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Ultra-small 1-2 nm gold nanoparticles (NP) were conjugated with a poorly-soluble peptide auto-antigen, associated with type 1 diabetes, to modify the peptide pharmacokinetics, following its intradermal delivery. Peptide distribution was characterized, in vivo, after delivery using either conventional intradermal injection or a hollow microneedle device. The poorly-soluble peptide was effectively presented in distant lymph nodes (LN), spleen and draining LN when conjugated to the nanoparticles, whereas peptide alone was only presented in the draining LN. By contrast, nanoparticle conjugation to a highly-soluble peptide did not enhance in vivo distribution. Transfer of both free peptide and peptide-NPs from the skin to LN was reduced in mice lacking lymphoid homing receptor CCR7, suggesting that both are actively transported by migrating dendritic cells to LN. Collectively, these data demonstrate that intradermally administered ultra-small gold nanoparticles can widen the distribution of poorly-soluble auto-antigenic peptides to multiple lymphoid organs, thus enhancing their use as potential therapeutics., (Copyright © 2020. Published by Elsevier Inc.)

Secretory intestinal IgA can protect from re-infection with rotavirus (RV), but very little is known about the mechanisms that induce IgA production during intestinal virus infections. Classical dendritic cells (cDCs) in the intestine can facilitate both T cell-dependent and -independent secretory IgA. Here, we show that BATF3-dependent cDC1, but not cDC2, are critical for the optimal induction of RV-specific IgA responses in the mesenteric lymph nodes. This depends on the selective expression of the TGFβ-activating integrin αvβ8 by cDC1. In contrast, αvβ8 on cDC1 is dispensible for steady state immune homeostasis. Given that cDC2 are crucial in driving IgA during steady state but are dispensable for RV-specific IgA responses, we propose that the capacity of DC subsets to induce intestinal IgA responses reflects the context, as opposed to an intrinsic property of individual DC subsets.

During foam cell formation and atherosclerosis development, the scavenger receptor CD36 plays critical roles in lipid uptake and triggering of atherogenicity via the activation of Vav molecules. The Vav family includes three highly conserved members known as Vav1, Vav2, and Vav3. As Vav1 and Vav3 were found to exert function in atherosclerosis development, it remains thus to decipher whether Vav2 also plays a role in the development of atherosclerosis. In this study we found that Vav2 deficiency in RAW264.7 macrophages significantly diminished oxidized LDL uptake and CD36 signaling, demonstrating that each Vav protein family member was required for foam cell formation. Genetic disruption of Vav2 in ApoE-deficient C57BL/6 mice significantly inhibited the severity of atherosclerosis. Strikingly, we further found that the genetic deletion of each member of the Vav protein family by CRISPR/Cas9 resulted in a similar alteration of transcriptomic profiles of macrophages. The three members of the Vav proteins were found to form complexes, and genetic ablation of each single Vav molecule was sufficient to prevent endocytosis of CD36. The functional interdependence of the three Vav family members in foam cell formation was due to their indispensable roles in transcriptomic programing, lipid uptake, and activation of the JNK kinase in macrophages., (Copyright © 2019 Huang et al.)

Intestinal macrophages (mφ) form one of the largest populations of mφ in the body and are vital for the maintenance of gut homeostasis. They have several unique properties and are derived from local differentiation of classical Ly6C hi monocytes, but the factors driving this tissue-specific process are not understood. Here we have used global transcriptomic analysis to identify a unique homeostatic signature of mature colonic mφ that is acquired as they differentiate in the mucosa. By comparing the analogous monocyte differentiation process found in the dermis, we identify TGFβ as an indispensable part of monocyte differentiation in the intestine and show that it enables mφ to adapt precisely to the requirements of their environment. Importantly, TGFβR signaling on mφ has a crucial role in regulating the accumulation of monocytes in the mucosa, via mechanisms that are distinct from those used by IL10.

