for MyD88, the adapter for all other TLRs and IL-1 family cytokines. Individual TLR3- and MyD88-mediated signaling was not required for the priming of CD8 T cell responses to RV and neither deficiency impacted on RV clearance. Surprisingly, the accumulation of RV-specific CD8 T cells was also not altered in the absence of type I IFN signaling, while their ability to produce IFNγ and granzyme were blunted. Together, this suggests a substantial level of redundancy in the sensing of RV infection and the translation of signals into protective CD8 T cell immunity.

ADP-ribosylating adjuvant reveals plasticity in cDC1 cells that drive mucosal Th17 cell development and protection against influenza virus infection.

Migratory dendritic cells expressing CD103 are the targets for mucosal vaccines. These belong to either of two lineage-restricted subsets, cDC1 or cDC2 cells, which have been linked to priming of functionally distinct CD4 T cells. However, recent studies have identified plasticity in cDC2 cells with overlapping functions with cDC1 cells, while the converse has not been reported. We genetically engineered a vaccine adjuvant platform that targeted the cholera toxin A1 (CTA1) ADP-ribosylating enzyme to CD103+ cDC1 and cDC2 cells using a single-chain antibody (scFv) to CD103. Unexpectedly, intranasal immunization with the CTA1-svFcCD103 adjuvant modified cDC1 cells to effectively prime Th17 cells, a function previously limited to cDC2 cells. In fact, cDC2 cells were dispensible, while cDC1 cells, lacking in Batf3-/- mice, were critical. Following intranasal immunizations isolated cDC1 cells from mLN exclusively promoted Rorgt+ T cells and IL-17, IL-21, and IL-22 production. Strong CD8 T cell responses through antigen cross presentation by cDC1 cells were also observed. Single-cell RNAseq analysis revealed upregulation of Th17-promoting gene signatures in sorted cDC1 cells. Gene expression in isolated cDC2 cells was largely unaffected. Our finding represents a major shift of paradigm as we have documented functional plasticity in cDC1 cells.

The C-terminal peptide of CCL21 drastically augments CCL21 activity through the dendritic cell lymph node homing receptor CCR7 by interaction with the receptor N-terminus.

The endogenous chemokines CCL19 and CCL21 signal via their common receptor CCR7. CCL21 is the main lymph node homing chemokine, but a weak chemo-attractant compared to CCL19. Here we show that the 41-amino acid positively charged peptide, released through C-terminal cleavage of CCL21, C21TP, boosts the immune cell recruiting activity of CCL21 by up to 25-fold and the signaling activity via CCR7 by ~ 100-fold. Such boosting is unprecedented. Despite the presence of multiple basic glycosaminoglycan (GAG) binding motifs, C21TP boosting of CCL21 signaling does not involve interference with GAG mediated cell-surface retention. Instead, boosting is directly dependent on O-glycosylations in the CCR7 N-terminus. As dictated by the two-step binding model, the initial chemokine binding involves interaction of the chemokine fold with the receptor N-terminus, followed by insertion of the chemokine N-terminus deep into the receptor binding pocket. Our data suggest that apart from a role in initial chemokine binding, the receptor N-terminus also partakes in a gating mechanism, which could give rise to a reduced ligand activity, presumably through affecting the ligand positioning. Based on experiments that support a direct interaction of C21TP with the glycosylated CCR7 N-terminus, we propose that electrostatic interactions between the positively charged peptide and sialylated O-glycans in CCR7 N-terminus may create a more accessible version of the receptor and thus guide chemokine docking to generate a more favorable chemokine-receptor interaction, giving rise to the peptide boosting effect.

Minor alterations in the intestinal microbiota composition upon Rotavirus infection do not affect susceptibility to DSS colitis.

