VIP/VPAC Axis Expression in Immune-Mediated Inflammatory Disorders: Associated miRNA Signatures.

Few studies have considered immune-mediated inflammatory disorders (IMID) together, which is necessary to adequately understand them given they share common mechanisms. Our goal was to investigate the expression of vasoactive intestinal peptide (VIP) and its receptors VPAC1 and VPAC2 in selected IMID, analyze the effect of biological therapies on them, and identify miRNA signatures associated with their expression. Serum VIP levels and mRNA of VPAC and miRNA expression in peripheral blood mononuclear cells were analyzed from 52 patients with psoriasis, rheumatoid arthritis, Graves' disease, or spondyloarthritis and from 38 healthy subjects. IMID patients showed higher levels of VIP and increased expression of VPAC2 compared to controls (p < 0.0001 and p < 0.0192, respectively). Receiver operating characteristic curve analysis showed that the levels of VIP or VPAC2 expression were adequate discriminators capable of identifying IMID. Treatment of IMID patients with anti-TNFα and anti-IL12/23 significantly affected serum VIP levels. We identified miRNA signatures associated with levels of serum VIP and VPAC2 expression, which correlated with IMID diagnosis of the patients. The results indicate that the expression of VIP/VPAC2 is able of identify IMIDs and open up a line of research based on the association between the VIP/VPAC axis and miRNA signatures in immune-mediated diseases.

Genetic LGALS1 Variants Are Associated with Heterogeneity in Galectin-1 Serum Levels in Patients with Early Arthritis.

Galectin 1 (Gal1) exerts immunomodulatory effects leading to therapeutic effects in autoimmune animal models. Patients with rheumatoid arthritis have been reported to show higher Gal1 serum levels than the healthy population. Our study aimed to find genetic variants on the Gal1 gene (LGALS1) modulating its expression and/or clinical features in patients with early arthritis (EA). LGALS1 was sequenced in 53 EA patients to characterize all genetic variants. Then, we genotyped rs9622682, rs929039, and rs4820293, which covered the main genetic variation in LGALS1, in 532 EA patients. Gal1 and IL-6 serum levels were measured by ELISA and Gal1 also by western blot (WB) in lymphocytes from patients with specific genotypes. Once disease activity improved with treatment, patients with at least one copy of the minor allele in rs9622682 and rs929039 or those with GG genotype in rs4820293 showed significantly higher Gal1 serum levels (p < 0.05). These genotypic combinations were also associated with higher Gal1 expression in lymphocytes by WB and lower IL-6 serum levels in EA patients. In summary, our study suggests that genetic variants studied in LGALS1 can explain heterogeneity in Gal1 serum levels showing that patients with higher Gal1 levels have lower serum IL-6 levels.

Human CD4+CD45RA+ T Cells Behavior after In Vitro Activation: Modulatory Role of Vasoactive Intestinal Peptide.

Naїve CD4+ T cells, which suffer different polarizing signals during T cell receptor activation, are responsible for an adequate immune response. In this study, we aimed to evaluate the behavior of human CD4+CD45RA+ T cells after in vitro activation by anti-CD3/CD28 bead stimulation for 14 days. We also wanted to check the role of the VIP system during this process. The metabolic biomarker Glut1 was increased, pointing to an increase in glucose requirement whereas Hif-1α expression was higher in resting than in activated cells. Expression of Th1 markers increased at the beginning of activation, whereas Th17-associated biomarkers augmented after that, showing a pathogenic Th17 profile with a possible plasticity to Th17/1. Foxp3 mRNA expression augmented from day 4, but no parallel increases were observed in IL-10, IL-2, or TGFß mRNA expression, meaning that these potential differentiated Treg could not be functional. Both VIP receptors were located on the plasma membrane, and expression of VPAC2 receptor increased significantly with respect to the VPAC1 receptor from day 4 of CD4+CD45RA+ T activation, pointing to a shift in VPAC receptors. VIP decreased IFNγ and IL-23R expression during the activation, suggesting a feasible modulation of Th17/1 plasticity and Th17 stabilization through both VPAC receptors. These novel results show that, without polarizing conditions, CD4+CD45RA+ T cells differentiate mainly to a pathogenic Th17 subset and an unpaired Treg subset after several days of activation. Moreover, they confirm the important immunomodulatory role of VIP, also on naїve Th cells, stressing the importance of this neuropeptide on lymphocyte responses in different pathological or non-pathological situations.

