Long-term tolerance to skin commensals is established neonatally through a specialized dendritic cell subgroup.

Early-life establishment of tolerance to commensal bacteria at barrier surfaces carries enduring implications for immune health but remains poorly understood. Here, we showed that tolerance in skin was controlled by microbial interaction with a specialized subset of antigen-presenting cells. More particularly, CD301b+ type 2 conventional dendritic cells (DCs) in neonatal skin were specifically capable of uptake and presentation of commensal antigens for the generation of regulatory T (Treg) cells. CD301b+ DC2 were enriched for phagocytosis and maturation programs, while also expressing tolerogenic markers. In both human and murine skin, these signatures were reinforced by microbial uptake. In contrast to their adult counterparts or other early-life DC subsets, neonatal CD301b+ DC2 highly expressed the retinoic-acid-producing enzyme, RALDH2, the deletion of which limited commensal-specific Treg cell generation. Thus, synergistic interactions between bacteria and a specialized DC subset critically support early-life tolerance at the cutaneous interface.

CD301b(+) Mononuclear Phagocytes Maintain Positive Energy Balance through Secretion of Resistin-like Molecule Alpha.

Mononuclear phagocytes (MNPs) are a highly heterogeneous group of cells that play important roles in maintaining the body's homeostasis. Here, we found CD301b (also known as MGL2), a lectin commonly used as a marker for alternatively activated macrophages, was selectively expressed by a subset of CD11b(+)CD11c(+)MHCII(+) MNPs in multiple organs including adipose tissues. Depleting CD301b(+) MNPs in vivo led to a significant weight loss with increased insulin sensitivity and a marked reduction in serum Resistin-like molecule (RELM) α, a multifunctional cytokine produced by MNPs. Reconstituting RELMα in CD301b(+) MNP-depleted animals restored body weight and normoglycemia. Thus, CD301b(+) MNPs play crucial roles in maintaining glucose metabolism and net energy balance.

Candida albicans morphology and dendritic cell subsets determine T helper cell differentiation.

Candida albicans is a dimorphic fungus responsible for chronic mucocutaneous and systemic infections. Mucocutaneous immunity to C. albicans requires T helper 17 (Th17) cell differentiation that is thought to depend on recognition of filamentous C. albicans. Systemic immunity is considered T cell independent. Using a murine skin infection model, we compared T helper cell responses to yeast and filamentous C. albicans. We found that only yeast induced Th17 cell responses through a mechanism that required Dectin-1-mediated expression of interleukin-6 (IL-6) by Langerhans cells. Filamentous forms induced Th1 without Th17 cell responses due to the absence of Dectin-1 ligation. Notably, Th17 cell responses provided protection against cutaneous infection while Th1 cell responses provided protection against systemic infection. Thus, C. albicans morphology drives distinct T helper cell responses that provide tissue-specific protection. These findings provide insight into compartmentalization of Th cell responses and C. albicans pathogenesis and have critical implications for vaccine strategies.

CD301b⁺ dermal dendritic cells drive T helper 2 cell-mediated immunity.

Unlike other types of T helper (Th) responses, whether the development of Th2 cells requires instruction from particular subset of dendritic cells (DCs) remains unclear. By using an in vivo depletion approach, we have shown that DCs expressing CD301b were required for the generation of Th2 cells after subcutaneous immunization with ovalbumin (OVA) along with papain or alum. CD301b⁺ DCs are distinct from epidermal or CD207⁺ dermal DCs (DDCs) and were responsible for transporting antigen injected subcutaneously with Th2-type adjuvants. Transient depletion of CD301b⁺ DCs resulted in less effective accumulation and decreased expression of CD69 by polyclonal CD4⁺ T cells in the lymph node. Moreover, despite intact cell division and interferon-γ production, CD301b⁺ DC depletion led to blunted interleukin-4 production by OVA-specific OT-II transgenic CD4⁺ T cells and significantly impaired Th2 cell development upon infection with Nippostrongylus brasiliensis. These results reveal CD301b⁺ DDCs as the key mediators of Th2 immunity.

