Mast cells help organize the Peyer's patch niche for induction of IgA responses.

Peyer's patches (PPs) are lymphoid structures situated adjacent to the intestinal epithelium that support B cell responses that give rise to many intestinal IgA-secreting cells. Induction of isotype switching to IgA in PPs requires interactions between B cells and TGFß-activating conventional dendritic cells type 2 (cDC2s) in the subepithelial dome (SED). However, the mechanisms promoting cDC2 positioning in the SED are unclear. Here, we found that PP cDC2s express GPR35, a receptor that promotes cell migration in response to various metabolites, including 5-hydroxyindoleacetic acid (5-HIAA). In mice lacking GPR35, fewer cDC2s were found in the SED, and frequencies of IgA+ germinal center (GC) B cells were reduced. IgA plasma cells were reduced in both the PPs and lamina propria. These phenotypes were also observed in chimeric mice that lacked GPR35 selectively in cDCs. GPR35 deficiency led to reduced coating of commensal bacteria with IgA and reduced IgA responses to cholera toxin. Mast cells were present in the SED, and mast cell-deficient mice had reduced PP cDC2s and IgA+ cells. Ablation of tryptophan hydroxylase 1 (Tph1) in mast cells to prevent their production of 5-HIAA similarly led to reduced PP cDC2s and IgA responses. Thus, mast cell-guided positioning of GPR35+ cDC2s in the PP SED supports induction of intestinal IgA responses.

Radial glia promote microglial development through integrin αVß8 -TGFß1 signaling.

Microglia diversity emerges from interactions between intrinsic genetic programs and environment-derived signals, but how these processes unfold and interact in the developing brain remains unclear. Here, we show that radial glia-expressed integrin beta 8 (ITGB8) expressed in radial glia progenitors activates microglia-expressed TGFß1, permitting microglial development. Domain-restricted deletion of Itgb8 in these progenitors establishes complementary regions with developmentally arrested "dysmature" microglia that persist into adulthood. In the absence of autocrine TGFß1 signaling, we find that microglia adopt a similar dysmature phenotype, leading to neuromotor symptoms almost identical to Itgb8 mutant mice. In contrast, microglia lacking the TGFß signal transducers Smad2 and Smad3 have a less polarized dysmature phenotype and correspondingly less severe neuromotor dysfunction. Finally, we show that non-canonical (Smad-independent) signaling partially suppresses disease and development associated gene expression, providing compelling evidence for the adoption of microglial developmental signaling pathways in the context of injury or disease.

Severe COVID-19 patients have impaired plasmacytoid dendritic cell-mediated control of SARS-CoV-2.

Type I and III interferons (IFN-I/λ) are important antiviral mediators against SARS-CoV-2 infection. Here, we demonstrate that plasmacytoid dendritic cells (pDC) are the predominant IFN-I/λ source following their sensing of SARS-CoV-2-infected cells. Mechanistically, this short-range sensing by pDCs requires sustained integrin-mediated cell adhesion with infected cells. In turn, pDCs restrict viral spread by an IFN-I/λ response directed toward SARS-CoV-2-infected cells. This specialized function enables pDCs to efficiently turn-off viral replication, likely via a local response at the contact site with infected cells. By exploring the pDC response in SARS-CoV-2 patients, we further demonstrate that pDC responsiveness inversely correlates with the severity of the disease. The pDC response is particularly impaired in severe COVID-19 patients. Overall, we propose that pDC activation is essential to control SARS-CoV-2-infection. Failure to develop this response could be important to understand severe cases of COVID-19.

Network analysis of large-scale ImmGen and Tabula Muris datasets highlights metabolic diversity of tissue mononuclear phagocytes.

The diversity of mononuclear phagocyte (MNP) subpopulations across tissues is one of the key physiological characteristics of the immune system. Here, we focus on understanding the metabolic variability of MNPs through metabolic network analysis applied to three large-scale transcriptional datasets: we introduce (1) an ImmGen MNP open-source dataset of 337 samples across 26 tissues; (2) a myeloid subset of ImmGen Phase I dataset (202 MNP samples); and (3) a myeloid mouse single-cell RNA sequencing (scRNA-seq) dataset (51,364 cells) assembled based on Tabula Muris Senis. To analyze such large-scale datasets, we develop a network-based computational approach, genes and metabolites (GAM) clustering, for unbiased identification of the key metabolic subnetworks based on transcriptional profiles. We define 9 metabolic subnetworks that encapsulate the metabolic differences within MNP from 38 different tissues. Obtained modules reveal that cholesterol synthesis appears particularly active within the migratory dendritic cells, while glutathione synthesis is essential for cysteinyl leukotriene production by peritoneal and lung macrophages.