Viral triggers at the intestinal mucosa can have multiple global effects on intestinal integrity, causing elevated intestinal barrier strength and relative protection from subsequent inflammatory bowel disease (IBD) induction in various models. As viruses can interfere with the intestinal immune system both directly and indirectly through commensal bacteria, cause-effect relationships are difficult to define. Due to the complexity of putatively causative factors, our understanding of such virus-mediated protection is currently very limited. We here set out to better understand the impact that adult enteric infection with rotavirus (RV) might have on the composition of the intestinal microbiome and on the severity of IBD. We found that RV infection neither induced significant long-lasting microbiota community changes in the small or large intestine nor affected the severity of subsequent dextran sulfate sodium-induced colitis. Hence, adult murine RV infection does not exert lasting effects on intestinal homeostasis.

Rotavirus infection causes mesenteric lymph node hypertrophy independently of type I interferon or TNF-α in mice.

Lymphoid organ hypertrophy is a characteristic feature of acute infection and is considered to enable efficient induction of adaptive immune responses. Accordingly, oral infection with rotavirus induced a robust increase in cellularity in the mesenteric LNs, whose kinetics correlated with viral load and was caused by halted lymphocyte egress and increased recruitment of cells without altered cellular proliferation. Lymphocyte sequestration and mesenteric LN hypertrophy were independent of type 1 IFN receptor signaling or the continuous presence of TNF-α. Our results support previous findings that adaptive immunity toward rotavirus is initiated primarily in the mesenteric LNs and show that type I IFN or TNF-α are not required to coordinate the events involved in the LN response.

Mononuclear phagocyte regulation by the transcription factor Blimp-1 in health and disease.

B lymphocyte-induced maturation protein-1 (Blimp-1), the transcription factor encoded by the gene Prdm1, plays a number of crucial roles in the adaptive immune system, which result in the maintenance of key effector functions of B- and T-cells. Emerging clinical data, as well as mechanistic evidence from mouse studies, have additionally identified critical functions of Blimp-1 in the maintenance of immune homeostasis by the mononuclear phagocyte (MNP) system. Blimp-1 regulation of gene expression affects various aspects of MNP biology, including developmental programmes such as fate decisions of monocytes entering peripheral tissue, and functional programmes such as activation, antigen presentation and secretion of soluble inflammatory mediators. The highly tissue-, subset- and state-specific regulation of Blimp-1 expression in MNPs suggests that Blimp-1 is a dynamic regulator of immune activation, integrating environmental cues to fine-tune the function of innate cells. In this review, we will discuss the current knowledge regarding Blimp-1 regulation and function in macrophages and dendritic cells.

Migration of murine intestinal dendritic cell subsets upon intrinsic and extrinsic TLR3 stimulation.

Initiation of adaptive immunity to particulate antigens in lymph nodes largely depends on their presentation by migratory dendritic cells (DCs). DC subsets differ in their capacity to induce specific types of immunity, allowing subset-specific DC-targeting to influence vaccination and therapy outcomes. Faithful drug design, however, requires exact understanding of subset-specific versus global activation mechanisms. cDC1, the subset of DCs that excel in supporting immunity toward viruses, intracellular bacteria, and tumors, express uniquely high levels of the pattern recognition receptor TLR3. Using various murine genetic models, we show here that both, the cDC1 and cDC2 subsets of cDCs are activated and migrate equally well in response to TLR3 stimulation in a cell extrinsic and TNF-α dependent manner, but that cDC1 show a unique requirement for type I interferon signaling. Our findings reveal common and differing pathways regulating DC subset migration, offering important insights for the design of DC-based vaccination and therapy approaches.

Divergent T follicular helper cell requirement for IgA and IgE production to peanut during allergic sensitization.

