B cell contribution to immunometabolic dysfunction and impaired immune responses in obesity.

Obesity increases the risk of type 2 diabetes mellitus, cardiovascular disease, fatty liver disease, and cancer. It is also linked with more severe complications from infections, including COVID-19, and poor vaccine responses. Chronic, low-grade inflammation and associated immune perturbations play an important role in determining morbidity in people living with obesity. The contribution of B cells to immune dysregulation and meta-inflammation associated with obesity has been documented by studies over the past decade. With a focus on human studies, here we consolidate the observations demonstrating that there is altered B cell subset composition, differentiation, and function both systemically and in the adipose tissue of individuals living with obesity. Finally, we discuss the potential factors that drive B cell dysfunction in obesity and propose a model by which altered B cell subset composition in obesity underlies dysfunctional B cell responses to novel pathogens.

Isolation of HLA-DR-naturally presented peptides identifies T-cell epitopes for rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) immunopathogenesis revolves around the presentation of poorly characterised self-peptides by human leucocyte antigen (HLA)-class II molecules on the surface of antigen-presenting cells to autoreactive CD4 +T cells. Here, we analysed the HLA-DR-associated peptidome of synovial tissue (ST) and of dendritic cells (DCs) pulsed with synovial fluid (SF) or ST, to identify potential T-cell epitopes for RA. METHODS: HLA-DR/peptide complexes were isolated from RA ST samples (n=3) and monocyte-derived DCs, generated from healthy donors carrying RA-associated shared epitope positive HLA-DR molecules and pulsed with RA SF (n=7) or ST (n=2). Peptide sequencing was performed by high-resolution mass spectrometry. The immunostimulatory capacity of selected peptides was evaluated on peripheral blood mononuclear cells from patients with RA (n=29) and healthy subjects (n=12) by flow cytometry. RESULTS: We identified between 103 and 888 HLA-DR-naturally presented peptides per sample. We selected 37 native and six citrullinated (cit)-peptides for stimulation assays. Six of these peptides increased the expression of CD40L on CD4 +T cells patients with RA, and specifically triggered IFN-γ expression on RA CD4 +T cells compared with healthy subjects. Finally, the frequency of IFN-γ-producing CD4 +T cells specific for a myeloperoxidase-derived peptide showed a positive correlation with disease activity. CONCLUSIONS: We significantly expanded the peptide repertoire presented by HLA-DR molecules in a physiologically relevant context, identifying six new epitopes recognised by CD4 +T cells from patients with RA. This information is important for a better understanding of the disease immunopathology, as well as for designing tolerising antigen-specific immunotherapies.

TNF-α Affects Signature Cytokines of Th1 and Th17 T Cell Subsets through Differential Actions on TNFR1 and TNFR2.

Tumor necrosis factor (TNF)-α is a pleiotropic cytokine implicated in the etiology of several autoimmune diseases, including rheumatoid arthritis (RA). TNF-α regulates diverse effector functions through the activation of TNF-α receptor (TNFR)1 and TNFR2. Although the detrimental role of this cytokine has been addressed in distinct disease settings, the effects of TNF-α on cytokine production by isolated CD4+ T helper type 1 (Th1) and Th17 cells, two T cell subpopulations that contribute to the pathogenesis of RA, have not been completely elucidated. Here, we show that TNF-α promotes a reduction and expansion in the frequency of both T cell subsets producing IFN-γ and IL-17, respectively. Selective blockade of TNFR1 or TNFR2 on Th1 and Th17 cells revealed that TNFR2 mediates the decrease in IFN-γ production, while signaling through both receptors augments IL-17 production. We also demonstrate that Th1, but not Th17 cells from RA patients present lower levels of TNFR1 compared to healthy controls, whereas TNFR2 expression on both T cell types is similar between patients and controls. Since TNF-α receptors levels in RA patients are not significantly changed by the therapeutic blockade of TNF-α, we propose that targeting TNFR2 may represent an alternative strategy to normalize the levels of key cytokines that contribute to RA pathogenesis.

Neuropilin-1+ regulatory T cells promote skin allograft survival and modulate effector CD4+ T cells phenotypic signature.