Inverted direct allorecognition triggers early donor-specific antibody responses after transplantation.

The generation of antibodies against donor-specific major histocompatibility complex (MHC) antigens, a type of donor-specific antibodies (DSAs), after transplantation requires that recipient's allospecific B cells receive help from T cells. The current dogma holds that this help is exclusively provided by the recipient's CD4+ T cells that recognize complexes of recipient's MHC II molecules and peptides derived from donor-specific MHC alloantigens, a process called indirect allorecognition. Here, we demonstrated that, after allogeneic heart transplantation, CD3ε knockout recipient mice lacking T cells generate a rapid, transient wave of switched alloantibodies, predominantly directed against MHC I molecules. This is due to the presence of donor CD4+ T cells within the graft that recognize intact recipient's MHC II molecules expressed by B cell receptor-activated allospecific B cells. Indirect evidence suggests that this inverted direct pathway is also operant in patients after transplantation. Resident memory donor CD4+ T cells were observed in perfusion liquids of human renal and lung grafts and acquired B cell helper functions upon in vitro stimulation. Furthermore, T follicular helper cells, specialized in helping B cells, were abundant in mucosa-associated lymphoid tissue of lung and intestinal grafts. In the latter, more graft-derived passenger T cells correlated with the detection of donor T cells in recipient's circulation; this, in turn, was associated with an early transient anti-MHC I DSA response and worse transplantation outcomes. We conclude that this inverted direct allorecognition is a possible explanation for the early transient anti-MHC DSA responses frequently observed after lung or intestinal transplantations.

A RORγt+ cell instructs gut microbiota-specific Treg cell differentiation.

The mutualistic relationship of gut-resident microbiota and the host immune system promotes homeostasis that ensures maintenance of the microbial community and of a largely non-aggressive immune cell compartment1,2. The consequences of disturbing this balance include proximal inflammatory conditions, such as Crohn's disease, and systemic illnesses. This equilibrium is achieved in part through the induction of both effector and suppressor arms of the adaptive immune system. Helicobacter species induce T regulatory (Treg) and T follicular helper (TFH) cells under homeostatic conditions, but induce inflammatory T helper 17 (TH17) cells when induced Treg (iTreg) cells are compromised3,4. How Helicobacter and other gut bacteria direct T cells to adopt distinct functions remains poorly understood. Here we investigated the cells and molecular components required for iTreg cell differentiation. We found that antigen presentation by cells expressing RORγt, rather than by classical dendritic cells, was required and sufficient for induction of Treg cells. These RORγt+ cells-probably type 3 innate lymphoid cells and/or Janus cells5-require the antigen-presentation machinery, the chemokine receptor CCR7 and the TGFß activator αv integrin. In the absence of any of these factors, there was expansion of pathogenic TH17 cells instead of iTreg cells, induced by CCR7-independent antigen-presenting cells. Thus, intestinal commensal microbes and their products target multiple antigen-presenting cells with pre-determined features suited to directing appropriate T cell differentiation programmes, rather than a common antigen-presenting cell that they endow with appropriate functions.

New perspectives on the regulation of germinal center reaction via αvß8- mediated activation of TGFß.

Transforming growth factor-ß (TGFß) is a long-known modulator of immune responses but has seemingly contradictory effects on B cells. Among cytokines, TGFß has the particularity of being produced and secreted in a latent form and must be activated before it can bind to its receptor and induce signaling. While the concept of controlled delivery of TGFß signaling via αvß8 integrin-mediated activation has gained some interest in the field of mucosal immunity, the role of this molecular mechanism in regulating T-dependent B cell responses is just emerging. We review here the role of TGFß and its activation, in particular by αvß8 integrin, in the regulation of mucosal IgA responses and its demonstrated and putative involvement in regulating germinal center (GC) B cell responses. We examine both the direct effect of TGFß on GC B cells and its ability to modulate the functions of helper cells, namely follicular T cells (Tfh and Tfr) and follicular dendritic cells. Synthetizing recently published works, we reconcile apparently conflicting data and propose an innovative and unified view on the regulation of the GC reaction by TGFß, highlighting the role of its activation by αvß8 integrin.

Regulatory T cells promote cancer immune-escape through integrin αvß8-mediated TGF-ß activation.

