Targeting the splicing of mRNA in autoimmune diseases: BAFF inhibition in Sjögren's syndrome as a proof of concept.

BAFF (B-cell-activating factor of the tumor necrosis factor family), a pivotal cytokine for B-cell activation, is overexpressed by salivary gland (SG) epithelial cells in primary Sjogren's syndrome (pSS). ΔBAFF, a physiological inhibitor of BAFF, is a minor alternative splice variant of BAFF. A U7 RNA was reengineered to deliver antisense sequences targeting BAFF splice regions. A major decrease of BAFF messenger RNA (mRNA) and protein secretion, concomitantly with the increase of ΔBAFF mRNA, was observed in vitro. In vivo, SG retrograd instillation of nonobese diabetic mice by the modified U7 cloned into an adeno-associated virus vector significantly decreased BAFF protein expression and lymphocytic infiltrates and improved salivary flow. This study offers a rationale for localized therapeutic BAFF inhibition in pSS and represents a proof of concept of the interest of exon skipping in autoimmune diseases.

FADD protein release mirrors the development and aggressiveness of human non-small cell lung cancer.

BACKGROUND: The need to unfold the underlying mechanisms of lung cancer aggressiveness, the deadliest cancer in the world, is of prime importance. Because Fas-associated death domain protein (FADD) is the key adaptor molecule transmitting the apoptotic signal delivered by death receptors, we studied the presence and correlation of intra- and extracellular FADD protein with development and aggressiveness of non-small cell lung cancer (NSCLC). METHODS: Fifty NSCLC patients were enrolled in this prospective study. Intracellular FADD was detected in patients' tissue by immunohistochemistry. Tumours and distant non-tumoural lung biopsies were cultured through trans-well membrane in order to analyse extracellular FADD. Correlation between different clinical/histological parameters with level/localisation of FADD protein has been investigated. RESULTS: Fas-associated death domain protein could be specifically downregulated in tumoural cells and FADD loss correlated with the presence of extracellular FADD. Indeed, human NSCLC released FADD protein, and tumoural samples released significantly more FADD than non-tumoural (NT) tissue (P=0.000003). The release of FADD by both tumoural and NT tissue increased significantly with the cancer stage, and was correlated with both early and late steps of the metastasis process. CONCLUSION: The release of FADD by human NSCLC could be a new marker of poor prognosis as it correlates positively with both tumour progression and aggressiveness.

Evidence of the contribution of the X chromosome to systemic sclerosis susceptibility: association with the functional IRAK1 196Phe/532Ser haplotype.

OBJECTIVE: Several autoimmune disorders, including systemic sclerosis (SSc), are characterized by a strong sex bias. To date, it is not known whether genes on the sex chromosomes influence SSc susceptibility. Recently, an IRAK1 haplotype that contains the 196Phe functional variant (rs1059702), located on Xq28, was found to confer susceptibility to systemic lupus erythematosus (SLE). This study was undertaken to test for an association between SSc and the IRAK1 SLE risk haplotype. METHODS: We tested for an association with the IRAK1 SLE risk haplotype in a discovery set of 849 SSc patients and 625 controls. IRAK1 rs1059702 was further genotyped in a replication set, which included Caucasian women from Italy (493 SSc patients and 509 controls) and Germany (466 SSc patients and 1,083 controls). RESULTS: An association between the IRAK1 haplotype and SSc was detected in the discovery set. In both the discovery and replication sets, the rs1059702 TT genotype was found to be associated with specific SSc subsets, highlighting a potential contribution to disease severity. A meta-analysis provided evidence of an association of both the T allele and TT genotype with the overall disease, with an odds ratio (OR) of 1.20 and 95% confidence interval (95% CI) of 1.06-1.35 for the T allele (P = 0.003) and an OR of 1.49 and 95% CI of 1.06-2.10 for the TT genotype (P = 0.023). However, the most notable associations were observed with the diffuse cutaneous, anti-topoisomerase I antibody positive, and SSc-related fibrosing alveolitis subsets (OR 2.35 [95% CI 1.51-3.66], P = 1.56 × 10(-4), OR 2.84 [95% CI 1.87-4.32], P = 1.07 × 10(-6), and OR 2.09 [95% CI 1.35-3.24], P = 9.05 × 10(-4), respectively). CONCLUSION: Our study provides the first evidence of an association between IRAK1 and SSc, demonstrating that a sex chromosome gene directly influences SSc susceptibility and its phenotypic heterogeneity.