CD4+ T cells support cytotoxic T lymphocyte priming by controlling lymph node input.

Rapid induction of CD8(+) cytotoxic T lymphocyte (CTL) responses is critical to combat acute infection with intracellular pathogens. CD4(+) T cells help prime antigen-specific CTLs in secondary lymphoid organs after infection in the periphery. Although the frequency of naïve precursors is very low, the immune system is able to efficiently screen for cognate CTLs through mechanisms that are not well understood. Here we examine the role of CD4(+) T cells in early phases of the immune response. We show that CD4(+) T cells help optimal CTL expansion by facilitating entry of naïve polyclonal CD8(+) T cells into the draining lymph node (dLN) early after infection or immunization. CD4(+) T cells also facilitate input of naïve B cells into reactive LNs. Such "help" involves expansion of the arteriole feeding the dLN and enlargement of the dLN through activation of dendritic cells. In an antigen- and CD40-dependent manner, CD4(+) T cells activate dendritic cells to support naïve lymphocyte recruitment to the dLN. Our results reveal a previously unappreciated mode of CD4(+) T-cell help, whereby they increase the input of naïve lymphocytes to the relevant LN for efficient screening of cognate CD8(+) T cells.

Microbiota regulates immune defense against respiratory tract influenza A virus infection.

Although commensal bacteria are crucial in maintaining immune homeostasis of the intestine, the role of commensal bacteria in immune responses at other mucosal surfaces remains less clear. Here, we show that commensal microbiota composition critically regulates the generation of virus-specific CD4 and CD8 T cells and antibody responses following respiratory influenza virus infection. By using various antibiotic treatments, we found that neomycin-sensitive bacteria are associated with the induction of productive immune responses in the lung. Local or distal injection of Toll-like receptor (TLR) ligands could rescue the immune impairment in the antibiotic-treated mice. Intact microbiota provided signals leading to the expression of mRNA for pro-IL-1ß and pro-IL-18 at steady state. Following influenza virus infection, inflammasome activation led to migration of dendritic cells (DCs) from the lung to the draining lymph node and T-cell priming. Our results reveal the importance of commensal microbiota in regulating immunity in the respiratory mucosa through the proper activation of inflammasomes.

Innate cells and STAT1-dependent signals orchestrate vaccine-induced protection against invasive Cryptococcus infection.

Fungal pathogens are underappreciated causes of significant morbidity and mortality worldwide. In previous studies, we determined that a heat-killed, Cryptococcus neoformans fbp1-deficient strain (HK-fbp1) is a potent vaccine candidate. We determined that vaccination with HK-fbp1 confers protective immunity against lethal Cryptococcosis in an interferon γ (IFNγ)-dependent manner. In this study, we set out to uncover cellular sources and relevant targets of the protective effects of IFNγ in response to the HK-fbp1 vaccine. We found that early IFNγ production peaks at day 3 and that monocytes and neutrophils are important sources of this cytokine after vaccination. Neutralization of IFNγ at day 3 results in impaired CCR2+ monocyte recruitment and reduced differentiation into monocyte-derived dendritic cells (Mo-DC). In turn, depletion of CCR2+ cells prior to immunization results in impaired activation of IFNγ-producing CD4 and CD8 T cells. Thus, monocytes are important targets of innate IFNγ and help promote further IFNγ production by lymphocytes. We employed monocyte-fate mapper and conditional STAT1 knockout mice to uncover that STAT1 activation in CD11c+ cells, including alveolar macrophages, Mo-DCs, and monocyte-derived macrophages (Mo-Mac) is essential for HK-fbp1 vaccine-induced protection. Altogether, our aggregate findings suggest critical roles for innate cells as orchestrators of vaccine-induced protection against Cryptococcus infection.IMPORTANCEThe number of patients susceptible to invasive fungal infections across the world continues to rise at an alarming pace yet current antifungal drugs are often inadequate. Immune-based interventions and novel antifungal vaccines hold the promise of significantly improving patient outcomes. In previous studies, we identified a Cryptococcus neoformans mutant strain (Fbp1-deficient) as a potent, heat-inactivated vaccine candidate capable of inducing homologous and heterologous antifungal protection. In this study, we used a combination of methods together with a cohort of conditional knockout mouse strains to interrogate the roles of innate cells in the orchestration of vaccine-induced antifungal protection. We uncovered novel roles for neutrophils and monocytes as coordinators of a STAT1-dependent cascade of responses that mediate vaccine-induced protection against invasive cryptococcosis. This new knowledge will help guide the future development of much-needed antifungal vaccines.