Comparative Study of Senescent Th Biomarkers in Healthy Donors and Early Arthritis Patients. Analysis of VPAC Receptors and Their Influence.

Pro-inflammatory CD4+CD28- T cells are characteristic of immunosenescence, but also of several autoimmune/inflammatory diseases. Vasoactive intestinal peptide (VIP) acts as an anti-inflammatory and immunomodulatory mediator on these cells. Our objective was to study the mutual influence between senescent Th cells and VIP axis in early arthritis (EA), comparing with non-EA donors. We characterized the correlation between senescent Th cells and clinic parameters of EA as well as the behavior of senescent Th biomarkers by real-time PCR. Clinical data were systematically recorded at baseline and after 6 months of follow-up. The number of CD4+CD28- T cells measured by sorting is higher in patients who initially meet ACR classification criteria for rheumatoid arthritis (RA) compared to those who were classified as undifferentiated arthritis (UA). A slight positive correlation between EA CD4+CD28- T cells and CRP or ESR and a negative correlation with bone mineral density were found. Th senescent biomarkers in EA CD4+CD28- T cells were similar to donors, however some of them increased after 6 months of follow-up. VPAC receptors were analyzed by real-time PCR and immunofluorescence, and CD4+CD28- T cells showed higher expression of VPAC2 and lower of VPAC1, VPAC2 showing a significant increased expression in EA cells. Sorted CD4+CD28- T cells were in vitro expanded in presence of VIP, wherein VIP increased senescent biomarker CD27, while it diminished CD57 or NKG2 senescent biomarkers. Our study demonstrates for the first time the existence of a link between senescent Th cells and the VIP axis.

A Clinical Approach for the Use of VIP Axis in Inflammatory and Autoimmune Diseases.

The neuroendocrine and immune systems are coordinated to maintain the homeostasis of the organism, generating bidirectional communication through shared mediators and receptors. Vasoactive intestinal peptide (VIP) is the paradigm of an endogenous neuropeptide produced by neurons and endocrine and immune cells, involved in the control of both innate and adaptive immune responses. Exogenous administration of VIP exerts therapeutic effects in models of autoimmune/inflammatory diseases mediated by G-protein-coupled receptors (VPAC1 and VPAC2). Currently, there are no curative therapies for inflammatory and autoimmune diseases, and patients present complex diagnostic, therapeutic, and prognostic problems in daily clinical practice due to their heterogeneous nature. This review focuses on the biology of VIP and VIP receptor signaling, as well as its protective effects as an immunomodulatory factor. Recent progress in improving the stability, selectivity, and effectiveness of VIP/receptors analogues and new routes of administration are highlighted, as well as important advances in their use as biomarkers, contributing to their potential application in precision medicine. On the 50th anniversary of VIP's discovery, this review presents a spectrum of potential clinical benefits applied to inflammatory and autoimmune diseases.

Methotrexate limits inflammation through an A20-dependent cross-tolerance mechanism.

OBJECTIVES: Methotrexate (MTX) is the anchor drug for treatment of rheumatoid arthritis (RA), but the mechanism of its anti-inflammatory action is not fully understood. In RA, macrophages display a proinflammatory polarisation profile that resembles granulocyte-macrophage colony-stimulating factor (GM-CSF)-differentiated macrophages and the response to MTX is only observed in thymidylate synthase+ GM-CSF-dependent macrophages. To determine the molecular basis for the MTX anti-inflammatory action, we explored toll-like receptor (TLR), RA synovial fluid (RASF) and tumour necrosis factor receptor (TNFR)-initiated signalling in MTX-exposed GM-CSF-primed macrophages. METHODS: Intracellular responses to TLR ligands, TNFα or RASF stimulation in long-term low-dose MTX-exposed human macrophages were determined through quantitative real-time PCR, western blot, ELISA and siRNA-mediated knockdown approaches. The role of MTX in vivo was assessed in patients with arthritis under MTX monotherapy and in a murine sepsis model. RESULTS: MTX conditioned macrophages towards a tolerant state, diminishing interleukin (IL)-6 and IL-1ß production in LPS, LTA, TNFα or RASF-challenged macrophages. MTX attenuated LPS-induced MAPK and NF-κB activation, and toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF1)-dependent signalling. Conversely, MTX increased the expression of the NF-κB suppressor A20 (TNFAIP3), itself a RA-susceptibility gene. Mechanistically, MTX-induced macrophage tolerance was dependent on A20, as siRNA-mediated knockdown of A20 reversed the MTX-induced reduction of IL-6 expression. In vivo, TNFAIP3 expression was significantly higher in peripheral blood cells of MTX-responsive individuals from a cohort of patients with arthritis under MTX monotherapy, whereas MTX-treated mice exhibited reduced inflammatory responses to LPS. CONCLUSIONS: MTX impairs macrophage proinflammatory responses through upregulation of A20 expression. The A20-mediated MTX-induced innate tolerance might limit inflammation in the RA synovial context, and positions A20 as a potential MTX-response biomarker.