Presence of TGFß in the tumor microenvironment is one of the most relevant cancer immune-escape mechanisms. TGFß is secreted in an inactive form, and its activation within the tumor may depend on different cell types and mechanisms than its production. Here we show in mouse melanoma and breast cancer models that regulatory T (Treg) cells expressing the ß8 chain of αvß8 integrin (Itgß8) are the main cell type in the tumors that activates TGFß, produced by the cancer cells and stored in the tumor micro-environment. Itgß8 ablation in Treg cells impairs TGFß signalling in intra-tumoral T lymphocytes but not in the tumor draining lymph nodes. Successively, the effector function of tumor infiltrating CD8+ T lymphocytes strengthens, leading to efficient control of tumor growth. In cancer patients, anti-Itgß8 antibody treatment elicits similar improved cytotoxic T cell activation. Thus, this study reveals that Treg cells work in concert with cancer cells to produce bioactive-TGFß and to create an immunosuppressive micro-environment.

Apoptotic cells induce CD103 expression and immunoregulatory function in myeloid dendritic cell precursors through integrin αv and TGF-ß activation.

In the mammalian gut CD103+ve myeloid DCs are known to suppress inflammation threatened by luminal bacteria, but stimuli driving DC precursor maturation towards this beneficial phenotype are incompletely understood. We isolated CD11+ve DCs from mesenteric lymph nodes (MLNs) of healthy mice; CD103+ve DCs were 8-24 fold more likely than CD103-ve DCs to exhibit extensive of prior phagocytosis of apoptotic intestinal epithelial cells. However, CD103+ve and CD103-ve MLN DCs exhibited similar ex vivo capacity to ingest apoptotic cells, indicating that apoptotic cells might drive immature DC maturation towards the CD103+ve phenotype. When cultured with apoptotic cells, myeloid DC precursors isolated from murine bone marrow and characterised as lineage-ve CD103-ve, displayed enhanced expression of CD103 and ß8 integrin and acquired increased capacity to induce T regulatory lymphocytes (Tregs) after 7d in vitro. However, DC precursors isolated from αv-tie2 mice lacking αv integrins in the myeloid line exhibited reduced binding of apoptotic cells and complete deficiency in the capacity of apoptotic cells and/or latent TGF-ß1 to enhance CD103 expression in culture, whereas active TGF-ß1 increased DC precursor CD103 expression irrespective of αv expression. Fluorescence microscopy revealed clustering of αv integrin chains and latent TGF-ß1 at points of contact between DC precursors and apoptotic cells. We conclude that myeloid DC precursors can deploy αv integrin to orchestrate binding of apoptotic cells, activation of latent TGF-ß1 and acquisition of the immunoregulatory CD103+ve ß8+ve DC phenotype. This implies that a hitherto unrecognised consequence of apoptotic cell interaction with myeloid phagocytes is programming that prevents inflammation.

Missing self triggers NK cell-mediated chronic vascular rejection of solid organ transplants.

Current doctrine is that microvascular inflammation (MVI) triggered by a transplant -recipient antibody response against alloantigens (antibody-mediated rejection) is the main cause of graft failure. Here, we show that histological lesions are not mediated by antibodies in approximately half the participants in a cohort of 129 renal recipients with MVI on graft biopsy. Genetic analysis of these patients shows a higher prevalence of mismatches between donor HLA I and recipient inhibitory killer cell immunoglobulin-like receptors (KIRs). Human in vitro models and transplantation of ß2-microglobulin-deficient hearts into wild-type mice demonstrates that the inability of graft endothelial cells to provide HLA I-mediated inhibitory signals to recipient circulating NK cells triggers their activation, which in turn promotes endothelial damage. Missing self-induced NK cell activation is mTORC1-dependent and the mTOR inhibitor rapamycin can prevent the development of this type of chronic vascular rejection.

Plasmacytoid dendritic cells control dengue and Chikungunya virus infections via IRF7-regulated interferon responses.

Type I interferon (IFN-I) responses are critical for the control of RNA virus infections, however, many viruses, including Dengue (DENV) and Chikungunya (CHIKV) virus, do not directly activate plasmacytoid dendritic cells (pDCs), robust IFN-I producing cells. Herein, we demonstrated that DENV and CHIKV infected cells are sensed by pDCs, indirectly, resulting in selective IRF7 activation and IFN-I production, in the absence of other inflammatory cytokine responses. To elucidate pDC immunomodulatory functions, we developed a mouse model in which IRF7 signaling is restricted to pDC. Despite undetectable levels of IFN-I protein, pDC-restricted IRF7 signaling controlled both viruses and was sufficient to protect mice from lethal CHIKV infection. Early pDC IRF7-signaling resulted in amplification of downstream antiviral responses, including an accelerated natural killer (NK) cell-mediated type II IFN response. These studies revealed the dominant, yet indirect role of pDC IRF7-signaling in directing both type I and II IFN responses during arbovirus infections.

