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Major histocompatibility complex class II (MHC-II) molecules bind to and display antigenic peptides on the surface of antigen-presenting cells (APCs). In the absence of infection, MHC-II molecules on APCs present self-peptides and interact with CD4(+) T cells to maintain tolerance and homeostasis. In the thymus, self-peptides bind to MHC-II molecules expressed by defined populations of APCs specialised for the different steps of T-cell selection. Cortical epithelial cells present peptides for positive selection, whereas medullary epithelial cells and dendritic cells are responsible for peptide presentation for negative selection. However, few data are available on the peptides presented by MHC molecules in the thymus. Here, we apply mass spectrometry to analyse and identify MHC-II-associated peptides from five fresh human thymus samples. The data show a diverse self-peptide repertoire, mostly consisting of predicted MHC-II high binders. Despite technical limitations preventing single cell population analyses of peptides, these data constitute the first direct assessment of the HLA-II-bound peptidome and provide insight into how this peptidome is generated and how it drives T-cell repertoire formation., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: Efferocytosis is a crucial process by which apoptotic cells are cleared by phagocytes, maintaining immune tolerance to self in the absence of inflammation. Peripheral tolerance, lost in autoimmune processes, may be restored by the administration of autologous dendritic cells loaded with islet apoptotic cells in experimental type 1 diabetes., Objective: To evaluate tolerogenic properties in dendritic cells induced by the clearance of apoptotic islet cells, thus explaining the re-establishment of tolerance in a context of autoimmunity., Methods: Bone marrow derived dendritic cells from non-obese diabetic mice, a model of autoimmune diabetes, were generated and pulsed with islet apoptotic cells. The ability of these cells to induce autologous T cell proliferation and to suppress mature dendritic cell function was assessed, together with cytokine production. Microarray experiments were performed using dendritic cells to identify differentially expressed genes after efferocytosis., Results: Molecular and functional changes in dendritic cells after the capture of apoptotic cells were observed. 1) Impaired ability of dendritic cells to stimulate autologous T cell proliferation after the capture of apoptotic cells even after proinflammatory stimuli, with a cytokine profile typical for immature dendritic cells. 2) Suppressive ability of mature dendritic cell function. 3) Microarray-based gene expression profiling of dendritic cells showed differential expression of genes involved in antigen processing and presentation after efferocytosis. 4) Prostaglandin E2 increased production was responsible for immunosuppressive mechanism of dendritic cells after the capture of apoptotic cells., Conclusions: The tolerogenic behaviour of dendritic cells after islet cells efferocytosis points to a mechanism of silencing potential autoreactive T cells in the microenvironment of autoimmunity. Our results suggest that dendritic cells may be programmed to induce specific immune tolerance using apoptotic cells; this is a viable strategy for a variety of autoimmune diseases.

Context: One of the side effects of interferon-alpha therapy is interferon-induced thyroiditis (IIT). The role of lymphocyte subpopulations in IIT melanoma patients remains to be defined., Objective: Our objective was to assess different peripheral blood lymphocyte subpopulations, mainly regulatory T cells (Tregs), in melanoma patients who developed IIT., Design, Patients and Methods: From 30 melanoma patients receiving high-dose interferon (HDI)-alpha 2b (IFN-α2b) treatment, those who developed IIT (IIT patients) were selected and compared with patients who did not develop IIT (Co-MM) and healthy controls (Co-H). Peripheral blood mononuclear cells were obtained before treatment (BT), mid-treatment (MT), end of treatment (ET), 24 weeks post-treatment and at appearance of IIT (TT)., Results: Nine patients developed IIT (30%): four Hashimoto's thyroiditis and five destructive thyroiditis. An increase in Tregs was observed in both melanoma groups during HDI treatment. A decrease in CD3(+) , NKT lymphocyte subpopulations and Bcl2 expression on B cells was also observed in both groups. However, no changes were observed in the percentage of CD4(+) , CD8(+) , CD3(+) γδ(+) , CD19(+) , transitional B cells (CD24(high) CD38(high) CD19(+) CD27(-) ), natural killer (NK), invariant NKT (iNKT) lymphocytes and Th1/Th2 balance when BT was compared with ET. At TT, IIT patients had a higher Tregs percentage than Co-MM (P = 0·012) and Co-H (P = 0·004), a higher iNKT percentage than Co-MM (P = 0·011), a higher transitional B cells percentage than Co-H (P = 0·015), a lower CD3(+) percentage than Co-H (P = 0·001) and a lower Bcl2 expression on B cells than Co-H (P < 0·001)., Conclusions: Our results point to the immunomodulatory effects of IFN-α on different lymphocyte subpopulations and a possible role of Tregs in melanoma patients who developed IIT., (© 2012 Blackwell Publishing Ltd.)