Dendritic cells (DC) are mononuclear phagocytes which exhibit a branching (dendritic) morphology and excel at naïve T cell activation. DC encompass several subsets initially identified by their expression of cell surface molecules and later shown to possess distinct functions. DC subset differentiation is orchestrated by transcription factors, growth factors and cytokines. Identifying DC subsets is challenging as very few cell surface molecules are uniquely expressed on any one of these cell populations. There is no standard consensus to identify mononuclear phagocyte subsets; varying antigens are employed depending on the tissue and animal species studied and between laboratories. This has led to confusion in how to accurately define and classify DCs across tissues and between species. Here we report a comparative genomics strategy that enables universal definition of DC and other mononuclear phagocyte subsets across species. We performed a meta-analysis of several public datasets of human and mouse mononuclear phagocyte subsets isolated from blood, spleen, skin or cutaneous lymph nodes, including by using a novel and user friendly software, BubbleGUM, which generates and integrates gene signatures for high throughput gene set enrichment analysis. This analysis demonstrates the equivalence between human and mouse skin XCR1(+) DCs, and between mouse and human Langerhans cells., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Dendritic cells (DCs) of the skin have an important role in skin-mediated immunity capable of promoting potent immune responses. We availed of polymeric dissolving microneedle (MN) arrays laden with nano-encapsulated antigen to specifically target skin DC networks. This modality of immunization represents an economic, efficient, and potent means of antigen delivery directly to skin DCs, which are inefficiently targeted by more conventional immunization routes. Following MN immunization, Langerhans cells (LCs) constituted the major skin DC subset capable of cross-priming antigen-specific CD8+ T cells ex vivo. Although all DC subsets were equally efficient in priming CD4+ T cells, LCs were largely responsible for orchestrating the differentiation of CD4+ IFN-γ- and IL-17-producing effectors. Importantly, depletion of LCs prior to immunization had a profound effect on CD8+ CTL responses in vivo, and vaccinated animals displayed reduced protective anti-tumor and viral immunity. Interestingly, this cross-priming bias was lost following MN immunization with soluble antigen, suggesting that processing and cross-presentation of nano-particulate antigen is favored by LCs. Therefore, these studies highlight the importance of LCs in skin immunization strategies and that targeting of nano-particulate immunogens through dissolvable polymeric MNs potentially provides a promising technological platform for improved vaccination strategies.

γδ T cells are resident in cerebrospinal fluid and central nervous system (CNS) lesions of multiple sclerosis (MS) patients, but as multifaceted cells exhibiting innate and adaptive characteristics, their function remains unknown. Previous studies in experimental autoimmune encephalomyelitis (EAE) are contradictory and identified these cells as either promoting or suppressing disease pathogenesis. This study examines distinct γδ T cell subsets during EAE and indicates they mediate differential functions in CNS inflammation and demyelination resulting in pathogenesis or protection. We identified two γδ subsets in the CNS, Vγ1(+) and Vγ4(+), with distinct cytokine profiles and tissue specificity. Anti-γδ T cell receptor (TCR) monoclonal antibody (mAb) administration results in activation and downregulation of surface TCR, rendering the cells undetectable, but with opposing effects: anti-Vγ4 treatment exacerbates disease whereas anti-Vγ1 treatment is protective. The Vγ4(+) subset produces multiple pro-inflammatory cytokines including high levels of IL-17, and accounts for 15-20% of the interleukin-17 (IL-17) producing cells in the CNS, but utilize a variant transcriptional program than CD4(+) Th17 cells. In contrast, the Vγ1 subset produces CCR5 ligands, which may promote regulatory T cell differentiation. γδ T cell subsets thus play distinct and opposing roles during EAE, providing an explanation for previous reports and suggesting selective targeting to optimize regulation as a potential therapy for MS., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Psoriasis is a common chronic inflammatory skin disease that involves dysregulated interplay between immune cells and keratinocytes. Recently, it has been reported that IL-23 induces CCR6+ γδ T cells, which have the pivotal role in psoriasis-like skin inflammation in mice of producing IL-17A and IL-22. Langerhans cells (LCs) are a subset of dendritic cells that reside in the epidermis and regulate immune responses. The role of LCs has been extensively investigated in contact hypersensitivity, but their role in psoriasis remains to be clarified. In this study, we focused on Th17-related factors and assessed the role of LCs and γδ T cells in the development of psoriasis using a mouse psoriasis model triggered by topical application of imiquimod (IMQ). LC depletion by means of diphtheria toxin (DT) in Langerin DT receptor-knocked-in mice suppressed hyperkeratosis, parakeratosis, and ear swelling in the IMQ-treated regions. In addition, LC-depleted mice showed decreased levels of Th17-related cytokines in IMQ-treated skin lesions. Moreover, the IMQ-treated skin of LC-depleted mice showed a decreased number of IL-17A-producing CCR6+ γδ T cells. These results suggest that LCs are required for the development of psoriasis-like lesions induced by IMQ in mice.