ß8 Integrin Expression and Activation of TGF-ß by Intestinal Dendritic Cells Are Determined by Both Tissue Microenvironment and Cell Lineage.

Activation of TGF-ß by dendritic cells (DCs) expressing αvß8 integrin is essential for the generation of intestinal regulatory T cells (Tregs) that in turn promote tolerance to intestinal Ags. We have recently shown that αvß8 integrin is preferentially expressed by CD103(+) DCs and confers their ability to activate TGF-ß and generate Tregs. However, how these DCs become specialized for this vital function is unknown. In this study, we show that ß8 expression is controlled by a combination of factors that include DC lineage and signals derived from the tissue microenvironment and microbiota. Specifically, our data demonstrate that TGF-ß itself, along with retinoic acid and TLR signaling, drives expression of αvß8 in DCs. However, these signals only result in high levels of ß8 expression in cells of the cDC1 lineage, CD8α(+), or CD103(+)CD11b(-) DCs, and this is associated with epigenetic changes in the Itgb8 locus. Together, these data provide a key illustrative example of how microenvironmental factors and cell lineage drive the generation of regulatory αvß8-expressing DCs specialized for activation of TGF-ß to facilitate Treg generation.

B Cells Loaded with Synthetic Particulate Antigens: A Versatile Platform To Generate Antigen-Specific Helper T Cells for Cell Therapy.

Adoptive cell therapy represents a promising approach for several chronic diseases. This study describes an innovative strategy for biofunctionalization of nanoparticles, allowing the generation of synthetic particulate antigens (SPAg). SPAg activate polyclonal B cells and vectorize noncognate proteins into their endosomes, generating highly efficient stimulators for ex vivo expansion of antigen-specific CD4+ T cells. This method also allows harnessing the ability of B cells to polarize CD4+ T cells into effectors or regulators.

Protein tyrosine phosphatase-PEST and ß8 integrin regulate spatiotemporal patterns of RhoGDI1 activation in migrating cells.

Directional cell motility is essential for normal development and physiology, although how motile cells spatiotemporally activate signaling events remains largely unknown. Here, we have characterized an adhesion and signaling unit comprised of protein tyrosine phosphatase (PTP)-PEST and the extracellular matrix (ECM) adhesion receptor ß8 integrin that plays essential roles in directional cell motility. ß8 integrin and PTP-PEST form protein complexes at the leading edge of migrating cells and balance patterns of Rac1 and Cdc42 signaling by controlling the subcellular localization and phosphorylation status of Rho GDP dissociation inhibitor 1 (RhoGDI1). Translocation of Src-phosphorylated RhoGDI1 to the cell's leading edge promotes local activation of Rac1 and Cdc42, whereas dephosphorylation of RhoGDI1 by integrin-bound PTP-PEST promotes RhoGDI1 release from the membrane and sequestration of inactive Rac1/Cdc42 in the cytoplasm. Collectively, these data reveal a finely tuned regulatory mechanism for controlling signaling events at the leading edge of directionally migrating cells.

Wiskott-Aldrich syndrome protein deficiency in innate immune cells leads to mucosal immune dysregulation and colitis in mice.

BACKGROUND & AIMS: Immunodeficiency and autoimmune sequelae, including colitis, develop in patients and mice deficient in Wiskott-Aldrich syndrome protein (WASP), a hematopoietic cell-specific intracellular signaling molecule that regulates the actin cytoskeleton. Development of colitis in WASP-deficient mice requires lymphocytes; transfer of T cells is sufficient to induce colitis in immunodeficient mice. We investigated the interactions between innate and adaptive immune cells in mucosal regulation during development of T cell-mediated colitis in mice with WASP-deficient cells of the innate immune system. METHODS: Naïve and/or regulatory CD4(+) T cells were transferred from 129 SvEv mice into RAG-2-deficient (RAG-2 KO) mice or mice lacking WASP and RAG-2 (WRDKO). Animals were observed for the development of colitis; effector and regulatory functions of innate immune and T cells were analyzed with in vivo and in vitro assays. RESULTS: Transfer of unfractionated CD4(+) T cells induced severe colitis in WRDKO, but not RAG-2 KO, mice. Naïve wild-type T cells had higher levels of effector activity and regulatory T cells had reduced suppressive function when transferred into WRDKO mice compared with RAG-2 KO mice. Regulatory T-cell proliferation, generation, and maintenance of FoxP3 expression were reduced in WRDKO recipients and associated with reduced numbers of CD103(+) tolerogenic dendritic cells and levels of interleukin-10. Administration of interleukin-10 prevented induction of colitis following transfer of T cells into WRDKO mice. CONCLUSIONS: Defective interactions between WASP-deficient innate immune cells and normal T cells disrupt mucosal regulation, potentially by altering the functions of tolerogenic dendritic cells, production of interleukin-10, and homeostasis of regulatory T cells.

