Activation of regulatory dendritic cells by Mertk coincides with a temporal wave of apoptosis in neonatal lungs.

Multiple mechanisms restrain inflammation in neonates, most likely to prevent tissue damage caused by overly robust immune responses against newly encountered pathogens. Here, we identify a population of pulmonary dendritic cells (DCs) that express intermediate levels of CD103 (CD103int) and appear in the lungs and lung-draining lymph nodes of mice between birth and 2 weeks of age. CD103int DCs express XCR1 and CD205 and require expression of the transcription factor BATF3 for development, suggesting that they belong to the cDC1 lineage. In addition, CD103int DCs express CCR7 constitutively and spontaneously migrate to the lung-draining lymph node, where they promote stromal cell maturation and lymph node expansion. CD103int DCs mature independently of microbial exposure and TRIF- or MyD88-dependent signaling and are transcriptionally related to efferocytic and tolerogenic DCs as well as mature, regulatory DCs. Correlating with this, CD103int DCs show limited ability to stimulate proliferation and IFN-γ production by CD8+ T cells. Moreover, CD103int DCs acquire apoptotic cells efficiently, in a process that is dependent on the expression of the TAM receptor, Mertk, which drives their homeostatic maturation. The appearance of CD103int DCs coincides with a temporal wave of apoptosis in developing lungs and explains, in part, dampened pulmonary immunity in neonatal mice. Together, these data suggest a mechanism by which DCs sense apoptotic cells at sites of noninflammatory tissue remodeling, such as tumors or the developing lungs, and limit local T cell responses.

B cell and antibody targeting of transplant alloantigen epitopes is supported by both self and allo-recognition.

Donor-specific antibody (DSA) responses against human leukocyte antigen (HLA) proteins mismatched between kidney transplant donors and recipients cause allograft loss. Using single-cell, molecular, structural, and proteomic techniques, we profiled the HLA-specific (alloreactive) B cell response in kidney and blood of a transplant recipient with antibody-mediated rejection (AMR). We identified 14 distinct alloreactive B cell lineages, which spanned the rejected organ and blood and expressed high-affinity anti-donor HLA-specific B cell receptors, many of which were clonally linked to circulating DSA. The alloreactive B cell response was focused on exposed, solvent-accessible mismatched HLA residues, while also demonstrating extensive contacts with self-HLA residues. Consistent with structural evidence of self-recognition, measurable self-reactivity by donor-specific B cells was common and positively correlated with anti-donor affinity maturation. Thus, allo- and self-reactive signatures appeared to converge, suggesting that during AMR, the recognition of non-self and breaches of tolerance conspire to produce a pathogenic donor-specific adaptive response.

Spiking SARS-CoV-2 antiviral immunity in the respiratory tract.

The COVID-19 pandemic enabled the successful launch of mRNA-based vaccines that, when given intramuscularly, elicit spike-specific antibodies and prevent severe disease, but do not promote mucosal immunity. New data suggest how to boost systemic immunity and elicit pulmonary immunity in a way that more effectively controls infection and impairs transmission.

A single intranasal administration of AdCOVID protects against SARS-CoV-2 infection in the upper and lower respiratory tracts.

The coronavirus disease 2019 (COVID-19) pandemic has illustrated the critical need for effective prophylactic vaccination to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Intranasal vaccination is an attractive approach for preventing COVID-19 as the nasal mucosa is the site of initial SARS-CoV-2 entry and viral replication prior to aspiration into the lungs. We previously demonstrated that a single intranasal administration of a candidate adenovirus type 5-vectored vaccine encoding the receptor-binding domain of the SARS-CoV-2 spike protein (AdCOVID) induced robust immunity in both the airway mucosa and periphery, and completely protected K18-hACE2 mice from lethal SARS-CoV-2 challenge. Here we show that a single intranasal administration of AdCOVID limits viral replication in the nasal cavity of K18-hACE2 mice. AdCOVID also induces sterilizing immunity in the lungs of mice as reflected by the absence of infectious virus. Finally, AdCOVID prevents SARS-CoV-2 induced pathological damage in the lungs of mice. These data show that AdCOVID not only limits viral replication in the respiratory tract, but it also prevents virus-induced inflammation and immunopathology following SARS-CoV-2 infection.

Circulating Tregs Accumulate in Omental Tumors and Acquire Adipose-Resident Features.