Cooperative events between DC subsets involve cell contact and soluble factors. Upon viral challenge, murine pDCs induce cDC cooperation through CD40-CD40L interactions and IL-15 secretion, whereas in humans, the same effect is mediated by IFN-α. Conversely, during bacterial infections, pDC maturation may be induced by activated cDCs, although no mechanisms had been described so far. Here, we investigate how human pDCs are "conditioned" by cDCs. Blood-borne DC subsets (cDCs and pDCs) were sorted from healthy donors. IL-3-maintained pDCs were cocultured with LPS-activated, poly (I:C)-activated, or control cDCs [cDC(LPS), cDC(P(I:C)), cDC(CTRL)]. Coculture experiments showed that cDC(LPS)-conditioned pDCs up-regulated maturation markers, such as CD25 and CD86, whereas SNs contained higher amounts of IL-6 and CCL19 compared with control conditions. Gene-expression analyses on sorted cDC(LPS) or cDC(P(I:C)) conditioned pDCs confirmed the induction of several genes, including IL-6 and CCL19 and remarkably, several Notch target genes. Further studies using the γ-secretase/Notch inhibitor DAPT and soluble Notch ligands resulted in a significantly reduced expression of canonical Notch target genes in conditioned pDCs. DAPT treatment also hampered the secretion of CCL19 (but not of IL-6) by cDC(LPS) conditioned pDCs. These results reveal the involvement of γ-secretase-mediated mechanisms, including the Notch pathway, in the cell contact-dependent communication between human DC subsets. The resulting partial activation of pDCs after encountering with mature cDCs endows pDCs with an accessory function that may contribute to T cell recruitment and activation.

cDCs and pDCs differ in multiple aspects. Among those, antigen capture is a recognized feature of cDCs, whereas pDCs display poor capacity to capture cell-derived antigens. However, animal models of organ transplantation suggested a role for pDCs in tolerance induction via phagocytosis of donor antigens. In a transplantation setting, microvesicles, such as apoptotic bodies and exosomes secreted by the graft, may be potential sources of alloantigen. Here, we tested the capacity of human pDCs to capture exosomes and apoptotic bodies from Jurkat T cells. Exosomes and apoptotic bodies were indeed captured by pDCs, although required longer times of incubation when compared with the highly endocytic cDCs. In cDCs and pDCs, exosome capture was more efficient than apoptotic bodies. Endocytosis inhibitors clearly impaired exosome capture by cDCs, although this could not be verified in pDCs as a result of cellular toxicity. Functionally, capture of Jurkat-derived exosomes did not induce nor prevent pDC maturation, and exosome-loaded pDCs induced T cell proliferation, suggesting a link between capture and presentation. Thus, exosomes and apoptotic bodies may be sources of antigen for human pDCs.