During allograft rejection, several immune cell types, including dendritic cells, CD4(+) and CD8(+) T cells among others, recirculate between the graft and the nearest draining lymph node, resulting in immunity against the 'foreign' tissue. Regulatory CD4(+) T cells are critical for controlling the magnitude of the immune response and may act to promote or maintain tolerance. They are characterized by the expression of CD25 and Foxp3, and more recently, Neuropilin-1 (Nrp1). The role of these suppressor cells during allograft rejection is not well understood. Our work shows that during graft rejection, there is an increase in the frequency of total CD4(+) T cells expressing Nrp1, but the expression of this molecule is downregulated in the regulatory CD4(+) T-cell compartment. Interestingly, the expression of the transcription factor Eos, which renders cell function stability, is also reduced. In adoptive transfer experiments, we observed that during allograft rejection: (i) natural regulatory CD4(+) T cells maintain high levels of Nrp1 expression, (ii) effector CD4(+) T cells (Nrp1(-)) become Nrp1(+)Eos(+) and (iii) the transfer of regulatory CD4(+) T cells (Nrp1(+)) can promote allograft survival, and also enhance the gain of Nrp1 and Eos on T-effector cells. Together, these data suggest that rejection occurs, at least in part, through the loss of Nrp1 expression on regulatory CD4(+) T cells, their stability or both. Additionally, the transfer of regulatory CD4(+) T cells (based on Nrp1 expression) permits the acceptance of the allograft, placing Nrp1 as a new target for immune therapy.

Blocking of p38 and transforming growth factor ß receptor pathways impairs the ability of tolerogenic dendritic cells to suppress murine arthritis.

OBJECTIVE: Dendritic cells (DCs) modulated with lipopolysaccharide (LPS) are able to reduce inflammation when therapeutically administered into mice with collagen-induced arthritis (CIA). The aim of this study was to uncover the mechanisms that define the tolerogenic effect of short-term LPS-modulated DCs on CIA. METHODS: Bone marrow-derived DCs were stimulated for 4 hours with LPS and characterized for their expression of maturation markers and their cytokine secretion profiles. Stimulated cells were treated with SB203580 or SB431542 to inhibit the p38 or transforming growth factor ß (TGFß) receptor pathway, respectively, or were left unmodified and, on day 35 after CIA induction, were used to inoculate mice. Disease severity was evaluated clinically. CD4+ T cell populations were counted in the spleen and lymph nodes from inoculated or untreated mice with CIA. CD4+ splenic T cells were transferred from mice with CIA treated with LPS-stimulated DCs or from untreated mice with CIA into other mice with CIA on day 35 of arthritis. RESULTS: Treatment with LPS-stimulated DCs increased the numbers of interleukin-10 (IL-10)-secreting and TGFß-secreting CD4+ T cells, but decreased the numbers of Th17 cells. Adoptive transfer of CD4+ T cells from treated mice with CIA reproduced the inhibition of active CIA accomplished with LPS-stimulated DCs. The therapeutic effect of LPS-stimulated DCs and their influence on T cell populations were abolished when the p38 and the TGFß receptor pathways were inhibited. CONCLUSION: DCs modulated short-term (4 hours) with LPS are able to confer a sustained cure in mice with established arthritis by re-educating the CD4+ T cell populations. This effect is dependent on the p38 and the TGFß receptor signaling pathways, which suggests the participation of IL-10 and TGFß in the recovery of tolerance.

Ketogenic diet administration later in life improves memory by modifying the synaptic cortical proteome via the PKA signaling pathway in aging mice.

Aging compromises brain function leading to cognitive decline. A cyclic ketogenic diet (KD) improves memory in aged mice after long-term administration; however, short-term effects later in life and the molecular mechanisms that govern such changes remain unclear. Here, we explore the impact of a short-term KD treatment starting at elderly stage on brain function of aged mice. Behavioral testing and long-term potentiation (LTP) recordings reveal that KD improves working memory and hippocampal LTP. Furthermore, the synaptosome proteome of aged mice fed a KD long-term evidence changes predominantly at the presynaptic compartment associated to the protein kinase A (PKA) signaling pathway. These findings were corroborated in vivo by western blot analysis, with high BDNF abundance and PKA substrate phosphorylation. Overall, we show that a KD modifies brain function even when it is administered later in life and recapitulates molecular features of long-term administration, including the PKA signaling pathway, thus promoting synaptic plasticity at advanced age.

The immunological response and post-treatment survival of DC-vaccinated melanoma patients are associated with increased Th1/Th17 and reduced Th3 cytokine responses.