Vasoactive intestinal peptide gene polymorphisms, associated with its serum levels, predict treatment requirements in early rheumatoid arthritis.

We previously reported that early arthritis (EA) patients with low vasoactive intestinal peptide (VIP) serum levels demonstrate a worse clinical disease course. In this study, we analysed whether variants in the VIP gene correlated with its serum levels and clinical EA parameters. The VIP gene was sequenced in patients with extremely high/low VIP levels, measured by enzyme immunoassay. Sixteen single nucleotide polymorphisms (SNPs) were differentially distributed between both groups, which were subsequently genotyped in two patients' sets. We observed that patients with rs688136 CC genotype showed higher VIP levels in both discovery (n = 91; p = 0.033) and validation populations (n = 131; p = 0.007). This effect was attenuated by the presence of minor alleles rs35643203 and rs12201140, which showed a clear trend towards low VIP level association (p = 0.118 and p = 0.049, respectively). Functional studies with miR-205-5p, which has a target site in the 3' UTR close to rs688136, revealed a miRNA-mediated regulatory mechanism explaining the higher VIP gene expression in homozygous patients. Moreover, patients with an rs688136 CC genotype and no minor alleles of the other polymorphisms required less treatment (p = 0.009). We concluded that the identification of polymorphisms associated with VIP serum levels would complement the clinical assessment of the disease severity in rheumatoid arthritis patients.

Clinical Relevance of VPAC1 Receptor Expression in Early Arthritis: Association with IL-6 and Disease Activity.

BACKGROUND: The vasoactive intestinal peptide (VIP) receptors VPAC1 and VPAC2 mediate anti-inflammatory and immunoregulatory responses in rheumatoid arthritis (RA). Data on the expression of these receptors could complement clinical assessment in the management of RA. Our goal was to investigate the correlation between expression of both receptors and the 28-Joint Disease Activity Score (DAS28) in peripheral blood mononuclear cells (PBMCs) from patients with early arthritis (EA). We also measured expression of IL-6 to evaluate the association between VIP receptors and systemic inflammation. METHODS: We analyzed 250 blood samples collected at any of the 5 scheduled follow-up visits from 125 patients enrolled in the Princesa Early Arthritis Register Longitudinal study. Samples from 22 healthy donors were also analyzed. Sociodemographic, clinical, and therapeutic data were systematically recorded. mRNA expression levels were determined using real-time PCR. Then, longitudinal multivariate analyses were performed. RESULTS: PBMCs from EA patients showed significantly higher expression of VPAC2 receptors at baseline compared to healthy donors (p<0.001). With time, however, VPAC2 expression tended to be significantly lower while VPAC1 receptor expression increased in correlation with a reduction in DAS28 index. Our results reveal that more severe inflammation, based on high levels of IL-6, is associated with lower expression of VPAC1 (p<0.001) and conversely with increased expression of VPAC2 (p<0.001). A major finding of this study is that expression of VPAC1 is lower in patients with increased disease activity (p = 0.001), thus making it possible to differentiate between patients with various degrees of clinical disease activity. CONCLUSION: Patients with more severe inflammation and higher disease activity show lower levels of VPAC1 expression, which is associated with patient-reported impairment. Therefore, VPAC1 is a biological marker in EA.

The Minor Allele of rs7574865 in the STAT4 Gene Is Associated with Increased mRNA and Protein Expression.