Multiple sclerosis (MS) is a chronic demyelinating autoimmune disease of the central nervous system. Current therapies decrease the frequency of relapses and limit, to some extent, but do not prevent disease progression. Hence, new therapeutic approaches that modify the natural course of MSneed to be identified. Tolerance induction to self-antigens using monocyte-derived dendritic cells (MDDCs) is a promising therapeutic strategy in autoimmunity. In this work, we sought to generate and characterize tolerogenic MDDCs (tolDCs) from relapsing-remitting (RR) MSpatients, loaded with myelin peptides as specific antigen, with the aim of developing immunotherapeutics for MS. MDDCs were generated from both healthy-blood donors and RR-MSpatients, and MDDCmaturation was induced with a proinflammatory cytokine cocktail in the absence or presence of 1α,25-dihydroxyvitamin-D(3) , a tolerogenicity-inducing agent. tolDCs were generated from monocytes of RR-MSpatients as efficiently as from monocytes of healthy subjects. The RR-MStolDCs expressed a stable semimature phenotype and an antiinflammatory profile as compared with untreated MDDCs. Importantly, myelin peptide-loaded tolDCs induced stable antigen-specific hyporesponsiveness in myelin-reactive T cells from RR-MS patients. These results suggest that myelin peptide-loaded tolDCs may be a powerful tool for inducing myelin-specific tolerance in RR-MS patients., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Context: One salient feature of autoimmune thyroid disease is the inappropriate expression of human leukocyte antigen (HLA) class II molecules by thyroid follicular cells. Metallothioneins (MT) are small proteins induced by tissue stress that can contribute to restoring homeostasis of tissue inflammation and have been found to be increased in a transcriptomic analysis of Graves' disease (GD) glands., Methodology: To assess the role of MT in the pathogenesis of GD, we analyzed MT-I and -II expression and distribution in GD-affected thyroid glands (n = 14) compared with other thyroid diseases (n = 20) and normal thyroid glands (n = 5). Two-color indirect immunofluorescence and semiquantitative morphometry were applied. The relationship between MT and HLA class II expression was analyzed by their degree of colocalization in GD sections, and in vitro induction kinetics and expression of these molecules on the HT93 thyroid cell line were compared by quantitative RT-PCR and flow cytometry using interferon-γ and zinc as stimuli., Results: MT were clearly overexpressed in nine of 14 GD glands. MT expression distribution in GD was almost reciprocal to that of HLA class II. In vitro analysis of MT and HLA class II demonstrated that MT is induced more slowly and at a lower level than HLA. Moreover, the main MT inducer, zinc, reduces interferon-γ-induced class II expression., Conclusions: These findings show that MT and HLA class II play very different roles in the autoimmune process by affecting the thyroid gland, thereby pointing to the possible role of MT as a marker of cell stress and homeostasis restoration in GD.

Immunotherapy using monocyte-derived dendritic cells (MDDC) is increasingly being considered as alternative therapeutic approach in cancer, infectious diseases and also in autoimmunity when patients are not responsive to conventional treatments. In general, generation of MDDC from monocytes is induced in the presence of GM-CSF and IL-4, and a maturation stimulus is added to the culture to obtain mature DCs suitable for therapy. For DC maturation, different combinations of pro-inflammatory mediators and Toll-like receptor ligands have been tested, obtaining DCs that differ in their properties and the type of immune response they promote. Therefore, it is necessary to find an optimal cytokine environment for DC maturation to obtain a cellular product suitable for DC-based immunotherapeutic protocols. In this study, we have evaluated in vitro the effects of different maturation stimuli on the viability, phenotype, cytokine profile, stability and functionality of immunogenic and tolerogenic (1α,25-dihydroxyvitamin D(3)-treated) MDDC. Maturation was induced using the clinical grade TLR4-agonist: monophosphoryl lipid A (LA), compared to the traditional cytokine cocktail (CC; clinical grade TNF-α, IL-1β, PGE2) and a combination of both. Our results showed the combination of CC+LA rendered a potent immunogenic DC population that induced the production of IFN-γ and IL-17 in allogeneic co-cultures, suggesting a Th17 polarization. Moreover, these immunogenic DCs showed a high surface expression of CD83, CD86, HLA-DR and secretion of IL-12p70. When aiming to induce tolerance, using LA to generate mature TolDC did not represent a clear advantage, and the stability and the suppressive capability exhibited by CC-matured TolDC may represent the best option. Altogether, these findings demonstrate the relevance of an appropriate maturation stimulus to rationally modulate the therapeutic potential of DCs in immunotherapy., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objective: One of the side effects of interferon-alpha (IFN-α) therapy is interferon-induced thyroiditis (IIT). The role of lymphocyte subpopulations in IIT remains to be defined. The aim of this study was to assess different peripheral blood lymphocyte subpopulations, mainly CD4(+) CD25(+) CD127low/-FoxP3(+) regulatory T cells (Tregs), in patients with chronic hepatitis C virus (HCV) infection who developed IIT., Design, Patients and Methods: From 120 patients with chronic HCV who started antiviral treatment, those who developed IIT (IIT patients) were selected and compared with patients who did not develop IIT (Co-HCV). Peripheral blood mononuclear cells were obtained before treatment (BT), mid-treatment (MT), end of treatment (ET), 24 weeks post-treatment (PT) and at appearance of IIT (TT)., Results: Eleven patients developed IIT: three Hashimoto's thyroiditis, one Graves'disease, one positive antithyroidal antibodies, one nonautoimmune hypothyroidism and five destructive thyroiditis. During antiviral treatment, an increase in CD8(+) and in Tregs was observed in both groups. A decrease in CD3(+) , CD19(+) and NKT lymphocyte subpopulations was also observed (all P < 0·05). However, no changes were observed in the percentage of CD4(+) , CD3(+) γδ(+) and iNKT lymphocytes, Th1/Th2 balance and Bcl2 expression on B cells when BT was compared with ET. At the appearance of IIT (TT), IIT patients had a higher Th1 response (CCR5(+) CCR7(-) ) (P < 0·01) and a higher Tregs percentage (P < 0·05) than Co-HCV., Conclusions: Our results point to the immunomodulatory effects of IFN-α on different lymphocyte subpopulations and a possible role of Th1 response and Tregs in patients with HCV who developed IIT., (© 2011 Blackwell Publishing Ltd.)