Macrophages (mφ) are essential for intestinal homeostasis and the pathology of inflammatory bowel disease (IBD), but it is unclear whether discrete mφ populations carry out these distinct functions or if resident mφ change during inflammation. We show here that most resident mφ in resting mouse colon express very high levels of CX3CR1, are avidly phagocytic and MHCII(hi), but are resistant to Toll-like receptor (TLR) stimulation, produce interleukin 10 constitutively, and express CD163 and CD206. A smaller population of CX3CR1(int) cells is present in resting colon and it expands during experimental colitis. Ly6C(hi)CCR2(+) monocytes can give rise to all mφ subsets in both healthy and inflamed colon and we show that the CX3CR1(int) pool represents a continuum in which newly arrived, recently divided monocytes develop into resident CX3CR1(hi) mφ. This process is arrested during experimental colitis, resulting in the accumulation of TLR-responsive pro-inflammatory mφ. Phenotypic analysis of human intestinal mφ indicates that analogous processes occur in the normal and Crohn's disease ileum. These studies show for the first time that resident and inflammatory mφ in the intestine represent alternative differentiation outcomes of the same precursor and targeting these events could offer routes for therapeutic intervention in IBD.

E-cadherin is best characterized as adherens junction protein, which through homotypic interactions contributes to the maintenance of the epithelial barrier function. In epithelial cells, the cytoplasmic tail of E-cadherin forms a dynamic complex with catenins and regulates several intracellular signal transduction pathways, including Wnt/β-catenin, PI3K/Akt, Rho GTPase, and NF-κB signaling. Recent progress uncovered a novel and critical role for this adhesion molecule in mononuclear phagocyte functions. E-cadherin regulates the maturation and migration of Langerhans cells, and its ligation prevents the induction of a tolerogenic state in bone marrow-derived dendritic cells (DCs). In this respect, the functionality of β-catenin could be instrumental in determining the balance between immunogenicity and tolerogenicity of DCs in vitro and in vivo. Fusion of alternatively activated macrophages and osteoclasts is also E-cadherin-dependent. In addition, the E-cadherin ligands CD103 and KLRG1 are expressed on DC-, T-, and NK-cell subsets and contribute to their interaction with E-cadherin-expressing DCs and macrophages. Here we discuss the regulation, function, and implications of E-cadherin expression in these central orchestrators of the immune system.

The stromal network of the thymus provides a unique environment that supports the development of mature CD4(+) and CD8(+) T cells expressing a very diverse repertoire of T cell receptors (TCR) with limited reactivity to self-antigens. Thymic cortical epithelial cells (cTECs) are specialized antigen-presenting cells (APCs) that promote the positive selection of developing thymocytes while medullary thymic epithelial cells (mTECs) and thymic dendritic cells (tDCs) induce central tolerance to self-antigens. Recent studies showed that cTECs express a unique set of proteases involved in the generation of self-peptides presented by major-histocompatibility encoded molecules (pMHC) and consequently may express a unique set of pMHC complexes. Conversely, the stromal cells of the medulla developed several mechanisms to mirror as closely as possible the constellation of self-peptides derived from peripheral tissues. Here, we discuss how these different features allow for the development of a highly diverse but poorly self-reactive repertoire of functional T cells., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Oral tolerance is a key feature of intestinal immunity, generating systemic tolerance to fed antigens. However, the molecular mechanism mediating oral tolerance remains unclear. In this study, we examined the role of the B7 family members of costimulatory molecules in the establishment of oral tolerance. Deficiencies of B7-H1 and B7-DC abrogated the oral tolerance, accompanied by enhanced antigen-specific CD4(+) T-cell response and IgG(1) production. Mesenteric lymph node (MLN) dendritic cells (DCs) displayed higher levels of B7-H1 and B7-DC than systemic DCs, whereas they showed similar levels of CD80, CD86, and B7-H2. MLN DCs enhanced the antigen-specific generation of CD4(+)Foxp3(+) inducible regulatory T cells (iT(regs)) from CD4(+)Foxp3(-) T cells rather than CD4(+) effector T cells (T(eff)) relative to systemic DCs, owing to the dominant expression of B7-H1 and B7-DC. Furthermore, the antigen-specific conversion of CD4(+)Foxp3(-) T cells into CD4(+)Foxp3(+) iT(regs) occurred in MLNs greater than in peripheral organs during oral tolerance under steady-state conditions, and such conversion required B7-H1 and B7-DC more than other B7 family members, whereas it was severely impaired under inflammatory conditions. In conclusion, our findings suggest that B7-H1 and B7-DC expressed on MLN DCs are essential for establishing oral tolerance through the de novo generation of antigen-specific CD4(+)Foxp3(+) iT(regs).