OBJECTIVE: The T allele of rs7574865 in STAT4 confers risk of developing autoimmune disorders. However, its functional significance remains unclear. Here we analyze how rs7574865 affects the transcription of STAT4 and its protein expression. METHODS: We studied 201 patients (80% female; median age, 54 years; median disease duration, 5.4 months) from PEARL study. Demographic, clinical, laboratory and therapeutic data were collected at each visit. IL-6 serum levels were measured by enzyme immune assay. The rs7574865 was genotyped using TaqMan probes. The expression levels of STAT4 mRNA were determined at 182 visits from 69 patients using quantitative real-time polymerase chain reaction. STAT4 protein was assessed by western blot in 62 samples from 34 patients. To determine the effect of different variables on the expression of STAT4 mRNA and protein, we performed multivariate longitudinal analyses using generalized linear models. RESULTS: After adjustment for age, disease activity and glucocorticoid dose as confounders, the presence of at least one copy of the T allele of rs7574865 was significantly associated with higher levels of STAT4 mRNA. Similarly, TT patients showed significantly higher levels of STAT4 protein than GG patients. IL-6 induced STAT4 and STAT5 phosphorylation in peripheral blood lymphocytes. Patients carrying at least one T allele of rs7574865 displayed lower levels of serum IL-6 compared to GG homozygous; by contrast the production of C-reactive protein was similar in both populations. CONCLUSION: Our data suggest that the presence of the rs7574865 T allele enhances STAT4 mRNA transcription and protein expression. It may enhance the signaling of molecules depending on the STAT4 pathway.

Vasoactive Intestinal Peptide in Early Spondyloarthritis: Low Serum Levels as a Potential Biomarker for Disease Severity.

Spondyloarthritis (SpA) is a family of inflammatory diseases sharing clinical, genetic, and radiological features. While crucial for tailoring early interventions, validated prognostic biomarkers are scarce in SpA. We analyze the correlation between serum levels of vasoactive intestinal peptide (VIP) and disease activity/severity in patients with early chronic inflammatory back pain. The study population comprised 54 patients enrolled in our early chronic inflammatory back pain register. We collected demographic information, clinical data, laboratory data, and imaging findings. VIP levels were measured by enzyme immunoassay in serum samples from 162 visits. The association between independent variables and VIP levels was analyzed using longitudinal multivariate analysis nested by patient and visit. No significant differences were observed in VIP levels between these two groups. Lower levels of VIP were significantly associated with a higher Bath Ankylosing Spondylitis Disease Activity Index (BASFI) score, presence of bone edema in magnetic resonance imaging (MRI) scan, and lower hemoglobin levels. Coexistence of cutaneous psoriasis was independently associated with lower VIP levels, and similar trend was observed for enthesitis. We conclude that SpA patients with low serum VIP levels had worse 2-year disease outcome, suggesting that serum VIP levels could be a valid prognostic biomarker.

Serum levels of vasoactive intestinal peptide as a prognostic marker in early arthritis.

OBJECTIVE: Suitable biomarkers are essential for the design of therapeutic strategies in personalized medicine. Vasoactive intestinal peptide (VIP) has demonstrated immunomodulatory properties in autoimmune murine and ex vivo human models. Our aim was to study serum levels of VIP during the follow-up of an early arthritis (EA) cohort and to analyze its value as a biomarker predicting severity and therapeutic requirements. METHODS: Data from 91 patients on an EA register were analyzed (76% rheumatoid arthritis (RA), 24% undifferentiated arthritis, 73% women, and median age 54 years; median disease duration at entry, 5.4 months). We collected per protocol sociodemographic, clinical, and therapeutic data. VIP levels were determined by enzyme immunoassay in sera harvested from the 91 patients (353 visits; 3.9 visit/patient) and from 100 healthy controls. VIP values below the 25(th) percentile of those assessed in healthy population were considered low. To determine the effect of independent variables on VIP levels, we performed a longitudinal multivariate analysis nested by patient and visit. A multivariate ordered logistic regression was modeled to determine the effect of low VIP serum levels on disease activity at the end of follow-up. RESULTS: VIP concentrations varied considerably across EA patients. Those fulfilling the criteria for RA had the lowest values in the whole sample, although no significant differences were observed compared with healthy donors. Disease activity, which was assessed using DAS28, inversely correlated with VIP levels. After a two-year follow-up, those patients with low baseline levels of VIP displayed higher disease activity and received more intensive treatment. CONCLUSION: Patients who are unable to up-regulate VIP seem to have a worse clinical course despite receiving more intense treatment. Therefore, measurement of VIP levels may be suitable as a prognostic biomarker.