Graves' disease (GD) is the paradigm of an anti-receptor autoimmune disease, with agonistic auto-antibodies against the thyrotropin receptor (TSHR-thyroid-stimulating hormone receptor) being the underlying pathogenic effector mechanism. The TSHR belongs to the category of tissue-restricted antigens, which are promiscuously expressed in the thymus and thereby induce central T cell tolerance. In order to understand the association between TSHR gene polymorphisms and GD, we tested the hypothesis that TSHR gene variants affect susceptibility to GD by influencing levels of TSHR transcription in the thymus. We show that thymic glands from non-autoimmune donors homozygous for the rs179247 SNP predisposing allele of TSHR had significantly fewer TSHR mRNA transcripts than carriers of the protective allele. In addition, in heterozygous individuals, the TSHR predisposing allele was expressed at a lower level than the protective one as demonstrated by allele-specific transcript quantification. This unbalanced allelic expression was detectable in both thymic epithelial cells and thymocytes. Since the level of self-antigen expression is known to influence the threshold of central tolerance, these results are compatible with the notion that defective central tolerance contributes to the pathogenesis of GD, a scenario already implicated in type 1 diabetes mellitus, myasthenia gravis and autoimmune myocarditis.

Psoriasis is a common inflammatory skin disease with key immunological and genetic components. Recruitment of leukocytes into the skin is a central step in its pathogenesis, mediated by cytokines. Among the cytokines expressed in psoriatic lesions, C-C chemokine ligand 4 (CCL4) and C-C chemokine ligand 4-like (CCL4L) chemokines appear to be pivotal elements for the skin recruitment of proinflammatory cells. The aim of this study is to evaluate the relationship between CCL4L polymorphisms (including single-nucleotide polymorphisms (SNPs) and copy number variation (CNV)) and the course and prognosis of psoriasis. We analyzed the CNV and the rs4796195 SNP in 211 psoriatic patients and 234 controls; sera from both populations were also quantified for CCL4/CCL4L protein. Our results showed that a high CNV (≥3 copies) is associated with psoriasis severity, whereas moderate disease correlated with a lower CNV (≤2 copies); specifically, the CCL4L1 allele frequency is higher in severe psoriasis, whereas CCL4L2 is more frequent in patients with a milder disease. In addition, we found a positive correlation between the CNV and sera protein levels. Our results suggest that CCL4L genotyping could not only allow a better understanding of the psoriatic pathogenesis but could also be used as a prognostic tool, even helping to modulate the efficacy of treatments.