Tumors that metastasize in the peritoneal cavity typically end up in the omental adipose tissue, a particularly immune-suppressive environment that includes specialized adipose-resident regulatory T cells (Treg). Tregs rapidly accumulate in the omentum after tumor implantation and potently suppress antitumor immunity. However, it is unclear whether these Tregs are recruited from the circulation or derived from preexisting adipose-resident Tregs by clonal expansion. Here we show that Tregs in tumor-bearing omenta predominantly have thymus-derived characteristics. Moreover, naïve tumor antigen-specific CD4+ T cells fail to differentiate into Tregs in tumor-bearing omenta. In fact, Tregs derived from the pretumor repertoire are sufficient to suppress antitumor immunity and promote tumor growth. However, tumor implantation in the omentum does not promote Treg clonal expansion, but instead leads to increased clonal diversity. Parabiosis experiments show that despite tissue-resident (noncirculating) characteristics of omental Tregs in naïve mice, tumor implantation promotes a rapid influx of circulating Tregs, many of which come from the spleen. Finally, we show that newly recruited Tregs rapidly acquire characteristics of adipose-resident Tregs in tumor-bearing omenta. These data demonstrate that most Tregs in omental tumors are recruited from the circulation and adapt to their environment by altering their homing, transcriptional, and metabolic properties.

Dissemination of Candida auris to deep organs in neonatal murine invasive candidiasis.

Candida auris is an emerging multidrug resistant fungal pathogen, which represents a major challenge for newborns systemic infections worldwide. Management of C. auris infections is complicated due to its intrinsic antifungal resistance and the limited information available on its pathogenesis, particularly during neonatal period. In this study, we developed a murine model of C. auris neonatal invasive infection. C. auris dissemination was evaluated by fungal burden and histopathological analysis of lung, brain, liver, kidney, and spleen at different time intervals. We found fungal cells in all the analyzed tissues, neonatal liver and brain were the most susceptible tissues to fungal invasion. This model will help to better understand pathogenesis mechanisms and facilitate strategies for control and prevention of C. auris infections in newborns.

Single-Dose Intranasal Administration of AdCOVID Elicits Systemic and Mucosal Immunity against SARS-CoV-2 and Fully Protects Mice from Lethal Challenge.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective prophylactic vaccination to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry. The current intramuscular vaccines elicit systemic immunity but not necessarily high-level mucosal immunity. Here, we tested a single intranasal dose of our candidate adenovirus type 5-vectored vaccine encoding the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (AdCOVID) in inbred, outbred, and transgenic mice. A single intranasal vaccination with AdCOVID elicited a strong and focused immune response against RBD through the induction of mucosal IgA in the respiratory tract, serum neutralizing antibodies, and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. A single AdCOVID dose resulted in immunity that was sustained for over six months. Moreover, a single intranasal dose completely protected K18-hACE2 mice from lethal SARS-CoV-2 challenge, preventing weight loss and mortality. These data show that AdCOVID promotes concomitant systemic and mucosal immunity and represents a promising vaccine candidate.

Distinct innate immune responses between sublethal and lethal models of disseminated candidiasis in newborn BALB/c mice.

Invasive candidiasis is associated with a high incidence and mortality rates in infants, especially in preterm newborns. The immunopathogenesis of the mycosis during the neonatal period is poorly understood. Although several in vivo models exist to study invasive candidiasis, the majority of studies employ distinct routes of infection and use 2 to 6 day-old mice that could be less comparable in studying candidiasis in preterm infants. In this study, by using 0-days-old mice we developed a new neonatal murine model of intravenous Candida albicans infection. Using different inoculums of Candida albicans we evaluated survival, dissemination of the fungus, frequency of CD45+ cells, and cytokine production in the liver, brain, and kidneys of newborn and adult BALB/c mice. Unexpectedly, the newborn mice infected with a low inoculum (1×105 cfu per mouse) of Candida albicans survive to the infection. Compared to adult mice, the liver and brain of newborn animals had the greatest fungal burden, fungal invasion and leukocyte infiltrate. A moderate production of TNFα, IL-1ß, IL-6 and IFNγ was detected in tissues of newborn mice infected with a non-lethal inoculum of Candida albicans. In contrast, overproduction of TNFα, IL-1ß, IL-6 and IL-10 was determined when injecting with a lethal inoculum. In agreement, flow cytometry of brain and liver showed an inoculum-dependent CD45+ leukocyte infiltration in newborn mice infected with Candida albicans. Overall, our data shows that Candida albicans infection in newborn mice affects mainly the brain and liver and a 2-fold increase of the inoculum rapidly becomes lethal probably due to massive fungal invasion and exacerbated CD45+ leukocyte infiltrate and cytokine production. This study is the first analysis of innate immune responses in different tissues during early neonatal disseminated candidiasis.