The metalloprotease ADAM8 is associated with and regulates the function of the adhesion receptor PSGL-1 through ERM proteins.

The P-selectin glycoprotein ligand-1 (PSGL-1) is involved in the initial contact of leukocytes with activated endothelium, and its adhesive function is regulated through its proteolytic processing. We have found that the metalloprotease ADAM8 is both associated with PSGL-1 through the ezrin­radixin­moesin actin-binding proteins and able to cause the proteolytic cleavage of this adhesion receptor. Accordingly, ADAM8 knockdown increases PSGL-1 expression, and functional assays show that ADAM8 is able to reduce leukocyte rolling on P-selectin and hence on activated endothelial cells. We conclude that ADAM8 modulates the expression and function of PSGL-1.

Characterization of serum interleukin-15 in healthy volunteers and patients with early arthritis to assess its potential use as a biomarker.

As interleukin-15 (IL-15) has been implicated in the pathophysiology of rheumatoid arthritis, we analysed the serum IL-15 (sIL-15) levels in healthy subjects and patients with early arthritis to establish a cut-off point that might serve to define elevated sIL-15. This is an initial step to determine whether sIL-15 has the potential for use as a biomarker for patients with early arthritis. The IL-15 concentration was measured in serum obtained from 161 healthy controls and from 174 patients with early arthritis, and the relationship between the expression of the two IL-15 mRNA variants and the sIL-15 levels was also assessed. In healthy controls, the median sIL-15 value was 0.83 [interquartile range (IQR) 0-8.68] pg/mL; there was no significant difference in the sIL-15 values according to gender [median level in males was 1.99 (IQR: 0-8.68) pg/mL and in females 0.50 (0-8.25) pg/mL: p = 0.821]. Moreover, sIL-15 levels did not correlate with age (r = 0.033, p = 0.685), and they did not display a clear circadian rhythm in healthy donors, with the median values for IL-15 close to zero at each time tested. In the light of these findings, we considered that sIL-15 was elevated if its concentration was above 20 pg/mL, since this cut-off point corresponded to the 90th percentile for this healthy population. We found that 30% of the patients with early arthritis had sIL-15 values > 20 pg/mL. The levels of sIL-15 did not correlate with disease duration in early arthritis patients, nor did they fluctuate with changes in disease activity over the follow-up period. In addition, the high level of sIL15 in patients was not associated with alterations in the alternative splicing of the IL-15 mRNA, favouring the variant that produces the protein with a long signal peptide for secretion. Serum IL-15 levels were increased in a subpopulation of patients with early arthritis, indicating that this measure may serve as a biomarker for this condition. Further studies will be necessary to determine whether the clinical evolution or response to treatment of patients with high sIL-15 levels differs.

F-actin-binding protein drebrin regulates CXCR4 recruitment to the immune synapse.

The adaptive immune response depends on the interaction of T cells and antigen-presenting cells at the immune synapse. Formation of the immune synapse and the subsequent T-cell activation are highly dependent on the actin cytoskeleton. In this work, we describe that T cells express drebrin, a neuronal actin-binding protein. Drebrin colocalizes with the chemokine receptor CXCR4 and F-actin at the peripheral supramolecular activation cluster in the immune synapse. Drebrin interacts with the cytoplasmic tail of CXCR4 and both proteins redistribute to the immune synapse with similar kinetics. Drebrin knockdown in T cells impairs the redistribution of CXCR4 and inhibits actin polymerization at the immune synapse as well as IL-2 production. Our data indicate that drebrin exerts an unexpected and relevant functional role in T cells during the generation of the immune response.

Identification of genes responsive to solar simulated UV radiation in human monocyte-derived dendritic cells.