INTRODUCTION: Immunization with autologous dendritic cells (DCs) loaded with a heat shock-conditioned allogeneic melanoma cell lysate caused lysate-specific delayed type hypersensitivity (DTH) reactions in a number of patients. These responses correlated with a threefold prolonged long-term survival of DTH(+) with respect to DTH(-) unresponsive patients. Herein, we investigated whether the immunological reactions associated with prolonged survival were related to dissimilar cellular and cytokine responses in blood. MATERIALS AND METHODS: Healthy donors and melanoma patient's lymphocytes obtained from blood before and after vaccinations and from DTH biopsies were analyzed for T cell population distribution and cytokine release. RESULTS/DISCUSSION: Peripheral blood lymphocytes from melanoma patients have an increased proportion of Th3 (CD4(+) TGF-ß(+)) regulatory T lymphocytes compared with healthy donors. Notably, DTH(+) patients showed a threefold reduction of Th3 cells compared with DTH(-) patients after DCs vaccine treatment. Furthermore, DCs vaccination resulted in a threefold augment of the proportion of IFN-γ releasing Th1 cells and in a twofold increase of the IL-17-producing Th17 population in DTH(+) with respect to DTH(-) patients. Increased Th1 and Th17 cell populations in both blood and DTH-derived tissues suggest that these profiles may be related to a more effective anti-melanoma response. CONCLUSIONS: Our results indicate that increased proinflammatory cytokine profiles are related to detectable immunological responses in vivo (DTH) and to prolonged patient survival. Our study contributes to the understanding of immunological responses produced by DCs vaccines and to the identification of follow-up markers for patient outcome that may allow a closer individual monitoring of patients.

A short protocol using dexamethasone and monophosphoryl lipid A generates tolerogenic dendritic cells that display a potent migratory capacity to lymphoid chemokines.

BACKGROUND: Generation of tolerogenic dendritic cells (TolDCs) for therapy is challenging due to its implications for the design of protocols suitable for clinical applications, which means not only using safe products, but also working at defining specific biomarkers for TolDCs identification, developing shorter DCs differentiation methods and obtaining TolDCs with a stable phenotype. We describe here, a short-term protocol for TolDCs generation, which are characterized in terms of phenotypic markers, cytokines secretion profile, CD4+ T cell-stimulatory ability and migratory capacity. METHODS: TolDCs from healthy donors were generated by modulation with dexamethasone plus monophosphoryl lipid A (MPLA-tDCs). We performed an analysis of MPLA-tDCs in terms of yield, viability, morphology, phenotypic markers, cytokines secretion profile, stability, allogeneic and antigen-specific CD4+ T-cell stimulatory ability and migration capacity. RESULTS: After a 5-day culture, MPLA-tDCs displayed reduced expression of costimulatory and maturation molecules together to an anti-inflammatory cytokines secretion profile, being able to maintain these tolerogenic features even after the engagement of CD40 by its cognate ligand. In addition, MPLA-tDCs exhibited reduced capabilities to stimulate allogeneic and antigen-specific CD4+ T cell proliferation, and induced an anti-inflammatory cytokine secretion pattern. Among potential tolerogenic markers studied, only TLR-2 was highly expressed in MPLA-tDCs when compared to mature and immature DCs. Remarkable, like mature DCs, MPLA-tDCs displayed a high CCR7 and CXCR4 expression, both chemokine receptors involved in migration to secondary lymphoid organs, and even more, in an in vitro assay they exhibited a high migration response towards CCL19 and CXCL12. CONCLUSION: We describe a short-term protocol for TolDC generation, which confers them a stable phenotype and migratory capacity to lymphoid chemokines, essential features for TolDCs to be used as therapeutics for autoimmunity and prevention of graft rejection.

Dexamethasone preconditioning improves the response of collagen-induced arthritis to treatment with short-term lipopolysaccharide-stimulated collagen-loaded dendritic cells.