Intestinal cDC1 drive cross-tolerance to epithelial-derived antigen via induction of FoxP3+CD8+ Tregs.

Although CD8+ T cell tolerance to tissue-specific antigen (TSA) is essential for host homeostasis, the mechanisms underlying peripheral cross-tolerance and whether they may differ between tissue sites remain to be fully elucidated. Here, we demonstrate that peripheral cross-tolerance to intestinal epithelial cell (IEC)-derived antigen involves the generation and suppressive function of FoxP3+CD8+ T cells. FoxP3+CD8+ Treg generation was dependent on intestinal cDC1, whose absence led to a break of tolerance and epithelial destruction. Mechanistically, intestinal cDC1-derived PD-L1, TGFß, and retinoic acid contributed to the generation of gut-tropic CCR9+CD103+FoxP3+CD8+ Tregs Last, CD103-deficient CD8+ T cells lacked tolerogenic activity in vivo, indicating a role for CD103 in FoxP3+CD8+ Treg function. Our results describe a role for FoxP3+CD8+ Tregs in cross-tolerance in the intestine for which development requires intestinal cDC1.

Specialized immune responses in the peritoneal cavity and omentum.

The peritoneal cavity is a fluid filled space that holds most of the abdominal organs, including the omentum, a visceral adipose tissue that contains milky spots or clusters of leukocytes that are organized similar to those in conventional lymphoid tissues. A unique assortment of leukocytes patrol the peritoneal cavity and migrate in and out of the milky spots, where they encounter Ags or pathogens from the peritoneal fluid and respond accordingly. The principal role of leukocytes in the peritoneal cavity is to preserve tissue homeostasis and secure tissue repair. However, when peritoneal homeostasis is disturbed by inflammation, infection, obesity, or tumor metastasis, specialized fibroblastic stromal cells and mesothelial cells in the omentum regulate the recruitment of peritoneal leukocytes and steer their activation in unique ways. In this review, the types of cells that reside in the peritoneal cavity, the role of the omentum in their maintenance and activation, and how these processes function in response to pathogens and malignancy will be discussed.

Single-dose intranasal administration of AdCOVID elicits systemic and mucosal immunity against SARS-CoV-2 in mice.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective preventive vaccination to reduce burden and spread of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) in humans. Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry before viral spread to the lung. Although SARS-CoV-2 vaccine development is rapidly progressing, the current intramuscular vaccines are designed to elicit systemic immunity without conferring mucosal immunity. Here, we show that AdCOVID, an intranasal adenovirus type 5 (Ad5)-vectored vaccine encoding the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, elicits a strong and focused immune response against RBD through the induction of mucosal IgA, serum neutralizing antibodies and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. Therefore, AdCOVID, which promotes concomitant systemic and local mucosal immunity, represents a promising COVID-19 vaccine candidate.

Inducible Bronchus-Associated Lymphoid Tissue (iBALT) Attenuates Pulmonary Pathology in a Mouse Model of Allergic Airway Disease.

Inducible Bronchus Associated Lymphoid Tissue (iBALT) is an ectopic lymphoid tissue associated with severe forms of chronic lung diseases, including chronic obstructive pulmonary disease, rheumatoid lung disease, hypersensitivity pneumonitis and asthma, suggesting that iBALT may exacerbate these clinical conditions. However, despite the link between pulmonary pathology and iBALT formation, the role of iBALT in pathogenesis remains unknown. Here we tested whether the presence of iBALT in the lung prior to sensitization and challenge with a pulmonary allergen altered the biological outcome of disease. We found that the presence of iBALT did not exacerbate Th2 responses to pulmonary sensitization with ovalbumin. Instead, we found that mice with iBALT exhibited delayed Th2 accumulation in the lung, reduced eosinophil recruitment, reduced goblet cell hyperplasia and reduced mucus production. The presence of iBALT did not alter Th2 priming, but instead delayed the accumulation of Th2 cells in the lung following challenge and altered the spatial distribution of T cells in the lung. These results suggest that the formation of iBALT and sequestration of effector T cells in the context of chronic pulmonary inflammation may be a mechanism by which the immune system attenuates pulmonary inflammation and prevents excessive pathology.

Role of iBALT in Respiratory Immunity.