Background: The identification of major immunogenic peptides in multiple sclerosis (MS) is of great importance for the development of antigen-specific therapies. Cellular reactivity against a selected mix of seven myelin peptides was evaluated in vitro. The evolution of this reactivity over time and its correlation with clinical variables was also analysed., Material and Methods:   Forty-two patients with MS, 15 with other demyelinating diseases and 40 healthy donors (HD) were studied. Cell proliferation was measured by 3[H] thymidine incorporation into samples obtained at 0, 3, 6 and 12months of MS patient follow-up., Results:   A positive reaction to the peptide mix was detected in 31 of the 42 patients (74%), 12 of the 40 HD (30%) and 6 of the 15 (40%) patients with other demyelinating diseases. Patients with positive proliferation had greater disability (EDSS score, 3 [1-5.5] vs. 1.0[1-2], P=0.021), higher number of relapses (7±4.1 vs. 3±1.2, P<0.001) and shorter time since the last relapse (9±7.5 vs. 32±12.3months, P=0.036). After 12months of follow-up, cell reactivity was maintained in 33 patients (78%)., Conclusion:   A high percentage of patients exhibit a significant and maintained reactivity to myelin peptides over time. Therefore, this mix may be useful as a source of antigen in the development of protocols aimed at inducing specific tolerance in MS., (© 2010 The Author(s). European Journal of Neurology © 2010 EFNS.)

Because splenectomy has been linked to overwhelming infection years ago, management of splenic traumatisms has become progressively conservative. To assess the immunological function of the spleen in patients with splenic traumatism of different intensity, 43 patients with splenic injury (grades I through V) undergoing either nonoperative management, splenectomy, splenectomy with autotransplantation, or splenic embolization were analyzed for lymphocyte subpopulations and antibody responses to Streptococcus pneumoniae and Haemophilus influenzae vaccinations. Patients treated with splenectomy exhibited a significant decrease in CD4+ T lymphocytes and in Immunoglobulin (Ig) M(high)IgD(low) B cells (related to T-cell independent responses). Median fluorescence intensity of CD54+ in B cells also was reduced. The percent of IgM(high)IgD(low) B cells-a marker of marginal zone function-was inversely correlated with the number of pitted-red blood cells-a marker of red pulp function loss. IgM anti-S pneumoniae identified those patients with a defective rapid response to polysaccharide antigens. These results reinforce the importance of conservative options in the treatment of splenic traumatism for even a severely damaged organ. Despite the significant differences among the groups reported, it remains difficult to predict the IgM response to S pneumoniae vaccine of the individual patients. Better markers to assess splenic function and vaccination response after severe splenic traumatism-even in patients with nonoperative management-might improve risk assessment for overwhelming postsplenectomy infection., (Copyright © 2011 Mosby, Inc. All rights reserved.)

Notch signaling is involved in multiple cellular processes. Recent data also support the prominent role of Notch signaling in the regulation of the immune response. In this study, we analyzed the expression and function of Notch receptors and ligands on both human blood conventional dendritic cells (cDCs) and plasmacytoid DCs (pDCs). The expression and modulation upon TLR activation of Notch molecules partially differed between cDCs and pDCs, but functional involvement of the Notch pathway in both cell types was clearly revealed by specific inhibition using DAPT. Beyond the induction of Notch target genes and modulation of maturation markers, Notch pathway was also involved in a differential secretion of some specific cytokines/chemokines by DC subsets. Whereas Notch ligation induced IL-10 and CCL19 secretion in cDCs, Notch inhibition resulted in a diminished production of these proteins. With regard to pDCs, Notch activation induced TNF-α whereas Notch inhibition significantly abrogated the secretion of CCL19, CXCL9, CXCL10, and TNF-α. Additionally, Notch modulation of DC subsets differentially affected Th polarization of allostimulated T cells. Our results suggest that the Notch pathway may function as an additional mechanism controlling human DC responses, with differential activity on cDCs and pDCs. This control mechanism may ultimately contribute to define the local milieu promoted by these cells under the particular conditions of the immune response.