Background. Pharmacologically modulated dendritic cells (DCs) have been shown to restore tolerance in type II collagen-(CII-) induced arthritis (CIA). We examined the effect of dexamethasone (DXM) administration as a preconditioning agent, followed by an injection of lipopolysaccharide-(LPS-) stimulated and CII-loaded DCs on the CIA course. Methods. After CIA induction, mice pretreated with DXM were injected with 4-hour LPS-stimulated DCs loaded with CII (DXM/4hLPS/CII/DCs). Results. Mice injected with DXM/4hLPS/CII/DCs displayed significantly less severe clinical disease compared to animals receiving 4hLPS/CII/DCs alone or those in which only DXM was administered. Cytokine profile evaluation showed that CD4+ T cells from DXM/4hLPS/CII/DCs and 4hLPS/CII/DCs groups release higher IL-10 levels than those from mice receiving DXM alone or CIA mice. CD4+ T cells from all DC-treated groups showed less IL-17 release when compared to the CIA group. On the contrary, CD4+ T cells from DXM/4hLPS/CII/DCs and 4hLPS/CII/DCs groups released higher IFN- γ levels than those from CIA group. Conclusion. A combined treatment, including DXM preconditioning followed by an inoculation of short-term LPS-stimulated CII-loaded DCs, provides an improved strategy for attenuating CIA severity. Our results suggest that this benefit is driven by a modulation in the cytokine profile secreted by CD4+ T cells.

Weak CD4+ T-cell responses to citrullinated vimentin in rheumatoid arthritis patients carrying HLA-DR9 alleles.

Citrullinated vimentin (cVIM) is one of the antigens specifically targeted by anti-citrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA) patients. The association between ACPA and certain HLA-DRB1 alleles, those coding for the shared epitope (SE), suggests that this response could be T-cell mediated. HLA-DR9 alleles, which do not code for the SE, have recently been associated with ACPA (+) RA. The objective of this work was to study CD4+ T cell responses to cVIM in RA patients and healthy controls carrying HLA-DR9 alleles. Fourteen RA patients and ten healthy controls previously genotyped for HLA-DRB1 were studied for the presence of serum anti-cVIM antibodies by Western blot and ELISA. Peripheral blood mononuclear cells were stimulated with native vimentin and cVIM, and CD4+ T cells proliferation was assessed by flow cytometry. Citrulline-specific CD4+ T cells proliferation was found not only in RA patients but also in healthy controls. Although most patients carrying HLA-DR9 alleles present anti-cVIM antibodies, HLA-DR9 alleles were associated with weaker cVIM-driven CD4+ T-cell responses among RA patients. These results suggest that HLA-DR9 alleles could exert a protective effect on the recognition of cVIM epitopes by CD4+ T cells. In this context, other citrullinated proteins may break T and B cell tolerance, with cVIM only acting as a cross-reactive target for ACPA.

Immunosuppressive Mechanisms of Regulatory B Cells.

Regulatory B cells (Bregs) is a term that encompasses all B cells that act to suppress immune responses. Bregs contribute to the maintenance of tolerance, limiting ongoing immune responses and reestablishing immune homeostasis. The important role of Bregs in restraining the pathology associated with exacerbated inflammatory responses in autoimmunity and graft rejection has been consistently demonstrated, while more recent studies have suggested a role for this population in other immune-related conditions, such as infections, allergy, cancer, and chronic metabolic diseases. Initial studies identified IL-10 as the hallmark of Breg function; nevertheless, the past decade has seen the discovery of other molecules utilized by human and murine B cells to regulate immune responses. This new arsenal includes other anti-inflammatory cytokines such IL-35 and TGF-ß, as well as cell surface proteins like CD1d and PD-L1. In this review, we examine the main suppressive mechanisms employed by these novel Breg populations. We also discuss recent evidence that helps to unravel previously unknown aspects of the phenotype, development, activation, and function of IL-10-producing Bregs, incorporating an overview on those questions that remain obscure.

Microbiota-Derived Metabolites Suppress Arthritis by Amplifying Aryl-Hydrocarbon Receptor Activation in Regulatory B Cells.

The differentiation of IL-10-producing regulatory B cells (Bregs) in response to gut-microbiota-derived signals supports the maintenance of tolerance. However, whether microbiota-derived metabolites can modulate Breg suppressive function remains unknown. Here, we demonstrate that rheumatoid arthritis (RA) patients and arthritic mice have a reduction in microbial-derived short-chain fatty acids (SCFAs) compared to healthy controls and that in mice, supplementation with the SCFA butyrate reduces arthritis severity. Butyrate supplementation suppresses arthritis in a Breg-dependent manner by increasing the level of the serotonin-derived metabolite 5-Hydroxyindole-3-acetic acid (5-HIAA), which activates the aryl-hydrocarbon receptor (AhR), a newly discovered transcriptional marker for Breg function. Thus, butyrate supplementation via AhR activation controls a molecular program that supports Breg function while inhibiting germinal center (GC) B cell and plasmablast differentiation. Our study demonstrates that butyrate supplementation may serve as a viable therapy for the amelioration of systemic autoimmune disorders.