Immunoglobulin A (IgA) is the dominant antibody isotype in the gut and has been shown to regulate microbiota. Mucosal IgA is also widely believed to prevent food allergens from penetrating the gut lining. Even though recent work has elucidated how bacteria-reactive IgA is induced, little is known about how IgA to food antigens is regulated. Although IgA is presumed to be induced in a healthy gut at steady state via dietary exposure, our data do not support this premise. We found that daily food exposure only induced low-level, cross-reactive IgA in a minority of mice. In contrast, induction of significant levels of peanut-specific IgA strictly required a mucosal adjuvant. Although induction of peanut-specific IgA required T cells and CD40L, it was T follicular helper (TFH) cell, germinal center, and T follicular regulatory (TFR) cell-independent. In contrast, IgG1 and IgE production to peanut required TFH cells. These data suggest an alternative paradigm in which the cellular mechanism of IgA production to food antigens is distinct from IgE and IgG1. We developed an equivalent assay to study this process in stool samples from healthy, nonallergic humans, which revealed substantial levels of peanut-specific IgA that were stable over time. Similar to mice, patients with loss of CD40L function had impaired titers of gut peanut-specific IgA. This work challenges two widely believed but untested paradigms about antibody production to dietary antigens: (i) the steady state/tolerogenic response to food antigens includes IgA production and (ii) TFH cells drive food-specific gut IgA.

Extraction and analysis of signatures from the Gene Expression Omnibus by the crowd.

Gene expression data are accumulating exponentially in public repositories. Reanalysis and integration of themed collections from these studies may provide new insights, but requires further human curation. Here we report a crowdsourcing project to annotate and reanalyse a large number of gene expression profiles from Gene Expression Omnibus (GEO). Through a massive open online course on Coursera, over 70 participants from over 25 countries identify and annotate 2,460 single-gene perturbation signatures, 839 disease versus normal signatures, and 906 drug perturbation signatures. All these signatures are unique and are manually validated for quality. Global analysis of these signatures confirms known associations and identifies novel associations between genes, diseases and drugs. The manually curated signatures are used as a training set to develop classifiers for extracting similar signatures from the entire GEO repository. We develop a web portal to serve these signatures for query, download and visualization.

Selective Treg reconstitution during lymphopenia normalizes DC costimulation and prevents graft-versus-host disease.

Regulatory T cells (Tregs) have been shown to enhance immune reconstitution and prevent graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation; however, it is unclear how Tregs mediate these effects. Here, we developed a model to examine the mechanism of Treg-dependent regulation of immune reconstitution. Lymphopenic mice were selectively reconstituted with Tregs prior to transfer of conventional CD4+ T cells. Full Treg reconstitution prevented the rapid oligoclonal proliferation that gives rise to pathogenic CD4 effector T cells, while preserving the slow homeostatic form of lymphopenia-induced peripheral expansion that repopulates a diverse peripheral T cell pool. Treg-mediated CTLA-4-dependent downregulation of CD80/CD86 on DCs was critical for inhibition of rapid proliferation and was a function of the Treg/DC ratio achieved by reconstitution. In an allogeneic BM transplant model, selective Treg reconstitution before T cell transfer also normalized DC costimulation and provided complete protection against GVHD. In contrast, cotransfer of Tregs was not protective. Our results indicate that achieving optimal recovery from lymphopenia should aim to improve early Treg reconstitution in order to increase the relative number of Tregs to DCs and thereby inhibit spontaneous oligoclonal T cell proliferation.

Selective Expression of the MAPK Phosphatase Dusp9/MKP-4 in Mouse Plasmacytoid Dendritic Cells and Regulation of IFN-ß Production.

Plasmacytoid dendritic cells (pDCs) efficiently produce large amounts of type I IFN in response to TLR7 and TLR9 ligands, whereas conventional DCs (cDCs) predominantly secrete high levels of the cytokines IL-10 and IL-12. The molecular basis underlying this distinct phenotype is not well understood. In this study, we identified the MAPK phosphatase Dusp9/MKP-4 by transcriptome analysis as selectively expressed in pDCs, but not cDCs. We confirmed the constitutive expression of Dusp9 at the protein level in pDCs generated in vitro by culture with Flt3 ligand and ex vivo in sorted splenic pDCs. Dusp9 expression was low in B220(-) bone marrow precursors and was upregulated during pDC differentiation, concomitant with established pDC markers. Higher expression of Dusp9 in pDCs correlated with impaired phosphorylation of the MAPK ERK1/2 upon TLR9 stimulation. Notably, Dusp9 was not expressed at detectable levels in human pDCs, although these displayed similarly impaired activation of ERK1/2 MAPK compared with cDCs. Enforced retroviral expression of Dusp9 in mouse GM-CSF-induced cDCs increased the expression of TLR9-induced IL-12p40 and IFN-ß, but not of IL-10. Conditional deletion of Dusp9 in pDCs was effectively achieved in Dusp9(flox/flox); CD11c-Cre mice at the mRNA and protein levels. However, the lack of Dusp9 in pDC did not restore ERK1/2 activation after TLR9 stimulation and only weakly affected IFN-ß and IL-12p40 production. Taken together, our results suggest that expression of Dusp9 is sufficient to impair ERK1/2 activation and enhance IFN-ß expression. However, despite selective expression in pDCs, Dusp9 is not essential for high-level IFN-ß production by these cells.