Helminth protein enhances wound healing by inhibiting fibrosis and promoting tissue regeneration.

Skin wound healing due to full thickness wounds typically results in fibrosis and scarring, where parenchyma tissue is replaced with connective tissue. A major advance in wound healing research would be to instead promote tissue regeneration. Helminth parasites express excretory/secretory (ES) molecules, which can modulate mammalian host responses. One recently discovered ES protein, TGF-ß mimic (TGM), binds the TGF-ß receptor, though likely has other activities. Here, we demonstrate that topical administration of TGM under a Tegaderm bandage enhanced wound healing and tissue regeneration in an in vivo wound biopsy model. Increased restoration of normal tissue structure in the wound beds of TGM-treated mice was observed during mid- to late-stage wound healing. Both accelerated re-epithelialization and hair follicle regeneration were observed. Further analysis showed differential expansion of myeloid populations at different wound healing stages, suggesting recruitment and reprogramming of specific macrophage subsets. This study indicates a role for TGM as a potential therapeutic option for enhanced wound healing.

Role of mouse dendritic cell subsets in priming naive CD4 T cells.

Conventional dendritic cells (cDCs) are potent antigen-presenting cells that consist of developmentally, phenotypically, and functionally distinct subsets. Following immunization, each subset of cDCs acquires the antigen and presents it to CD4T (CD4+ T (cells)) cells with distinct spatiotemporal kinetics in the secondary lymphoid organs, often causing multiple waves of antigen presentation to CD4T cells. Here, we review the current understanding of the kinetics of antigen presentation by each cDC subset and its functional consequences in priming naive CD4T cells, and discuss its implications in the differentiation of CD4T cells.

Rapid activation of IL-2 receptor signaling by CD301b+ DC-derived IL-2 dictates the outcome of helper T cell differentiation.

Effector T helper (Th) cell differentiation is fundamental to functional adaptive immunity. Different subsets of dendritic cells (DCs) preferentially induce different types of Th cells, but the fate instruction mechanism for Th type 2 (Th2) differentiation remains enigmatic, as the critical DC-derived cue has not been clearly identified. Here, we show that CD301b+ DCs, a major Th2-inducing DC subset, drive Th2 differentiation through cognate interaction by 'kick-starting' IL-2 receptor signaling in CD4T cells. Mechanistically, CD40 engagement induces IL-2 production selectively from CD301b+ DCs to maximize CD25 expression in CD4 T cells, which is required specifically for the Th2 fate decision. On the other hand, CD25 in CD301b+ DCs facilitates directed action of IL-2 toward cognate CD4T cells. Furthermore, CD301b+ DC-derived IL-2 skews CD4T cells away from the T follicular helper fate. These results highlight the critical role of DC-intrinsic CD40-IL-2 axis in bifurcation of Th cell fate.

Protocol to quantify and characterize contact between T cells and antigen-presenting cells in the antigen-draining lymph nodes of mice using flow cytometry.

Physical contact between T cells and antigen-presenting cells (APCs) is essential for priming antigen-specific T cells, but quantitating the antigen-dependent T cell-APC contact can be laborious. Here, we present a simple flow-cytometry-based protocol for quantitating T cell-APC contacts in the antigen-draining lymph node in mice immunized with ovalbumin (OVA). This protocol quantifies the contact between adoptively transferred OVA-specific TCR transgenic CD4T (OT-II) cells and dendritic cell (DC) subsets. This approach can be applied to other types of intercellular interactions between T cells and APCs. For complete details on the use and execution of this protocol, please refer to Tatsumi et al. (2021).1.