Regulation of Tolerogenic Features on Dexamethasone-Modulated MPLA-Activated Dendritic Cells by MYC.

The potential of tolerogenic dendritic cells (tolDCs) to shape immune responses and restore tolerance has turn them into a promising therapeutic tool for cellular therapies directed toward immune regulation in autoimmunity. Although the cellular mechanisms by which these cells can exert their regulatory function are well-known, the mechanisms driving their differentiation and function are still poorly known, and the variety of stimuli and protocols applied to differentiate DCs toward a tolerogenic phenotype makes it even more complex to underpin the molecular features involved in their function. Through transcriptional profiling analysis of monocyte-derived tolDCs modulated with dexamethasone (Dex) and activated with monophosphoryl lipid A (MPLA), known as DM-DCs, we were able to identify MYC as one of the transcriptional regulators of several genes differentially expressed on DM-DCs compared to MPLA-matured DCs (M-DCs) and untreated/immature DCs (DCs) as revealed by Ingenuity Pathway Analysis (IPA) upstream regulators evaluation. Additionally, MYC was also amidst the most upregulated genes in DM-DCs, finding that was confirmed at a transcriptional as well as at a protein level. Blockade of transactivation of MYC target genes led to the downregulation of tolerance-related markers IDO1 and JAG1. MYC blockade also led to downregulation of PLZF and STAT3, transcription factors associated with immune regulation and inhibition of DC maturation, further supporting a role of MYC as an upstream regulator contributing to the regulatory phenotype of DM-DCs. On the other hand, we had previously shown that fatty acid oxidation, oxidative metabolism and zinc homeostasis are amongst the main biological functions represented in DM-DCs, and here we show that DM-DCs exhibit higher intracellular expression of ROS and Zinc compared to mature M-DCs and DCs. Taken together, these findings suggest that the regulatory profile of DM-DCs is partly shaped by the effect of the transcriptional regulation of tolerance-inducing genes by MYC and the modulation of oxidative metabolic processes and signaling mediators such as Zinc and ROS.

Inflammation, synovial angiogenesis and chondroid apoptosis in the evolution of type II collagen-induced arthritis.

Using the murine model of type II collagen-induced arthritis (CIA), we studied its evolution over time by histopathological, immunohistochemical and clinical evaluations. The first clinical symptoms appeared 28 days post-inoculation (dpi), with bovine type II collagen, with an average arthritic index of 1.00 +/- 0.48 corresponding to erythema of the articulation. The disease progressed, and by 70 dpi showed an average arthritic index of 3.83 +/- 0.27 corresponding to edema and maximum deformation, with ankylosis. Computed morphometry demonstrated that, in comparison to controls, the induction of CIA, produces a significant and increasing accumulation of inflammatory cells, fibrosis (p < 0.0001) and cartilage destruction (p = 0.0029). Likewise, the area of von Willebrand factor (vWF) immunostaining, as an indicator of endothelial proliferation, increased significantly from 28 dpi (p < 0.0001), in CIA mice compared to controls. However, the effective synovial vascularization, calculated as the synovial vascular bed area index, significantly increased by 42 dpi (p = 0.0014). This indicates that the activation and proliferation of endothelium becomes significant before an effective vascularization area is formed. The apoptosis index was also an earlier indicator of cartilage damage, becoming significant from 28 dpi in comparison to controls (p < 0.0001). Finally, it was observed that the increase in the arthritic index showed a strong correlation with the increase in both angiogenesis (r = 0.95; p = 0.0021) and apoptosis (r = 0.90; p = 0.0015). In conclusion, a robust correlation between synovial membrane inflammation, angiogenesis and chondrocyte apoptosis, with respect to the increase in the clinical severity of CIA, has been demonstrated by a quantitative computer-assisted immunomorphometric analysis.

Short-term lipopolysaccharide stimulation induces differentiation of murine bone marrow-derived dendritic cells into a tolerogenic phenotype.