Effects of a low-dose IL-2 treatment in HLA-B27 transgenic rat model of spondyloarthritis.

INTRODUCTION/AIM: HLA-B27/human ß2m transgenic rats (B27-rats) develop an inflammatory disorder resembling spondyloarthritis (SpA) with dysregulated IL-10/IL-17 production by regulatory T cells (Treg). Treg plays a major role in controlling pathogenic inflammatory processes. Interleukin 2 (IL-2), a cytokine which promotes Treg cell survival and function, may thus have therapeutic efficacy in SpA. Here, we tested this hypothesis using a low dose of IL-2 treatment in B27-rat. MATERIAL AND METHODS: B27-rats aged 4 weeks (before disease onset) and nontransgenic (NTG) littermates were administered intraperitoneally recombinant human IL-2 (Sanofi®; 2,000IU/injection) or PBS, 3 days per week during 6 weeks. Assessment of treatment effect was performed, based on clinical (weight, diarrhea, arthritis), histological (proximal and distal colon, caecum, ileum and tarsal/ankle joint) scores, and frequency of Treg in the spleen and lymph nodes (LN). RESULTS: IL-2 administration had no effect on weight gain, either in B27- or NTG-rats. Over the 6 weeks of treatment, the clinical disease score worsened similarly in both IL-2-treated and control groups of B27-rats. The macroscopic and histological evaluation of gut and joints showed marked inflammation in B27-rats; however, no change related to IL-2 treatment was observed. In the B27-rats, the percentage of Treg was moderately increased after IL-2 treatment in the spleen, but neither in mesenteric nor peripheral LN in those rats. CONCLUSION: Our data demonstrate that a low dose of IL-2 administered before disease onset was moderately effective for boosting Treg but failed to prevent SpA development in B27-rat.

Circulating endothelial progenitor cells in systemic sclerosis: association with disease severity.

BACKGROUND: Heterogeneous data have been reported regarding the detection and number of circulating endothelial progenitor cells (EPCs) in systemic sclerosis (SSc). OBJECTIVE: We investigated the number of circulating EPCs using recent recommendations and we quantified their late outgrowth in patients with SSc and healthy controls. PATIENTS AND METHODS: EPCs, defined as Lin-/7AAD-/CD34+/CD133+/VEGFR-2+ cells, were quantified in 50 patients with SSc (mean age: 55 (16) years, disease duration: 9 (9) years) and 26 controls (mean age: 53 (19) years) by cell sorting/flow cytometry and by counting late outgrowth colony-forming units (CFU). RESULTS: Patients with SSc displayed higher circulating EPC counts than controls (median 86 (5-282) vs 49 (5-275)) EPCs for 1 million Lin- mononuclear cells; p = 0.01). Lower EPC counts were associated with the higher Medsger's severity score (p = 0.01) and with the presence of past and/or current digital ulcers (p = 0.026). There was no difference for the number of late outgrowth EPC-CFUs between patients with SSc and controls in cell culture evaluation. The formation of colonies was associated with higher levels of circulating EPCs (p = 0.02) and the number of colonies correlated with levels of EPCs (R = 0.73, p = 0.0004), validating our combination of fluorescence-activated cell sorter surface markers. CONCLUSIONS: We quantified circulating EPCs with an accurate combination of markers herein validated. Our data demonstrate increased circulating EPC levels in SSc, supporting their mobilisation from bone marrow. Furthermore, the subset of patients with digital vascular lesions and high severity score displayed low EPC counts, suggesting increased homing at this stage. The predictive value of this biomarker now warrants further evaluation.

Lack of association between Tenascin-C gene and spondyloarthritis.