Distribution and function of macrophage galactose-type C-type lectin 2 (MGL2/CD301b): efficient uptake and presentation of glycosylated antigens by dendritic cells.

Dendritic cells (DCs) express cell surface lectins that are potentially involved in the recognition, uptake, and presentation of glycosylated foreign substances. A unique calcium-type (C-type) lectin, the macrophage galactose (Gal)-type C-type lectin (MGL/CD301) expressed on DCs, is thought to participate in the recognition of molecules from both altered self and pathogens due to its monosaccharide specificity for Gal and N-acetylgalactosamine (GalNAc). Although mice have two MGL genes, Mgl1 and Mgl2, their distinct roles have not been previously explored. The present report characterizes the properties of MGL2 by examining its distribution and its role in antigen presentation by DCs. We generated an MGL2-specific monoclonal antibody and examined MGL2 expression in tissues by immunohistochemistry and in isolated cells by flow cytometry. The cells reactive with this antibody were shown to be a portion of MGL1-expressing cells, mostly conventional DCs. Internalization of soluble polyacrylamide polymers (PAA) with alpha-GalNAc residues (GalNAc-PAA) by bone marrow-derived DCs (BM-DCs) was mediated by MGL2, as revealed by a comparison of Mgl1(-/-) and Mgl2(-/-) BM-DCs with wild-type BM-DCs. Biotinylated GalNAc-PAA conjugated to streptavidin (SAv) was more efficiently presented to SAv-primed T cells by BM-DCs than beta-N-acetylglucosamine-PAA conjugated to SAv or SAv alone as shown by thymidine uptake and cytokine production. This is the first report that demonstrates the involvement of GalNAc residues in antigen uptake and presentation by DCs that lead to CD4(+) T cell activation.

Effective CD4 T cell priming requires repertoire scanning by CD301b+ migratory cDC2 cells upon lymph node entry.

During the initiation of adaptive immune responses, millions of lymphocytes must be scanned to find the few cognate clones. The activation mechanisms of CD4 T cells have been extensively studied, but the cellular mechanisms that drive selection of cognate clones are not completely understood. Here, we show that recently homed naïve polyclonal CD4 T cells are temporarily retained before leaving the lymph node. This stop-and-go traffic of CD4 T cells provides an adequate time window for efficient scanning and timely priming of antigen-specific cognate clones. CD301b+ DCs, a major subset of migratory cDC2 cells, localize to the areas around high endothelial venules, where they retain incoming polyclonal CD4 T cells through MHCII-dependent but antigen-independent mechanisms, while concurrently providing cognate stimuli for priming. These results indicate that CD301b+ DCs function as an immunological "display window" for CD4 T cells to efficiently scan their antigen specificity.

Regulation of immature dendritic cell migration by RhoA guanine nucleotide exchange factor Arhgef5.

There are a large number of Rho guanine nucleotide exchange factors, most of which have no known functions. Here, we carried out a short hairpin RNA-based functional screen of Rho-GEFs for their roles in leukocyte chemotaxis and identified Arhgef5 as an important factor in chemotaxis of a macrophage phage-like RAW264.7 cell line. Arhgef5 can strongly activate RhoA and RhoB and weakly RhoC and RhoG, but not Rac1, RhoQ, RhoD, or RhoV, in transfected human embryonic kidney 293 cells. In addition, Gbetagamma interacts with Arhgef5 and can stimulate Arhgef5-mediated activation of RhoA in an in vitro assay. In vivo roles of Arhgef5 were investigated using an Arhgef-5-null mouse line. Arhgef5 deficiency did not affect chemotaxis of mouse macrophages, T and B lymphocytes, and bone marrow-derived mature dendritic cells (DC), but it abrogated MIP1alpha-induced chemotaxis of immature DCs and impaired migration of DCs from the skin to lymph node. In addition, Arhgef5 deficiency attenuated allergic airway inflammation. Therefore, this study provides new insights into signaling mechanisms for DC migration regulation.