Background: The use of tolerogenic DCs is a promising therapeutic strategy for transplantation and autoimmune disorders. Immunomodulatory DCs are primarily generated from monocytes (MDDCs) for in vitro experiments following protocols that fail to fulfil the strict regulatory rules of clinically applicable products. Here, we compared the efficacy of three different tolerance-inducing agents, dexamethasone, rapamycin and vitamin D3, on DC biology using GMP (Good Manufacturing Practice) or clinical grade reagents with the aim of defining their use for human cell therapy., Methods: Tolerogenic MDDCs were generated by adding tolerogenic agents prior to the induction of maturation using TNF-α, IL-β and PGE2. We evaluated the effects of each agent on viability, efficiency of differentiation, phenotype, cytokine secretion and stability, the stimulatory capacity of tol-DCs and the T-cell profiles induced., Results: Differences relevant to therapeutic applicability were observed with the cellular products that were obtained. VitD3-induced tol-DCs exhibited a slightly reduced viability and yield compared to Dexa-and Rapa-tol-DCs. Phenotypically, while Dexa-and VitD3-tol-DCs were similar to immature DCs, Rapa-tol-DCs were not distinguishable from mature DCs. In addition, only Dexa-and moderately VitD3-tol-DCs exhibited IL-10 production. Interestingly, in all cases, the cytokine secretion profiles of tol-DCs were not modified by a subsequent TLR stimulation with LPS, indicating that all products had stable phenotypes. Functionally, clearly reduced alloantigen T cell proliferation was induced by tol-DCs obtained using any of these agent. Also, total interferon-gamma (IFN-γ) secretion by T cells stimulated with allogeneic tol-DCs was reduced in all three cases, but only T cells co-cultured with Rapa-tol-DCs showed impaired intracellular IFN-γ production. In addition, Rapa-DCs promoted CD4+ CD127 low/negative CD25high and Foxp3+ T cells., Conclusions: Our results demonstrate contrasting influences of different clinical-grade pharmacological agents on human tol-DC generation. This should be taken into account for decisions on the use of a specific agent for the appropriate cellular therapy in the context of a particular disease.