OBJECTIVES: We previously identified a new susceptibility region linked to SpA in 9q31-34. Tenascin-C (TNC) appears as one of the best positional and functional candidate genes lying within this SPA2 locus. The objectives of the present study were to identify TNC polymorphisms, and to examine their putative association with SpA. METHODS: We first performed variants screening in 20 independent SpA patients from families with high linkage score to the SPA2 locus, and three unrelated controls: TNCs coding regions (28 exons), intron-exon boundaries and 5'- and 3'-flank regions were fully re-sequenced to identify polymorphisms. Then we genotyped selected variants in 183 independent trios, and assessed their intrafamilial association with SpA by transmission disequilibrium test. RESULTS: Variants screening allowed us to identify 26 polymorphisms, 7 of which were selected for further study, in addition to an intronic polymorphism previously reported as associated with Achilles tendon injuries. In intrafamilial association test, none of the variants showed significant transmission disequilibrium. Results from analysis restricted to AS were not different from those obtained on the whole SpA group. CONCLUSIONS: TNC was one of the best positional and functional candidate genes within the SPA2 locus. Nevertheless, we found no association between polymorphisms in this gene and SpA. However, we cannot exclude that variants located in intronic regions or in the vicinity of TNC, which were not tested in the present study, could be implicated in the predisposition to SpA.

Highly sensitive method to detect mRNAs in individual cells by direct RT-PCR using Tth DNA polymerase.

A new method for detecting the expression of low-abundance mRNA molecules has been developed that combines the sensitivity of PCR, the high efficiency and specificity of reverse transcription (RT) using Tth DNA polymerase at high temperature, and the enhancement of sensitivity and specificity of nested PCR. This method is highly sensitive, reproducible and allows the detection of mRNAs in individual cells by direct RT-PCR.

T cell-targeted immunotherapy in murine collagen-induced arthritis.

Counteracting the effect of autoimmunity can be achieved by elimination or inactivation of autoreactive T cells. We have focused on two approaches targeting on autoaggressive T cells in the model of collagen-induced arthritis (CIA) in mice. First, type II collagen (CII) primed DBA/1 mice were treated with various monoclonal antibodies (mAb) specific for the beta chains of the T cell receptor (TCR) using a protocol resulting in a long-term elimination of the target T cells. Indeed, CIA could be suppressed by injection of anti-V beta 8.1, 2 mAb and down-regulated by that of anti-V beta 2 and/or anti-V beta 5, presumably by deleting pathogenic T cell clones. In contrast, treatment with either anti-V beta 6 or anti-V beta 11 mAb did not alter CIA. Second, we generated CII-specific T cell hybrid clones that recognize the antigenic peptides in association with Kq and IA(q) molecules respectively for CD8+ and CD4+ cells. Vaccination with the irradiated hybrid clones, 3 weeks prior to immunization, was effective in preventing the development of arthritis. Furthermore, this suppression was antigen and disease specific. Most importantly, one CD8+ clone could reverse the ongoing disease. These new therapeutic approaches derived from animal models may offer a hope of more selective interventions for the treatment of human autoimmune diseases.

[What place for DNA microarray in inflammatory diseases?]. / Quel place pour les puces à ADN dans les maladies inflammatoires?

PURPOSE: DNA chip is a recently developed technique allowing analysis of thousands of genes at the same time in multiple biological samples. In few years it has become an obligatory step in massive gene expression study. The enormous quantity of results generated and the new way of thinking allowed make this kind of study a true revolution. KEY MESSAGE AND RECENT FACTS: The enormous discovery potential permitted by the accomplishment of multiple genomes sequencing and the advent of technologies allowing massive gene expression analyses have totally modified the diseases approach. Considering the obtainment of a real full picture of the transcriptional activity in an organ, tissue or cell is now legitimate. DNA microarray is obviously not the only technique allowing such type of analysis but it is without contest the technology which is the most popular and the one which has been recently the subject of the most important developments. It is certainly the technology which brought the main advances in tumour classification and discovery of new biomarkers. The first results based on this technology in inflammatory diseases have recently been reported. PERSPECTIVE AND PROJECTS: The optimal use of DNA microarrays will necessitate a powerful statistical analysis and an high quality biological experimentation. Strict standard and quality criteria are developing. Obviously, the DNA chips have a role to play in multifactorial inflammatory diseases mainly through their potential to bring new answers to diagnostic and pathophysiological problems. One potential development of the technique in such diseases will be the definition of disease specific gene profiles and the generation of chips allowing the detection of few targeted genes with all the known mutations of these genes. The correlation of global or targeted gene expression with clinical and pathological data will allow a new step forward in the understanding and taking care of inflammatory diseases.