Small intestinal CD103(+) dendritic cells (DCs) have the selective ability to promote de novo generation of regulatory T cells via the production of retinoic acid (RA). Considering that aldehyde dehydrogenase (ALDH) activity controls the production of RA, we used a flow cytometry-based assay to measure ALDH activity at the single-cell level and to perform a comprehensive analysis of the RA-producing DC populations present in lymphoid and nonlymphoid mouse tissues. RA-producing DCs were primarily of the tissue-derived, migratory DC subtype and can be readily found in the skin and in the lungs as well as in their corresponding draining lymph nodes. The RA-producing skin-derived DCs were capable of triggering the generation of regulatory T cells, a finding demonstrating that the presence of RA-producing, tolerogenic DCs is not restricted to the intestinal tract as previously thought. Unexpectedly, the production of RA by skin DCs was restricted to CD103(-) DCs, indicating that CD103 expression does not constitute a "universal" marker for RA-producing mouse DCs. Finally, Toll-like receptor (TLR) triggering or the presence of a commensal microflora was not essential for the induction of ALDH activity in the discrete ALDH(+) DC subsets that characterize tissues constituting environmental interfaces.

Fms-like tyrosine kinase 3 ligand (FLT3L) plays a major role in dendritic cell (DC) biology. Deficiency of FLT3L causes a dramatic decrease in DC numbers, whereas increasing its availability (by repetitive injections for 7-10 days) leads to a 10-fold increase in DC numbers. In this study, we show that FLT3L treatment indirectly leads to an expansion of peripheral naturally occurring T regulatory cells (NTregs). The FLT3L-induced increase in NTregs was still observed in thymectomized mice, ruling out the role of the thymus in this mechanism. Instead, the increased number of NTregs was due to proliferation of preexisting NTregs, most likely due to favored interactions with increased number of DCs. In vitro, we show that DCs induce regulatory T-cell (Treg) proliferation by direct cell contact and in an interleukin-2-dependent, T-cell receptor-independent manner. FLT3L could prevent death induced by acute graft-versus-host disease (GVHD). This study demonstrates unique aspects in the regulation of Treg homeostasis by DCs, which were unappreciated until now. It also reinforces the relevance of FLT3L treatment in GVHD by its ability to increase both the number of tolerizing DCs and NTregs.

The regulation of CD4 T-cell numbers during an immune response should take account of the amount of antigen (Ag), the initial frequency of Ag-specific T cells, the mix of naive versus experienced cells, and (ideally) the diversity of the repertoire. Here we describe a novel mechanism of T-cell regulation that potentially deals with all of these parameters. We found that CD4 T cells establish a negative feedback loop by capturing their cognate major histocompatibility class (MHC)/peptide complexes from Ag-presenting cells and presenting them to Ag-experienced CD4 T cells, thereby inhibiting their recruitment into the response while allowing recruitment of naive T cells. The inhibition is Ag specific, begins at day 2 (long before Ag disappearance), and cannot be overcome by providing new Ag-loaded dendritic cells. In this way, CD4 T-cell proliferation is regulated in a functional relationship to the amount of Ag, while allowing naive T cells to generate repertoire variety.