Dendritic cells (DCs) are professional, antigen-presenting cells, which induce and regulate T cell reactivity. DCs are crucial in innate and adaptive immune responses, and are also involved in central and peripheral tolerance induction. Tolerance can be mediated by immature and semi-mature DCs expressing low levels of co-stimulator and major histocompatibility complex (MHC) molecules. The aim of this study was to investigate the ability of short-term lipopolysaccharide (LPS) stimulation to modulate the stage of differentiation of bone marrow-derived DCs. For this purpose, DCs obtained from DBA1/lacJ mice were stimulated for four (4hLPS/DCs) or 24 (24hLPS/DCs) hours with LPS, using DCs without stimulation (0hLPS/DCs) as a control. Flow cytometry analysis of 4hLPS/DCs showed intermediate CD40 and MHC class II expression, lower than that of 24hLPS/DCs (fully mature), and greater than that of 0hLPS/DCs (immature). A functional assay showed that 4hLPS/DCs displayed increased endocytotic ability compared to 24hLPS/DCs, indicating a semi-mature state. 4hLPS/DCs were greater producers of IL-10 protein and TGFbeta1 mRNA than 24hLPS/DCs and immature DCs, displaying a cytokine production pattern that is characteristic of tolerogenic DCs. An assay for antigen-presenting capacity demonstrated that 4hLPS/DCs induced secretion of IL-2 from an OTH4 T cell hybridoma, indicating a functional presenting activity. Finally, the tolerogenic phenotype of 4hLPS/DCs was demonstrated by their ability to interfere with the progression of bovine type II collagen (bII)-induced arthritis (CIA) when they were loaded with bCII antigen and injected into mice with established CIA. We conclude that the stimulation of murine bone marrow-derived DCs with LPS for four hours generates semi-mature DCs with tolerogenic capability.

Analysis of the HLA-DR peptidome from human dendritic cells reveals high affinity repertoires and nonconventional pathways of peptide generation.

Dendritic cells (DCs) are the major professional APCs of the immune system; however, their MHC-II-associated peptide repertoires have been hard to analyze, mostly because of their scarce presence in blood and tissues. In vitro matured human monocyte-derived DCs (MoDCs) are widely used as professional APCs in experimental systems. In this work, we have applied mass spectrometry to identify the HLA-DR-associated self-peptide repertoires from small numbers of mature MoDCs (∼5 × 106 cells), derived from 7 different donors. Repertoires of 9 different HLA-DR alleles were defined from analysis of 1319 peptides, showing the expected characteristics of MHC-II-associated peptides. Most peptides identified were predicted high binders for their respective allele, formed nested sets, and belonged to endo-lysosomal pathway-degraded proteins. Approximately 20% of the peptides were derived from cytosolic and nuclear proteins, a recurrent finding in HLA-DR peptide repertoires. Of interest, most of these peptides corresponded to single sequences, did not form nested sets, and were located at the C terminus of the parental protein, which suggested alternative processing. Analysis of cleavage patterns for terminal peptides predominantly showed aspartic acid before the cleavage site of both C- and N-terminal peptides and proline immediately after the cleavage site in C-terminal peptides. Proline was also frequent next to the cut sites of internal peptides. These data provide new insights into the Ag processing capabilities of DCs. The relevance of these processing pathways and their contribution to response to infection, tolerance induction, or autoimmunity deserve further analysis.

TIM-1 defines a human regulatory B cell population that is altered in frequency and function in systemic sclerosis patients.

BACKGROUND: Systemic sclerosis (SSc) is a systemic autoimmune disease characterized by excessive production of extracellular matrix by fibroblasts on skin and internal organs. Although Th2 cells have been involved in fibroblast stimulation, hyperactivated B cells may also play an important role. Regulatory B cells (Bregs) are cells capable of inhibiting inflammatory responses and controlling autoimmune diseases. Although many Breg populations have in common the ability to produce high amounts of IL-10, a unique surface marker defining most human Bregs is lacking. It has been described in mice that T cell Ig and mucin domain protein 1 (TIM-1) is an inclusive marker for Bregs, and that TIM-1+ B cells are able to prevent the development of autoimmunity. The aim of this work was to evaluate TIM-1 as a marker for human IL-10+ Bregs, and to determine whether TIM-1+ B cells are defective in SSc patients. METHODS: SSc patients (n = 39) and 53 healthy subjects were recruited. TIM-1 and IL-10 expression was assessed in resting or activated peripheral blood CD19+ B cells by flow cytometry. The regulatory function of TIM-1+ or activated B cells from SSc patients and healthy subjects was assessed in autologous and allogenic co-cultures with CD4+ T cells, where T cell proliferation and IFN-γ, IL-17, TNF-α and IL-4 production by T cells was measured by flow cytometry. RESULTS: TIM-1 and IL-10 were preferentially expressed in transitional B cells, but were upregulated in naïve and memory B cells upon stimulation. The frequency of transitional TIM-1+ IL-10+ B cells was significantly decreased in SSc patients compared to healthy controls. In addition, activated B cells from SSc patients induced stronger allogenic Th1 and Th2 responses than activated B cells from healthy controls. Finally, TIM-1+ B cells, including transitional and non-transitional cells, exhibited a higher CD4+ T cell suppressive ability than TIM-1- B cells in healthy controls, but not in SSc patients. CONCLUSIONS: TIM-1 is a unique marker for the identification of a human IL-10+ Breg subpopulation which is partially superimposed with transitional B cells. Alterations in TIM-1+ B cells could contribute to the development of autoimmune diseases such as SSc.