Angiogenesis in systemic sclerosis: impaired expression of vascular endothelial growth factor receptor 1 in endothelial progenitor-derived cells under hypoxic conditions.

OBJECTIVE: To assess angiogenesis and explore the expression and regulation of vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGFR-1), and VEGFR-2, the leading mediators of angiogenesis, in SSc patients and controls. METHODS: Late-outgrowth endothelial progenitor cells (EPCs), isolated from the peripheral blood of systemic sclerosis (SSc) patients and controls, and human umbilical vein endothelial cells (HUVECs) were assessed under normal and hypoxic conditions. Genomic background was evaluated in a large case-control study (including 659 patients with SSc and 511 controls) using tag single-nucleotide polymorphisms on VEGFR1 and VEGFR2 genes. RESULTS: EPCs from SSc patients had the phenotype of genuine endothelial cells and displayed in vitro angiogenic properties similar to those of HUVECs and control EPCs under basal conditions, as determined by flow cytometry, tube formation, and migration assay. However, after 6 hours of hypoxic exposure, EPCs from SSc patients exhibited lower induced expression of VEGFR-1 at the messenger RNA and protein levels, but similar VEGF and VEGFR-2 expression, compared with HUVECs or EPCs from healthy controls. There was no evidence of defective expression of hypoxia-inducible factor 1alpha. These results were supported by the lower serum levels of soluble VEGFR-1 found in SSc patients (n = 187) compared with healthy controls (n = 48) (mean +/- SD 163.7 +/- 98.5 versus 210.4 +/- 109.5 pg/ml; P = 0.0042). These abnormalities did not seem to be related to genomic background. CONCLUSION: Our findings shed new light on the possible role of VEGFR-1 in the main vascular disturbances that occur in SSc and lead to more severe disease.

Effect of catecholamines on deformability of red cells from trout: relative roles of cyclic AMP and cell volume.

1. In the presence of catecholamine the nucleated red blood cells of trout show a large increase in cell volume as a result of an accumulation of sodium and chloride due to activation of an amiloride-sensitive, cyclic AMP-dependent Na+-H+ exchanger allowing Na+ to enter in exchange for internal H+. 2. The activation of this cyclic AMP-dependent Na+-H+ exchange is considered to be involved in an adaptive response to hypoxia by increasing the oxygen-carrying capacity of erythrocytes. But cell swelling could increase resistance to blood flow and thus impair the expected physiological advantages for oxygen transport. The effect of catecholamine on the deformability properties of the red blood cells has been studied by measuring the rate at which blood flows through a Nucleopore filter (5 microM). 3. The results show that stimulation by catecholamine in fact increases the erythrocyte deformability, a response which must favour the supply of oxygen at the tissue level. 4. Hormonal stimulation increases the cellular cyclic AMP content (and cyclic AMP-dependent phosphorylation of cytoskeleton proteins could influence cell deformability) and the cell volume. It has been shown that when cellular cyclic AMP content is increased under conditions where the cell cannot swell, the erythrocyte becomes more rigid and not more deformable. Conversely the results show a systematic coincidence between cell swelling and deformability increase. The precise way in which volume change and deformability are interrelated needs more study.

Characteristics of cytotoxic thyroglobulin-specific T cell hybridomas.