Graves' disease (GD) is a chronic autoimmune process in the thyroid gland and involves IFN and IFN driven immune activation. Assuming the thyroid gland is the main site stimulating the autoimmune response, we investigated the role of IFNs and other factors in the chronic evolution of GD by comparing the transcriptomic profiles of thyroid glands from short clinical course (SC), long clinical course (LC) cases, and control glands (C). Over 200 differentially expressed genes of the immune system were identified. Results were extensively analyzed bioinformatically and validated by qPCR in 31 glands. The analysis indicated that GD involved a progressive accumulation of changes with clearly distinct profiles in the SC and LC glands. Humoral response, antigen presentation and chemokines & cytokines were overall the most represented gene ontology categories in LC cases. Ingenuity Pathway Analysis pointed to a few inflammatory pathways in SC cases whereas LC cases involved numerous complex pathways, such us "communication between innate and adaptive immune cells" and "autoimmune thyroid signaling". A broad IFN signature consisted of the over-expression of 74 and 84 type I and type II IFN responsive genes respectively (overall 96 out of 211, 45%), but many of these genes can also be directly activated through cytoplasmic viral receptors. For the first time, plasmocytoid dendritic cells were identified in GD thyroid, but surprisingly, the main producers of IFN-alpha were cells with a myeloid cell phenotype. In addition, cells with the phenotype of alternatively activated macrophages were detected in abundance in GD thyroids, confirming data from the transcriptomic analysis. Collectively, these results confirmed the role of IFNs, suggested other natural immunity triggers, identified new cell types in the local disease process, and expanded our knowledge of the processes that may determine the chronicity of GD., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Type 1 diabetes (T1D) is an autoimmune disease caused by the selective destruction of the insulin-producing β cells. Research into the pathogenesis of T1D has been hindered by the lack of detection of the autoimmune process during the asymptomatic period and by the inaccessibility to the target tissue. Therefore current understanding of the immunological phenomena that take place in the pancreas of the patients is very limited and much of the current knowledge on T1D has been obtained using animal models. Microarray technology and bioinformatics allow the comparison of the gene expression profile - transcriptome - in normal and pathological conditions, creating a global picture of altered processes. Microarray experiments have defined new transcriptional alterations associated with several autoimmune diseases, and are focused on the identification of specific biomarkers. In this review we summarize current data on gene expression profiles in T1D from an immunological point of view. Reported transcriptome studies have been performed in T1D patients and Non-Obese Diabetic mouse models analyzing peripheral blood, lymphoid organs and pancreas/islets. In the periphery, the distinctive profiles are inflammatory pathways inducible by IL-1β and IFNs that can help in the identification of new biomarkers. In the target organ, a remarkable finding is the overexpression of inflammatory and innate immune response genes and the active autoimmune response at longstanding stages, contrary to the pre-existing concept of acute autoimmune process in T1D., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Dendritic cells (DCs) are powerful antigen-presenting cells capable of maintaining peripheral tolerance. The possibility to generate tolerogenic DCs opens new therapeutic approaches in the prevention or remission of autoimmunity. There is currently no treatment inducing long-term tolerance and remission in type 1 diabetes (T1D), a disease caused by autoimmunity towards beta cells. An ideal immunotherapy should inhibit the autoimmune attack, avoid systemic side effects and allow islet regeneration. Apoptotic cells--a source of autoantigens--are cleared rapidly by macrophages and DCs through an immunologically silent process that contributes to maintaining tolerance. Our aims were to prevent T1D and to evaluate the re-establishment of peripheral tolerance using autologous DCs pulsed in vitro with apoptotic bodies from beta cells. Immature DCs derived from bone marrow of non-obese diabetic (NOD) mice were obtained and pulsed with antigen-specific apoptotic bodies from the beta cell line NIT-1. Those DCs that phagocytosed apoptotic cells diminished the expression of co-stimulatory molecules CD40 and CD86 and reduced secretion of proinflammatory cytokines. Moreover, these cells were resistant to increase the expression of co-stimulatory molecules after lipopolysaccharide activation. The administration of these cells to NOD transgenic mice expressing interferon-beta in their insulin-producing cells, a model of accelerated autoimmune diabetes, decreased diabetes incidence significantly and correlated positively with insulitis reduction. DCs pulsed with apoptotic cells that express disease-associated antigens constitutes a promising strategy to prevent T1D.

Type 1 diabetes (T1D) is caused by the selective destruction of the insulin-producing beta cells of the pancreas by an autoimmune response. Due to ethical and practical difficulties, the features of the destructive process are known from a small number of observations, and transcriptomic data are remarkably missing. Here we report whole genome transcript analysis validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and correlated with immunohistological observations for four T1D pancreases (collected 5 days, 9 months, 8 and 10 years after diagnosis) and for purified islets from two of them. Collectively, the expression profile of immune response and inflammatory genes confirmed the current views on the immunopathogenesis of diabetes and showed similarities with other autoimmune diseases; for example, an interferon signature was detected. The data also supported the concept that the autoimmune process is maintained and balanced partially by regeneration and regulatory pathway activation, e.g. non-classical class I human leucocyte antigen and leucocyte immunoglobulin-like receptor, subfamily B1 (LILRB1). Changes in gene expression in islets were confined mainly to endocrine and neural genes, some of which are T1D autoantigens. By contrast, these islets showed only a few overexpressed immune system genes, among which bioinformatic analysis pointed to chemokine (C-C motif) receptor 5 (CCR5) and chemokine (CXC motif) receptor 4) (CXCR4) chemokine pathway activation. Remarkably, the expression of genes of innate immunity, complement, chemokines, immunoglobulin and regeneration genes was maintained or even increased in the long-standing cases. Transcriptomic data favour the view that T1D is caused by a chronic inflammatory process with a strong participation of innate immunity that progresses in spite of the regulatory and regenerative mechanisms.