Dexamethasone and Monophosphoryl Lipid A Induce a Distinctive Profile on Monocyte-Derived Dendritic Cells through Transcriptional Modulation of Genes Associated With Essential Processes of the Immune Response.

There is growing interest in the use of tolerogenic dendritic cells (tolDCs) as a potential target for immunotherapy. However, the molecular bases that drive the differentiation of monocyte-derived DCs (moDCs) toward a tolerogenic state are still poorly understood. Here, we studied the transcriptional profile of moDCs from healthy subjects, modulated with dexamethasone (Dex) and activated with monophosphoryl lipid A (MPLA), referred to as Dex-modulated and MPLA-activated DCs (DM-DCs), as an approach to identify molecular regulators and pathways associated with the induction of tolerogenic properties in tolDCs. We found that DM-DCs exhibit a distinctive transcriptional profile compared to untreated (DCs) and MPLA-matured DCs. Differentially expressed genes downregulated by DM included MMP12, CD1c, IL-1B, and FCER1A involved in DC maturation/inflammation and genes upregulated by DM included JAG1, MERTK, IL-10, and IDO1 involved in tolerance. Genes related to chemotactic responses, cell-to-cell signaling and interaction, fatty acid oxidation, metal homeostasis, and free radical scavenging were strongly enriched, predicting the activation of alternative metabolic processes than those driven by counterpart DCs. Furthermore, we identified a set of genes that were regulated exclusively by the combined action of Dex and MPLA, which are mainly involved in the control of zinc homeostasis and reactive oxygen species production. These data further support the important role of metabolic processes on the control of the DC-driven regulatory immune response. Thus, Dex and MPLA treatments modify gene expression in moDCs by inducing a particular transcriptional profile characterized by the activation of tolerance-associated genes and suppression of the expression of inflammatory genes, conferring the potential to exert regulatory functions and immune response modulation.

Dexamethasone and Monophosphoryl Lipid A-Modulated Dendritic Cells Promote Antigen-Specific Tolerogenic Properties on Naive and Memory CD4+ T Cells.

Tolerogenic dendritic cells (DCs) are a promising tool to control T cell-mediated autoimmunity. Here, we evaluate the ability of dexamethasone-modulated and monophosphoryl lipid A (MPLA)-activated DCs [MPLA-tolerogenic DCs (tDCs)] to exert immunomodulatory effects on naive and memory CD4+ T cells in an antigen-specific manner. For this purpose, MPLA-tDCs were loaded with purified protein derivative (PPD) as antigen and co-cultured with autologous naive or memory CD4+ T cells. Lymphocytes were re-challenged with autologous PPD-pulsed mature DCs (mDCs), evaluating proliferation and cytokine production by flow cytometry. On primed-naive CD4+ T cells, the expression of regulatory T cell markers was evaluated and their suppressive ability was assessed in autologous co-cultures with CD4+ effector T cells and PPD-pulsed mDCs. We detected that memory CD4+ T cells primed by MPLA-tDCs presented reduced proliferation and proinflammatory cytokine expression in response to PPD and were refractory to subsequent stimulation. Naive CD4+ T cells were instructed by MPLA-tDCs to be hyporesponsive to antigen-specific restimulation and to suppress the induction of T helper cell type 1 and 17 responses. In conclusion, MPLA-tDCs are able to modulate antigen-specific responses of both naive and memory CD4+ T cells and might be a promising strategy to "turn off" self-reactive CD4+ effector T cells in autoimmunity.