Clones of cytotoxic thyroid-specific T cell hybridomas were generated by fusion of thyroglobulin-primed, "in vitro"-boosted CBA lymph node cells with the AKR-derived lymphoma cell line BW 5147. One hundred and thirty one clones were obtained. Among them, 15 were able to induce the lysis of 51Cr-labeled syngeneic thyroid epithelial cells after 5 h of incubation at 37 degrees C. Two T cell clones, HTC1 and HTC2, were further studied. These clones, which exhibit cell surface characteristics of cytotoxic cells, were specific for only syngeneic thyroid cells (allogeneic thyroid cells or syngeneic epithelial cells were never lysed by these hybridomas). Moreover, by using Ag-pulsed syngeneic macrophages as targets, syngeneic cytotoxicity was shown to be specific for thyroglobulin and not for a nonrelated Ag. The lysis obtained with these autoreactive thyroid-specific T cell clones is restricted to class I major histocompatibility Ag. This property is assessed by both the blocking of syngeneic cytotoxicity toward thyroid epithelial cells or thyroglobulin-pulsed macrophages only by anti-class I mAb and by the detection of specific lysis of target cells exclusively when effector hybrid cells and target thyroid epithelial cells or thyroglobulin-pulsed macrophages shared at least class I major histocompatibility Ag.

Selective increased presentation of type II collagen by leupeptin.

Type II collagen (CII) is an arthritogenic self antigen in DBA/1 (H-2q) mice. To analyze the intracellular processing of this fibrillar protein in the context of I-Aq molecules, we have generated hybrid antigen-presenting cells (APC) by fusion of B lymphoma (A20 and M12) cells with CII-primed spleen cells from DBA/1 mice. Efficient presentation of CII by these APC to specific T cell hybridomas required prior cleavage of the antigen and intracellular handling of the peptides. Inhibition of protein transport by brefeldin A prevented the presentation of CII peptides to T cell hybridomas, indicating that the intracellular presentation of CII was dependent on neo-synthesis of I-Aq molecules. In contrast, exposure of hybrid B lymphomas to leupeptin, a protease inhibitor, induced a dose-dependent increase of CII-specific T cell response, while abrogating the I-Aq-restricted presentation of ovalbumin. The enhancing effect of leupeptin was also observed when immune B cells were used as APC. In contrast, leupeptin inhibited the presentation of CII peptides by macrophages or total spleen cells. Pulse-chase analysis of metabolically labeled hybrid APC and immunoprecipitation with antibodies specific for class II molecules or invariant (li) chain revealed that leupeptin did not affect the li chain processing or the formation of stable class II dimers. The stimulatory effect of leupeptin observed on CII presentation suggests that leupeptin protects CII epitopes by interfering with proteases involved in the intracellular degradation of CII.

Pattern of humoral reactivity to type II collagen in rheumatoid arthritis.

Humoral immunity directed against type II collagen (CII) is a common although not specific feature of rheumatoid arthritis (RA). We have shown that 10 to 15% of the sera either from RA patients (n = 88) or from healthy controls (n = 149) reacted with native human CII. Conversely, autoantibodies to the alpha-1 (II) chains were significantly more frequent in the RA group (26.1% versus 6.0%, P<0.001), suggestingthatdenaturedCII may bean autoantigenin RA. Thus, human CII was cleaved with cyanogen bromide (CB), and immunoblotting techniques were performed on 19 RA and 21 normal sera. Among the four major CB peptides, CB10 and CB11 were recognized by most of the sera tested without distinction between normal or RA sera. Inhibition experiments using an ELISA have shown that: (i) antibodies to the native CII molecule did not cross-react with those recognizing the CB peptides, and vice-versa; (ii) the binding of the sera to native CII was partially inhibited by pre-incubation with alpha-1 (II) chains, and vice-versa; (iii) pre-incubation of the sera with CB peptides partially blocked the binding to alpha-1 (II) chains, whereas pre-incubation of the sera with alpha-1 (II) chains totally inhibited the reactivity against CB peptides; and (iv) a substantial proportion of the epitopes recognized by anti-CII autoantibodies was neither species specific nor type specific. Taken together, these findings reveal the existence of several populations of anti-CII autoantibodies: some antibodies react exclusively with conformational determinants of the CII molecule, and others are directed towards linear structures of alpha-1 (II) chains.