Apoptotic cells can deliver chemotherapeutics to engulfing macrophages and suppress inflammatory cytokine production.

Immunosuppression via cell-cell contact with apoptotic cells is a well studied immunological phenomenon. Although the original studies of immune repression used primary cells, which undergo spontaneous cell death or apoptosis in response to irradiation, more recent studies have relied on chemotherapeutic agents to induce apoptosis in cell lines. In this work, we demonstrate that Jurkat cells induced to die with actinomycin D suppressed inflammatory cytokine production by macrophages, whereas cells treated with etoposide did not. This immune repression mediated by actinomycin D-treated cells did not require phagocytosis or cell-cell contact and thus occurs through a different mechanism from that seen with primary apoptotic neutrophils. Moreover, cells induced to die with etoposide and then treated for a short time with actinomycin D also suppressed macrophage responses, indicating that suppression was mediated by actinomycin D independent of the mechanism of cell death. Finally, phagocytosis of actinomycin D-treated cells caused apoptosis in macrophages, and suppression could be blocked by inhibition of caspase activity in the target macrophage. Together, these data indicate that apoptotic cells act as "Trojan horses," delivering actinomycin D to engulfing macrophages. Suppression of cytokine production by macrophages is therefore due to exposure to actinomycin D from apoptotic cells and is not the result of cell-receptor interactions. These data suggest that drug-induced death may not be an appropriate surrogate for the immunosuppressive activity of apoptotic cells. Furthermore, these effects of cytotoxic drugs on infiltrating immune phagocytes may have clinical ramifications for their use as antitumor therapies.

Preferential expression of integrin αvß8 promotes generation of regulatory T cells by mouse CD103+ dendritic cells.

BACKGROUND & AIMS: Immune responses in the intestine are controlled by regulatory T cells (Tregs), which prevent inflammation in response to commensal bacteria. A specific population of intestinal dendritic cells (DCs), marked by expression of CD103, generate Tregs more efficiently than other DC populations through mechanisms that involve retinoic acid and transforming growth factor (TGF)-ß. However, it is not clear how CD103(+) DCs are specialized for this function. We investigated the ability of CD103(+) DCs to promote Treg generation through activation of TGF-ß and the role of integrins with the αv subunit in this process. METHODS: Naïve T cells were cultured with purified DCs from mesenteric lymph nodes (MLNs) or intestines of wild-type and αv conditional knockout mice to assess generation of Tregs. Antigens were administered orally to mice, and antigen-specific generation of Tregs was measured in intestinal tissues. Expression of the integrin αv subunit was measured in purified subpopulations of DCs by quantitative polymerase chain reaction and immunoblot analyses. RESULTS: In vitro, CD103(+) DCs generated more Tregs in the presence of latent TGF-ß than other MLN DCs. Efficient generation of Tregs required expression of the integrin αv subunit by DCs; mice that lacked αv in immune cells did not convert naïve T cells to intestinal Tregs in response to oral antigen. CD103(+) DCs derived from the MLNs selectively expressed high levels of integrin αvß8 compared with other populations of DCs. CONCLUSIONS: Expression of αvß8 is required for CD103(+) DCs to become specialized and activate latent TGF-ß and generate Tregs during the induction of tolerance to intestinal antigens in mice.

X-ray structure of the human calreticulin globular domain reveals a peptide-binding area and suggests a multi-molecular mechanism.

In the endoplasmic reticulum, calreticulin acts as a chaperone and a Ca(2+)-signalling protein. At the cell surface, it mediates numerous important biological effects. The crystal structure of the human calreticulin globular domain was solved at 1.55 Å resolution. Interactions of the flexible N-terminal extension with the edge of the lectin site are consistently observed, revealing a hitherto unidentified peptide-binding site. A calreticulin molecular zipper, observed in all crystal lattices, could further extend this site by creating a binding cavity lined by hydrophobic residues. These data thus provide a first structural insight into the lectin-independent binding properties of calreticulin and suggest new working hypotheses, including that of a multi-molecular mechanism.