Ultraviolet (UV) irradiation has profound effects on the skin and the systemic immune system. Several effects of UV radiation on Dendritic cells (DCs) functions have been described. However, gene expression changes induced by UV radiation in DCs have not been addressed before. In this report, we irradiated human monocyte-derived DCs with solar-simulated UVA/UVB and analyzed regulated genes on human whole genome arrays. Results were validated by RT-PCR and further analyzed by Gene Set Enrichment Analysis (GSEA). Solar-simulated UV radiation up-regulated expression of genes involved in cellular stress and inflammation, and down-regulated genes involved in chemotaxis, vesicular transport and RNA processing. Twenty four genes were selected for comparison by RT-PCR with similarly treated human primary keratinocytes and human melanocytes. Several genes involved in the regulation of the immune response were differentially regulated in UVA/UVB irradiated human monocyte-derived DCs, such as protein tyrosine phosphatase, receptor type E (PTPRE), thrombospondin-1 (THBS1), inducible costimulator ligand (ICOSL), galectins, Src-like adapter protein (SLA), IL-10 and CCR7. These results indicate that UV-exposure triggers the regulation of a complex gene repertoire involved in human-DC-mediated immune responses.

Functional role of P-selectin glycoprotein ligand 1/P-selectin interaction in the generation of tolerogenic dendritic cells.

Dendritic cells (DCs) have a key role in both the generation of the immune response and the induction of tolerance to self-Ags. In this work, the possible role of P-selectin glycoprotein ligand 1 (PSGL-1) on the tolerogenic activity of human DCs was explored. We found that the engagement of PSGL-1 by P-selectin on DCs induced the expression of c-Fos, IDO, IL-10, and TGF-beta genes. Remarkably, stimulation of DCs through PSGL-1 with P-selectin enhanced their capability to generate CD4(+)CD25(+)Foxp3(+) regulatory T cells, which expressed high levels of TGF-beta1 mRNA, synthesized IL-10, and suppressed the proliferation of autologous CD4(+)CD25(-) T cells. Accordingly, we found that DCs from PSGL-1(-/-) mice expressed higher levels of MHC class II molecules, and exhibited an enhanced immunogenicity compared with wild-type mice. In addition, the percentage of CD4(+)CD25(+)Foxp3(+) regulatory T cells in the thymus of PSGL-1-deficient animals was significantly reduced. Our data reveal an unexpected role of PSGL-1 on the tolerogenic function of DCs, and the regulation of the immune response.

The role of CD69 in acute neutrophil-mediated inflammation.

The leukocyte activation marker CD69 functions as a negative regulator of the immune response, both in NK-dependent tumor rejection and in the inflammation associated with lymphocyte-dependent collagen-induced arthritis. In contrast, it has been reported that CD69-deficient mice are refractory to the neutrophil-dependent acute inflammatory response associated with anti-type II collagen antibody-induced arthritis (CAIA), suggesting a positive regulatory role for CD69 in neutrophil function during arthritis induction. To clarify this discrepancy, the CAIA response was independently analyzed in our CD69-deficient mice. In these experiments, the inflammatory response was unaffected by CD69 deficiency. Additionally, the in vivo down-regulation of CD69 expression by treatment of wild-type mice with the anti-CD69 mAb 2.2, which mimics the CD69-deficient phenotype, did not affect the course of arthritis in this model. Moreover, down-regulation of CD69 expression increased expression in arthritic joints of key inflammatory mediators, including IL-1beta, IL-6 and the chemokine MCP-1. Neutrophil accumulation in zymosan-treated air pouches and in thioglycolate-treated peritoneal cavities was also unaffected in CD69-deficient mice. In addition, CD69 expression was absent in activated neutrophils. Taken together, these results rule out a significant stimulatory role for CD69 in acute inflammatory responses mediated by neutrophils.

Synaptic clusters of MHC class II molecules induced on DCs by adhesion molecule-mediated initial T-cell scanning.

Initial adhesive contacts between T lymphocytes and dendritic cells (DCs) facilitate recognition of peptide-MHC complexes by the TCR. In this report, we studied the dynamic behavior of adhesion and Ag receptors on DCs during initial contacts with T-cells. Adhesion molecules LFA-1- and ICAM-1,3-GFP as well as MHC class II-GFP molecules were very rapidly concentrated at the DC contact area. Binding of ICAM-3, and ICAM-1 to a lesser extent, to LFA-1 expressed by mature but not immature DC, induced MHC-II clustering into the immune synapse. Also, ICAM-3 binding to DC induced the activation of the Vav1-Rac1 axis, a regulatory pathway involved in actin cytoskeleton reorganization, which was essential for MHC-II clustering on DCs. Our results support a model in which ICAM-mediated MHC-II clustering on DC constitutes a priming mechanism to enhance antigen presentation to T-cells.