Combination of cyclosporine A and calcitriol in the treatment of adjuvant arthritis.

We investigated combination treatment with cyclosporine A (CsA) and calcitriol (CT), 2 immunomodulatory agents, in vivo in the adjuvant arthritis (AA) model in rats. Treatment initiated at priming with complete Freund's adjuvant consisted of daily administration of CsA and CT during the prearthritic phase (14 days). CsA given alone delayed the onset of the disease, whereas the severity was either not affected or paradoxically enhanced. Injection of CT alone reduced the severity of arthritis. The association of low doses of CsA (5 mg/kg) and CT (0.2 micrograms/kg) further delayed the onset of AA and abolished the aggravating effect. Our findings suggest that low doses of CsA and CT in association exhibit the additive beneficial effects of either agent alone.

[Role of collagen conformation in type II anticollagen immunity in rheumatoid polyarthritis]. / Rôle de la conformation du collagène dans l'immunité anti-collagène de type II au cours de la polyarthrite rhumatoïde.

Type II anticollagen (CII) autoimmunity is a frequently reported, but non-specific, phenomenon in rheumatoid arthritis (RA). The authors show that in 88 sera samples from patients suffering from RA, the incidence of antibodies targeted against endogenous human CII was the same as that found for 149 control blood donors (14.8% versus 11.4%). However, a significant difference was found for the incidence of antibodies targeted against the alpha-chains of CII (26.1% versus 6.0%, p less than 0.001). As a result of investigating the specificity of the anti-CII antibodies in greater detail by means of an immunoprinting of the CII peptide fragments obtained after splitting the molecule by cyanogen bromide, the authors have demonstrated that the largest CII peptides (CB10 and CB11) were better recognized than the smaller peptides (CB8, CB9.7), with no significant difference between PR and control plasmas. Using competitive methods, evidence was obtained in support of heterogeneous recognition by the anti-CII antibodies: some recognize conformational determinants only, whereas others are targeted against the primary sequences of the alpha-1 (II) chain.

Therapy against murine collagen-induced arthritis with T cell receptor V beta-specific antibodies.

Immunization with native type II collagen (CII) of susceptible strains of mice (H-2q) induces a rheumatoid arthritis-like disease. Collagen-induced arthritis (CIA) is an experimental model for T cell-mediated autoimmune disease. To investigate the T cell receptor (TcR) repertoire involved in the pathogenesis of CIA, CII-primed DBA/1 mice were treated with various TcR V beta-specific monoclonal antibodies (mAb) using a protocol resulting in a long-term elimination of the target T cells. In vivo treatment with anti-CD4 mAb led to nearly complete protection against CIA. Mice injected with anti-V beta 8.1, 2 or anti-V beta 5.1, 2 mAb had a reduced incidence of arthritis (respectively 28.6% and 50% vs 84.6% for the control group). Administration of anti-V beta 2 mAb delayed the onset of the disease whereas injection of anti-V beta 6 or anti-V beta 11 mAb did not alter CIA. Moreover, the combined treatment with anti-V beta 2 and anti-V beta 5 mAb efficiently reduced the development of CIA. The humoral response to CII was down-regulated only in the groups of mice that were improved by the treatment. In vitro proliferative response to CII of lymph node cells from primed DBA/1 was partially blocked by addition of several anti-V beta mAb. Thus, our findings suggest that the overall T cell response to CII may be polyclonal while the T cell clones involved in the pathogenesis of CIA express a limited number of V beta chains.

T cell regulation of collagen-induced arthritis in mice. II. Immunomodulation of arthritis by cytotoxic T cell hybridomas specific for type II collagen.