Pulmonary respiration inevitably exposes the mucosal surface of the lung to potentially noxious stimuli, including pathogens, allergens, and particulates, each of which can trigger pulmonary damage and inflammation. As inflammation resolves, B and T lymphocytes often aggregate around large bronchi to form inducible Bronchus-Associated Lymphoid Tissue (iBALT). iBALT formation can be initiated by a diverse array of molecular pathways that converge on the activation and differentiation of chemokine-expressing stromal cells that serve as the scaffolding for iBALT and facilitate the recruitment, retention, and organization of leukocytes. Like conventional lymphoid organs, iBALT recruits naïve lymphocytes from the blood, exposes them to local antigens, in this case from the airways, and supports their activation and differentiation into effector cells. The activity of iBALT is demonstrably beneficial for the clearance of respiratory pathogens; however, it is less clear whether it dampens or exacerbates inflammatory responses to non-infectious agents. Here, we review the evidence regarding the role of iBALT in pulmonary immunity and propose that the final outcome depends on the context of the disease.

Defining Genetic Variation in Widely Used Congenic and Backcrossed Mouse Models Reveals Varied Regulation of Genes Important for Immune Responses.

Genetic variation influences how the genome is interpreted in individuals and in mouse strains used to model immune responses. We developed approaches to utilize next-generation sequencing datasets to identify sequence variation in genes and enhancer elements in congenic and backcross mouse models. We defined genetic variation in the widely used B6-CD45.2 and B6.SJL-CD45.1 congenic model, identifying substantial differences in SJL genetic content retained in B6.SJL-CD45.1 strains on the basis of the vendor source of the mice. Genes encoding PD-1, CD62L, Bcl-2, cathepsin E, and Cxcr4 were within SJL genetic content in at least one vendor source of B6.SJL-CD45.1 mice. SJL genetic content affected enhancer elements, gene regulation, protein expression, and amino acid content in CD4+ T helper 1 cells, and mice infected with influenza showed reduced expression of Cxcr4 on B6.SJL-CD45.1 T follicular helper cells. These findings provide information on experimental variables and aid in creating approaches that account for genetic variables.

Tertiary Lymphoid Structures Among the World of Noncanonical Ectopic Lymphoid Organizations.

Tertiary lymphoid structures (TLOs), also known as ectopic lymphoid structures, are associated with chronic infections and inflammatory diseases. Despite their association with pathology, these structures are actually a normal, albeit transient, component of the immune system and facilitate local immune responses that are meant to mitigate inflammation and resolve infection. Many of the mechanisms controlling the formation and function of tertiary lymphoid structures have been identified, in part by experimentally triggering their formation using defined stimuli under controlled conditions. Here, we introduce the experimental and pathological conditions in which tertiary lymphoid tissues are formed, describe the mechanisms linked to their formation, and discuss their functions in the context of both infection and inflammation.

Immunization of Newborn Mice Accelerates the Architectural Maturation of Lymph Nodes, But AID-Dependent IgG Responses Are Still Delayed Compared to the Adult.

Lymph nodes (LNs) have evolved to maximize antigen (Ag) collection and presentation as well as lymphocyte proliferation and differentiation-processes that are spatially regulated by stromal cell subsets, including fibroblastic reticular cells (FRCs) and follicular dendritic cells (FDCs). Here, we showed that naïve neonatal mice have poorly organized LNs with few B and T cells and undetectable FDCs, whereas adult LNs have numerous B cells and large FDC networks. Interestingly, immunization on the day of birth accelerated B cell accumulation and T cell recruitment into follicles as well as FDC maturation and FRC organization in neonatal LNs. However, compared to adults, the formation of germinal centers was both delayed and reduced following immunization of neonatal mice. Although immunized neonates poorly expressed activation-induced cytidine deaminase (AID), they were able to produce Ag-specific IgGs, but with lower titers than adults. Interestingly, the Ag-specific IgM response in neonates was similar to that in adults. These results suggest that despite an accelerated structural maturation of LNs in neonates following vaccination, the B cell response is still delayed and reduced in its ability to isotype switch most likely due to poor AID expression. Of note, naïve pups born to Ag-immunized mothers had high titers of Ag-specific IgGs from day 0 (at birth). These transferred antibodies confirm a mother-derived coverage to neonates for Ags to which mothers (and most likely neonates) are exposed, thus protecting the neonates while they produce their own antibodies. Finally, the type of Ag used in this study and the results obtained also indicate that T cell help would be operating at this stage of life. Thus, neonatal immune system might not be intrinsically immature but rather evolutionary adapted to cope with Ags at birth.

Fugue G Minor: Getting the Lymph Node Ensemble Together with Circadian Rhythm.

A fugue is characterized by the systematic repetition of a principal theme in simultaneous melodic lines. In this issue of Immunity, Druzd et al. (2017) show that a similar phenomenon occurs in lymph nodes (LNs), in which lymphocyte entry and exit is governed by repetitive circadian rhythms.