Linker for activation of T cells (LAT) is an adaptor protein required for organization of the signaling machinery downstream of the platelet collagen receptor, the glycoprotein VI (GPVI). Here, we investigated the effect of LAT mutations on specific signaling pathways and on platelet functions in response to GPVI triggering by convulxin (Cvx). Using mice containing tyrosine to phenylalanine mutations of the adaptor, we show the crucial role played by the tyrosine residues at positions 175, 195, and 235 in the phosphorylation of LAT and in the whole pattern of protein tyrosine phosphorylation in response to Cvx. These 3 C-terminal tyrosine residues are important to recruit the tyrosine kinase Fyn, which may be involved in LAT phosphorylation. Efficient phosphoinositide 3-kinase (PI3K) activation requires the 3 C-terminal tyrosine residues of LAT but not its tyrosine 136. Interestingly, single mutation of the tyrosine 136 results in the loss of phospholipase C gamma2 (PLCgamma2) activation without affecting its PI3K-dependent membrane association, and is sufficient to impair platelet responses to Cvx. Thus, activation of PLCgamma2 via GPVI is dependent on 2 complementary events: its interaction with the tyrosine 136 of LAT and its membrane location, which itself requires events mediated by the 3 C-terminal tyrosines of LAT.

Natural killer (NK) cells express an array of activating receptors that associate with DAP12 (KARAP), CD3zeta, and/or FcRgamma ITAM (immunoreceptor tyrosine-based activation motif)-bearing signaling subunits. In T and mast cells, ITAM-dependent signals are integrated by critical scaffolding elements such as LAT (linker for activation of T cells) and NTAL (non-T-cell activation linker). Using mice that are deficient for ITAM-bearing molecules, LAT or NTAL, we show that NK cell cytotoxicity and interferon-gamma secretion are initiated by ITAM-dependent and -independent as well as LAT/NTAL-dependent and -independent pathways. The role of these various signaling circuits depends on the target cell as well as on the activation status of the NK cell. The multiplicity and the plasticity of the pathways that initiate NK cell effector functions contrast with the situation in T cells and B cells and provide an explanation for the resiliency of NK cell effector functions to various pharmacologic inhibitors and genetic mutations in signaling molecules.

Thrombospondin-1 (TSP1) is abundantly secreted during platelet activation and plays a role in irreversible platelet aggregation. A peptide derived from the C-terminal domain of TSP1, RFYVVMWK (RFY) can activate human platelets at least in part via its binding to integrin-associated protein. Although integrin-associated protein is known to physically interact with alphaIIb/beta3, we found that this major platelet integrin had only a partial implication in RFY-mediated platelet aggregation. Accordingly, RFY induced a significant Glanzmann type I thrombasthenic platelet aggregation. The alphaIIb/beta3-dependent part of platelet aggregation induced by RFY was mainly due to secreted ADP and thromboxane A2. In the absence of alphaIIb/beta3 and fibrinogen, RFY stimulated a rapid tyrosine phosphorylation of a set of proteins, including Syk, linker for activation of T cells (LAT) and phospholipase Cgamma2. This signaling pathway was critical for RFY-mediated platelet activation as revealed by the use of pharmacological inhibitors as well as LAT-deficient mouse platelets. Phosphoinositide 3-kinase activation was also required for RFY-mediated platelet aggregation. Our results unravel a new alphaIIb/beta3 and fibrinogen-independent mechanism for platelet aggregation in response to the active peptide from the C-terminal domain of TSP1.