Injection of native type II collagen (CII) to susceptible strains of mice (H-2q) induces a rheumatoid arthritis-like disease. To study the role of CD8+ T cells in the collagen-induced arthritis (CIA), we generated CII-specific T cell hybridomas by fusion of cells from arthritic C3H. Q mice and an AKR thymoma. Two hybrid clones (P3G8 and P2D9) were selected for their ability to lyse syngeneic CII-pulsed macrophages and recognize different antigenic epitopes in association with Kq molecules. When these T cell clones were irradiated and inoculated into (C3H.Q x AKR)F1 mice 21 days prior to priming with native CII/complete Freund's adjuvant, the incidence and the duration of CIA were significantly reduced in comparison to groups receiving saline or control T cell hybridoma. Furthermore, both anti-CII T cell hybridomas were able to attenuate CIA in highly susceptible inbred strains of mice and this suppression was antigen and disease specific. The protective activity seems to require intact cells as neither membrane fractions nor cytosolic preparations of the hybridoma T cells retained the vaccinating activity. Most importantly, one of the hybrid clones (P3G8) had a therapeutic effect on CIA since its administration to arthritic DBA/1 mice on day 30 after priming down-regulated the ongoing disease. Taken together, these findings suggest that anti-CII cytotoxic T cell clones can vaccinate against CIA and even reverse the disease.

Processing and presentation of type II collagen, a fibrillar autoantigen, by H-2q antigen-presenting cells.

Native type II collagen (CII) is a high molecular-weight fibrillar molecule which induces a chronic polyarthritis in mice expressing the H-2q haplotype. The present study was initiated to analyze the processing and the presentation of this nonglobular protein by H-2q antigen-presenting cells (APC). Efficiency of presentation was assessed by the ability of antigen-pulsed APC to activate collagen-specific CD4+ T cell hybridomas. Fixation of APC or the presence of chloroquine completely blocked the reactivity of the T cell hybrids to native, denatured and cyanogen bromide (CB) degraded CII, thus indicating the requirement of intracellular processing for adequate presentation of CII peptides to T cells. In the presence of various processing inhibitors (brefeldin A, leupeptin and N-tosyl-L-phenylalanine chloromethylketone) stimulation of T hybrids by CII-pulsed APC was reduced, pointing to the need of newly synthesized class II molecules, the use of several intracellular compartments and the implications of different proteases in the generation of CII peptides. Peritoneal macrophages and, to a lesser extent, total spleen cells, presented native and denatured CII with higher efficiency than purified splenic dendritic cells, naive or even immune B cells from CII-primed mice. In contrast, these dendritic and B cells were fully competent to present intact ovalbumin to a specific T cell hybrid. The stimulation by dendritic cells and immune B cells was greater when CB peptides of CII were added instead of the native molecule. Similarly, the cleavage of CII was an absolute requirement for its presentation by epidermal cells and B cell lymphomas to the T cell hybridomas. Taken together, these findings emphasize the crucial role of intracellular processing for recognition of soluble CII, similar to most antigens. However, in contrast to ovalbumin, the size and fibrillar nature of the native CII molecule influences its capture by the APC, thus limiting the type of APC able to present this antigen.

Transgenic expression of Fas ligand on thyroid follicular cells prevents autoimmune thyroiditis.

"Immune privilege" is defined as tissue resistance to aggression by specifically activated lymphocytes, and involves the interaction between Fas expressed on infiltrating cells and Fas ligand (FasL) constitutively expressed on the target tissue. To test whether ectopic expression of FasL on thyrocytes could prevent autoimmune aggression of the thyroid by activated lymphoid cells, three lines of transgenic mice expressing low, intermediate, and high levels of functional FasL on thyroid follicular cells were generated. Experimental autoimmune thyroiditis was induced by immunization with mouse thyroglobulin. In all of the experiments, the effects were dependent on the level of FasL expression. Low and intermediate expression had no or only weak preventive effects, respectively, whereas high FasL expression strongly inhibited lymphocytic infiltration of the thyroid. Anti-mouse thyroglobulin-proliferative and cytotoxic T cell responses, as well as autoantibody production, were diminished in transgenic mice expressing high levels of FasL relative to controls. Furthermore, in these latter mice Th1 responses to mouse thyroglobulin were profoundly down-regulated, uncovering a new potential role for FasL in peripheral tolerance to organ-specific Ags. In sum, the prevention of experimental autoimmune thyroiditis by FasL on thyrocytes is dependent on the level of FasL expression.