Advantages of Foxp3(+) regulatory T cell depletion using DEREG mice.

Several mechanisms enable immunological self-tolerance. Regulatory T cells (Tregs) are a specialized T cell subset that prevents autoimmunity and excessive immune responses, but can also mediate detrimental tolerance to tumors and pathogens in a Foxp3-dependent manner. Genetic tools exploiting the foxp3 locus including bacterial artificial chromosome (BAC)-transgenic DEREG mice have provided essential information on Treg biology and the potential therapeutic modulation of tolerance. In DEREG mice, Foxp3(+) Tregs selectively express eGFP and diphtheria toxin (DT) receptor, allowing for the specific depletion of Tregs through DT administration. We here provide a detailed overview about important considerations such as DT toxicity, which affects any mouse strain treated with DT, and Treg rebound after depletion. Additionally, we point out the specific advantages of BAC-transgenic DEREG mice including their suitability to study organ-specific autoimmunity such as type I diabetes. Moreover, we discuss recent insights into the role of Tregs in viral infections. In summary, DEREG mice are an important tool to study Treg-mediated tolerance and its therapeutic circumvention.

Orphan chemoattractant receptor GPR15 mediates dendritic epidermal T-cell recruitment to the skin.

Homing of murine dendritic epidermal T cells (DETCs) from the thymus to the skin is regulated by specific trafficking receptors during late embryogenesis. Once in the epidermis, Vγ3δ1 TCR DETCs are maintained through self-renewal and participate in wound healing. GPR15 is an orphan G protein-linked chemoattractant receptor involved in the recruitment of regulatory T cells to the colon. Here we show that GPR15 is highly expressed on fetal thymic DETC precursors and on recently recruited DETCs, and mediates the earliest seeding of the epidermis, which occurs at the time of establishment of skin barrier function. DETCs in GPR15(-/-) mice remain low at birth, but later participation of CCR10 and CCR4 in DETC homing allows DETCs to reach near normal levels in adult skin. Our findings establish a role for GPR15 in skin lymphocyte homing and suggest that it may contribute to lymphocyte subset targeting to diverse epithelial sites.

Comparative transcriptional and functional profiling defines conserved programs of intestinal DC differentiation in humans and mice.

Dendritic cells (DCs) that orchestrate mucosal immunity have been studied in mice. Here we characterized human gut DC populations and defined their relationship to previously studied human and mouse DCs. CD103(+)Sirpα(-) DCs were related to human blood CD141(+) DCs and to mouse intestinal CD103(+)CD11b(-) DCs and expressed markers of cross-presenting DCs. CD103(+)Sirpα(+) DCs aligned with human blood CD1c(+) DCs and mouse intestinal CD103(+)CD11b(+) DCs and supported the induction of regulatory T cells. Both CD103(+) DC subsets induced the TH17 subset of helper T cells, while CD103(-)Sirpα(+) DCs induced the TH1 subset of helper T cells. Comparative analysis of transcriptomes revealed conserved transcriptional programs among CD103(+) DC subsets and identified a selective role for the transcriptional repressors Bcl-6 and Blimp-1 in the specification of CD103(+)CD11b(-) DCs and intestinal CD103(+)CD11b(+) DCs, respectively. Our results highlight evolutionarily conserved and divergent programming of intestinal DCs.