CD301b+ dendritic cells stimulate tissue-resident memory CD8+ T cells to protect against genital HSV-2.

Tissue-resident memory CD8+ T (CD8 TRM) cells are an essential component of protective immune responses at barrier tissues, including the female genital tract. However, the mechanisms that lead to the initiation of CD8 TRM-mediated protective immunity after viral infection are unclear. Here we report that CD8 TRM cells established by 'prime and pull' method confer protection against genital HSV-2 infection, and that IFN-γ produced by CD8 TRM cells is required for this protection. Furthermore, we find that CD8 TRM-cell restimulation depends on a population of CD301b+ antigen-presenting cells (APC) in the lamina propria. Elimination of MHC class I on CD301b+ dendritic cells abrogates protective immunity, suggesting the requirement for cognate antigen presentation to CD8 TRM cells by CD301b+ dendritic cells. These results define the requirements for CD8 TRM cells in protection against genital HSV-2 infection and identify the population of APC that are responsible for activating these cells.

CD301b+ dendritic cells suppress T follicular helper cells and antibody responses to protein antigens.

Strong antibody response is considered a hallmark of a successful vaccine. While dendritic cells (DCs) are important for T follicular helper (Tfh) cell priming, how this process is regulated in vivo is unclear. We show here that the depletion of CD301b+ DCs specifically enhanced the development of Tfh cells, germinal center B cells and antibody responses against protein antigens. Exaggerated antibody responses in mice depleted of CD301b+ DCs occurred in the absence of any adjuvants, and resulting antibodies had broader specificity and higher affinity to the immunogen. CD301b+ DCs express high levels of PD-1 ligands, PD-L1 and PD-L2. Blocking PD-1 or PD-L1 during priming in wild-type mice partially mimicked the phenotype of CD301b+ DC-depleted animals, suggesting their role in Tfh suppression. Transient depletion of CD301b+ DC results in the generation of autoreactive IgG responses. These results revealed a novel regulatory mechanism and a key role of CD301b+ DCs in blocking autoantibody generation.

CD301b+ Macrophages Are Essential for Effective Skin Wound Healing.

Regeneration of skin's barrier function after injury requires temporally coordinated cellular interactions between multiple cell types. Macrophages are essential inflammatory cells in skin wound regeneration. These cells switch their phenotype from inflammatory in the early regenerative stages to anti-inflammatory in the midstages of healing to coordinate skin repair. However, little is known about how different subsets of anti-inflammatory macrophages contribute to skin wound healing. Here, we characterize midstage macrophages (CD45(+)/CD11b(+)/F4-80(+)) and identify two major populations: CD206(+)/CD301b(+) and CD206(+)/CD301b(-). The numbers of CD206(+)/CD301b(+) macrophages increased concomitantly with repair, when the anti-inflammatory phenotype switch occurs in midstage healing. Using diphtheria toxin-mediated depletion models in mice, we show that selective depletion of midstage CD301b-expressing macrophages phenocopied wound healing defects observed in mice where multiple myeloid lineages are depleted. Additionally, when FACS-isolated subpopulations of myeloid cells were transplanted into 3-day wounds of syngeneic mice, only CD206(+)/CD301b(+) macrophages significantly increased proliferation and fibroblast repopulation. These data show that the CD301b-expressing subpopulation of macrophages is critical for activation of reparative processes during the midstage of cutaneous repair.

Unique features of antiviral immune system of the vaginal mucosa.

A vast majority of human vaccines rely on neutralizing antibodies for protection. With the exception of vaccines against human papillomavirus, despite a great amount of dedicated effort by the scientific community, development of vaccines against sexually transmitted viruses has generally been unsuccessful. Understanding the immunobiology of the genital tract is key to designing vaccines that prevent spreading of these viruses. Recent studies demonstrate that adaptive immunity in the vaginal mucosa is uniquely regulated compared to other mucosal organs. In particular, development of virus-specific CD4+ and CD8+ T cells is critically important for antiviral defense in vagina. In this review, we provide an overview of our current understanding of a wide spectrum of immune responses in vagina--from innate viral sensing to memory development.