Psoriasis is a common T-cell-mediated skin disease with 2-3% prevalence worldwide. Psoriasis is considered to be an autoimmune disease, but the precise nature of the autoantigens triggering T-cell activation remains poorly understood. Here we find that two-thirds of patients with moderate-to-severe plaque psoriasis harbour CD4 þ and/or CD8 þ T cells specific for LL37, an antimicrobial peptide (AMP) overexpressed in psoriatic skin and reported to trigger activation of innate immune cells. LL37-specific T cells produce IFN-g, and CD4 þ T cells also produce Th17 cytokines. LL37-specific T cells can infiltrate lesional skin and may be tracked in patients blood by tetramers staining. Presence of circulating LL37-specific T cells correlates significantly with disease activity, suggesting a contribution to disease pathogenesis. Thus, we uncover a role of LL37 as a T-cell autoantigen in psoriasis and provide evidence for a role of AMPs in both innate and adaptive immune cell activation.

Chemical inhibition of the mitochondrial electron transport chain (mtETC) by antimycin A (AA) or the TCA cycle by monofluoroacetate (MFA) causes increased expression of nucleus-encoded alternative oxidase (AOX) genes in plants. In order to better understand the mechanisms of this mitochondrial retrograde regulation (MRR) of gene expression, constructs containing deleted and mutated versions of a promoter region of the Arabidopsis thaliana AOX1a gene (AtAOX1a) controlling expression of the coding region of the enhanced firefly luciferase gene were employed to identify regions of the AtAOX1a promoter important for induction in response to mtETC or TCA cycle inhibition. Transient transformation coupled with in vitro and in vivo assays as well as plants containing transgenes with truncated promoter regions were used to identify a 93 base pair portion of the promoter, termed the MRR region, that was necessary for induction. Further mutational analyses showed that most of the 93 bp MRR region is important for both AA and MFA induction. Sub-regions within the MRR region that are especially important for strong induction by both AA or MFA were identified. Specific mutations in a W-box and Dof motifs in the MRR region indicate that these specific motifs are not important for induction. Recent evidence suggests that MRR of AOX genes following inhibition of the mtETC is via a separate signaling pathway from MRR resulting from metabolic shifts, such as those that result from MFA treatment. Our data suggest that these signaling pathways share regulatory regions in the AtAOX1a promoter. Arabidopsis proteins interacted specifically with a probe containing the MRR region, as shown by electrophoretic mobility shift assays and Southwestern blotting. These interactions were eliminated under reducing conditions.

MHC-peptide tetramers have become essential tools for T-cell analysis, but few MHC class II tetramers incorporating peptides from human tumor and self-antigens have been developed. Among limiting factors are the high polymorphism of class II molecules and the low binding capacity of the peptides. Here, we report the generation of molecularly defined tetramers using His-tagged peptides and isolation of folded MHC/peptide monomers by affinity purification. Using this strategy we generated tetramers of DR52b (DRB3*0202), an allele expressed by approximately half of Caucasians, incorporating an epitope from the tumor antigen NY-ESO-1. Molecularly defined tetramers avidly and stably bound to specific CD4 + T cells with negligible background on nonspecific cells. Using molecularly defined DR52b/ NY-ESO-1 tetramers, we could demonstrate that in DR52b + cancer patients immunized with a recombinant NY-ESO-1 vaccine, vaccineinduced tetramer-positive cells represent ex vivo in average 1:5,000 circulating CD4 + T cells, include central and transitional memory polyfunctional populations, and do not include CD4 + CD25 + CD127 − regulatory T cells. This approach may significantly accelerate the development of reliable MHC class II tetramers to monitor immune responses to tumor and self-antigens.S oluble MHC-peptide tetramers, allowing the direct visualization, characterization, and isolation of antigen-specific T cells, have become essential tools for T-cell analysis. MHC class I tetramers incorporating short CTL peptide epitopes, originally developed by Altman and Davis (1) have been generated for a large number of murine and human alleles, incorporating a variety of peptides of microbial, tumor, and self-antigen origin (2). The development of MHC class II tetramers, however, and particularly of those incorporating peptides from tumor and self-antigens, has been far less successful (3-5). One limiting factor is the high polymorphism of the human MHC class II molecules, especially those encoded by the DRB1 locus, the most frequently studied. Another limiting factor is the binding affinity of antigenic peptides derived from tumor and self-antigens, which is generally lower than that of peptides from pathogens.NY-ESO-1 (ESO), a tumor-specific antigen of the cancer/testis group frequently expressed in tumors of different histological types (6), is an important candidate for the development of generic cancer vaccines (7). In a recent vaccination trial using a recombinant ESO protein (rESO) administered with Montanide ISA 51 and CpG ODN 7909, we have observed induction of CD4 + T cell responses in all vaccinated patients (8). By assessing vaccineinduced CD4 + T cells, we have identified an immunodominant epitope , core region ESO 123-137 ) restricted by HLADR52b (DRB3*0202), an allele expressed by half of Caucasians (9). DRB3-, DRB4-, and DRB5-encoded molecules are less polymorphic than those encoded by DRB1, and are therefore attractive candidates for the development of generic MHC class II tetramers.Our initial attempts to const...

Purpose: Cancer vaccines aim to generate and maintain antitumor immune responses. We designed a phase I/IIa clinical trial to test a vaccine formulation composed of Montanide ISA-51 (Incomplete Freund's Adjuvant), LAG-3Ig (IMP321, a non-Toll like Receptor agonist with adjuvant properties), and five synthetic peptides derived from tumor-associated antigens (four short 9/10-mers targeting CD8 T-cells, and one longer 15-mer targeting CD4 T-cells). Primary endpoints were safety and T-cell responses.Experimental Design: Sixteen metastatic melanoma patients received serial vaccinations. Up to nine injections were subcutaneously administered in three cycles, each with three vaccinations every 3 weeks, with 6 to 14 weeks interval between cycles. Blood samples were collected at baseline, 1-week after the third, sixth and ninth vaccination, and 6 months after the last vaccination.Circulating T-cells were monitored by tetramer staining directly ex vivo, and by combinatorial tetramer and cytokine staining on in vitro stimulated cells.Results: Side effects were mild to moderate, comparable to vaccines with Montanide alone. Specific CD8 T-cell responses to at least one peptide formulated in the vaccine preparation were found in 13 of 16 patients. However, two of the four short peptides of the vaccine formulation did not elicit CD8 T-cell responses. Specific CD4 T-cell responses were found in all 16 patients.Conclusions: We conclude that vaccination with IMP321 is a promising and safe strategy for inducing sustained immune responses, encouraging further development for cancer vaccines as components of combination therapies.