CC chemokine receptor (CCR) 9, the receptor for the CC-chemokine CCL25/thymus-expressed chemokine (TECK), is mainly expressed by thymocytes and by intraepithelial (IEL) and lamina propria lymphocytes of the small intestine. To study the biologic role of CCR9, a mouse strain was generated in which the CCR9 gene was deleted. In spite of the high level of CCR9 found in double- and single-positive thymocytes and of the expression of its corresponding ligand on thymic stromal cells, CCR9 deletion had no major effect on intrathymic T-cell development. It was noted that there was only a one-day lag in the appearance of double-positive cells during fetal ontogeny in CCR9(-/-) thymi. When tested in chemotaxis assay, thymocytes isolated from CCR9(-/-) mice failed to respond to TECK/CCL25. Taken together, these results suggest that in thymocytes, CCR9 is the only physiologic receptor for TECK/CCL25, and that it is dispensable for proper T-cell development. Bone marrow pre-pro-B cells migrate in response to TECK/CCL25, but more mature B cells do not. Consistent with this observation, it was shown that there are fewer pre-pro-B cells in CCR9(-/-) mice than in wild-type mice. However, this diminution does not appear to have a detectable effect on the generation of a normal complement of mature B cells. Finally, it was shown that in the small intestine of CCR9-deficient mice, the intraepithelial T-cell-to-epithelial cell ratio is decreased, an observation that can be accounted for by a marked diminution of the T-cell receptor gammadelta(+) compartment.

The crystal structure of the complex between a single chain Fv fragment of the KB5-C20 T-cell antigen receptor (TCR) and the specific anti-clonotypic antibody (Ab) Désiré-1 provides the first description of the interface between a clonotype and an anti-clonotype. In the four idiotype/anti-idiotype complexes of known three-dimensional structures, the interacting Fv fragments associate largely through their complementarity-determining regions (CDRs). In marked contrast, Désiré-1 binds to a face of the KB5-C20 TCR that is almost perpendicular to the TCR antigen binding site, and recognizes discontinuous stretches of TCR Valpha and Vbeta residues that belong to both the CDRs and the framework. Despite this peculiar mode of interaction, Désiré-1 constitutes a genuine anti-clonotypic Ab. Moreover, in spite of the fact that the Désiré-1 contact residues do not constitute a molecular mimic of the physiological ligand normally recognized by the KB5-C20 TCR, the bivalent Désiré-1 Ab is capable of efficiently activating T-cells expressing the KB5-C20 TCR., (Copyright 1999 Academic Press.)

Recent gene-targeting experiments have highlighted the existence of checkpoints that ensure that alpha beta T cells do not complete intrathymic differentiation if they have not attained certain landmark events. These 'proofreading' mechanisms operate by way of the pre-TCR and TCR complexes, which are sequentially expressed during T-cell development. These complexes are likely to signal via their associated CD3 subunits. By activating intracellular effectors, the CD3 subunits probably modulate gene expression profiles and drive the maturing alpha beta T cells through a precise developmental sequence.

Recent data support the existence of activation motifs within different subunits of the T-cell-receptor-CD3 complex. This architecture generates a receptor composed of discrete modules, each capable of being coupled to an effector pathway. Although new T-cell specific protein tyrosine kinases have recently been identified, the nature of the proximal non-receptor protein tyrosine kinase linking the T-cell receptor complex to essential signalling effectors remains unknown. Developmentally regulated differences in T-cell-receptor-CD3 assembly or stability may lead to the expression of isoforms displaying different sets of activation motifs. Whether this may be the basis of differential signalling during T-cell development is still a matter of speculation.

In the present experiments, we attempted to obtain evidence that T-cell receptors (Tcr) and immunoglobulins can react against the same antigen-peptide/MHC class II complexes on antigen-presenting cells (APC). Use was made of monoclonal APC, i.e. I-Ak alpha/beta gene-transfected L929 fibroblasts, monoclonal anti-insulin antibodies and selected insulin-specific T-cell lines. Evidence for similarities in antibody and T-cell recognition of insulin presented by I-Ak alpha/beta gene-transfected L-cell fibroblasts was not obtained after serious attempts. Furthermore, we found no evidence for synthesis of antibodies specific for insulin-peptide/MHC class II complexes.

A solid-phase radioimmunoanalysis of plasma membrane molecules solubilized in the presence of detergent was developed. From a crude cell lysate, membrane molecules were specifically immobilized by solid-phase adsorbed monoclonal antibodies. Analysis of these molecules was easily performed with radiolabeled monoclonal antibodies of different specificities. This technique was used to analyze the HLA-DR and HLA-A,B,C molecules expressed by RAJI cells. It was possible (a) to determine which epitopes are expressed on the same molecules; (b) to define subsets of HLA molecules according to the epitopes which they express, and (c) to perform quantitative analysis of HLA molecules on a crude cell lysate.