Inducible Bronchus-Associated Lymphoid Tissue: Taming Inflammation in the Lung.

Following pulmonary inflammation, leukocytes that infiltrate the lung often assemble into structures known as inducible Bronchus-Associated Lymphoid Tissue (iBALT). Like conventional lymphoid organs, areas of iBALT have segregated B and T cell areas, specialized stromal cells, high endothelial venules, and lymphatic vessels. After inflammation is resolved, iBALT is maintained for months, independently of inflammation. Once iBALT is formed, it participates in immune responses to pulmonary antigens, including those that are unrelated to the iBALT-initiating antigen, and often alters the clinical course of disease. However, the mechanisms that govern immune responses in iBALT and determine how iBALT impacts local and systemic immunity are poorly understood. Here, we review our current understanding of iBALT formation and discuss how iBALT participates in pulmonary immunity.

HIV-1 gp140 epitope recognition is influenced by immunoglobulin DH gene segment sequence.

Complementarity Determining Region 3 of the immunoglobulin (Ig) H chain (CDR-H3) lies at the center of the antigen-binding site where it often plays a decisive role in antigen recognition and binding. Amino acids encoded by the diversity (DH) gene segment are the main component of CDR-H3. Each DH has the potential to rearrange into one of six DH reading frames (RFs), each of which exhibits a characteristic amino acid hydrophobicity signature that has been conserved among jawed vertebrates by natural selection. A preference for use of RF1 promotes the incorporation of tyrosine into CDR-H3 while suppressing the inclusion of hydrophobic or charged amino acids. To test the hypothesis that these evolutionary constraints on DH sequence influence epitope recognition, we used mice with a single DH that has been altered to preferentially use RF2 or inverted RF1. B cells in these mice produce a CDR-H3 repertoire that is enriched for valine or arginine in place of tyrosine. We serially immunized this panel of mice with gp140 from HIV-1 JR-FL isolate and then used enzyme-linked immunosorbent assay (ELISA) or peptide microarray to assess antibody binding to key or overlapping HIV-1 envelope epitopes. By ELISA, serum reactivity to key epitopes varied by DH sequence. By microarray, sera with Ig CDR-H3s enriched for arginine bound to linear peptides with a greater range of hydrophobicity but had a lower intensity of binding than sera containing Ig CDR-H3s enriched for tyrosine or valine. We conclude that patterns of epitope recognition and binding can be heavily influenced by DH germ line sequence. This may help explain why antibodies in HIV-infected patients must undergo extensive somatic mutation in order to bind to specific viral epitopes and achieve neutralization.

ESAT-6 Targeting to DEC205+ Antigen Presenting Cells Induces Specific-T Cell Responses against ESAT-6 and Reduces Pulmonary Infection with Virulent Mycobacterium tuberculosis.

Airways infection with Mycobacterium tuberculosis (Mtb) is contained mostly by T cell responses, however, Mtb has developed evasion mechanisms which affect antigen presenting cell (APC) maturation/recruitment delaying the onset of Ag-specific T cell responses. Hypothetically, bypassing the natural infection routes by delivering antigens directly to APCs may overcome the pathogen's naturally evolved evasion mechanisms, thus facilitating the induction of protective immune responses. We generated a murine monoclonal fusion antibody (α-DEC-ESAT) to deliver Early Secretory Antigen Target (ESAT)-6 directly to DEC205+ APCs and to assess its in vivo effects on protection associated responses (IFN-γ production, in vivo CTL killing, and pulmonary mycobacterial load). Treatment with α-DEC-ESAT alone induced ESAT-6-specific IFN-γ producing CD4+ T cells and prime-boost immunization prior to Mtb infection resulted in early influx (d14 post-infection) and increased IFN-γ+ production by specific T cells in the lungs, compared to scarce IFN-γ production in control mice. In vivo CTL killing was quantified in relevant tissues upon transferring target cells loaded with mycobacterial antigens. During infection, α-DEC-ESAT-treated mice showed increased target cell killing in the lungs, where histology revealed cellular infiltrate and considerably reduced bacterial burden. Targeting the mycobacterial antigen ESAT-6 to DEC205+ APCs before infection expands specific T cell clones responsible for early T cell responses (IFN-γ production and CTL activity) and substantially reduces lung bacterial burden. Delivering mycobacterial antigens directly to APCs provides a unique approach to study in vivo the role of APCs and specific T cell responses to assess their potential anti-mycobacterial functions.