Diabetes mellitus is a debilitating disease and alternative methods of treatment are a priority if the short-term and long term sequelae are to be avoided.  Here the authors manipulate NES2Y cells, which have the potential to be used as 'fusion partners' to produce human insulin-producing glucose-responsive hybrids.  The fusion experiments were carried out using polyethylene glycol (PEG) and electroporation.  Human insulin production of the resulting hybrids (in response to glucose) was measured using ELISA. Our results showed that it is possible to engineer human glucose-responsive insulin-producing (hGRIPs) hybrid cells by the manipulation of two different cell types. The resulting hybrids continuously grow in culture and are insulin-secreting and glucose-responsive for a period of time.  Immortalised cells with the characteristics of human beta cells could provide an important resource for experimental studies in Type 1 diabetes, such as an improved understanding of the fundamental mechanisms of glucose-responsive insulin processing and secretion, transplantation and drug screening programs.

In the field of organ transplantation, the state of thymic function has not been a major concern but data from bone marrow transplantation studies have unravel the persistence of some thymopoiesis in the adult and, more importantly, the possibility of reinducing it. Given the central role of the thymus in tolerance, these facts have stimulated the interest in the biology of the thymus in humans. Contemporarily, basic research has provided new tools, if imperfect, to monitor thymic function, that is, T-cell receptor excision circles, markers for lymphocytes recently emigrated from the thymus and new imaging techniques. The deployment of these new tools is already changing some paradigms and has now established that re-enactment of thymic activity in the course of bone marrow transplantation or in patients with human immunodeficiency virus on highly active anti-retroviral therapy is beneficial and that can be achieved in the adult. Clinical trials using thymopoiesis-stimulating factors are underway. On the other hand, the discovery that the thymus contains a broad representation of self-antigens and that this depends on the expression of the product of the gene AIRE by the medullary thymic epithelial cells opens the possibility of manipulating central tolerance. Current protocols inducing microchimerism to generate tolerance to solid organ grafts suggest that this could be a feasible therapeutic goal. Therefore, there are many signs indicating that a period of translational research applying the principles of thymic biology and central tolerance to transplantation has already started.

Patients with Type 1 diabetes mellitus (T1D) have an increased prevalence of associated organ-specific autoimmune diseases such as pernicious anemia whose histological substrate is a chronic atrophic gastritis (CAG). Latent pernicious anemia precedes clinically-manifest pernicious anemia and may be difficult to detect solely on simple analytical grounds. We recently described an increased prevalence of clinically-latent pernicious anemia in T1D using low concentrations of pepsinogen I, a zymogen of pepsin present in gastric mucosa, as a useful additional diagnostic marker, besides parietal cell antibodies, for screening latent pernicious anemia in T1D. The failure of peripheral tolerance mechanisms such as regulatory T cells (Treg) might be involved in CAG development in T1D patients. Indeed, functional defects in Tregs have been described in T1D patients. To this end, the percentage of Tregs in peripheral blood of T1D-CAG patients was analyzed and compared with those of a group of T1D without associated autoantibodies and a healthy control group. Tregs levels were also analyzed in gastric biopsies of T1D-CAG patients. The results obtained have led to new questions regarding the pathogenic mechanisms implicated in the development of associated autoimmune diseases in T1D.

The development of specific therapies for organ-specific autoimmune diseases requires the identification of relevant immunogenic epitopes, recognized by both pathogenic T cells and autoantibodies. Here, we review the most relevant studies focused in the identification of peptides in multiple sclerosis (MS) and the distinct T cell reactivity induced in patients compared to controls. Only a few studies reported significant differences in terms of T cell reactivity to them. The current knowledge on this issue, and the diagnostic and therapeutic possibilities opened by the identification of pathogenic MS epitopes are discussed in this paper.

Lung transplantation (LT) has become an accepted therapy for selected patients with advanced lung disease. One of the main limitations to successful LT is rejection of the transplanted organ where chemokines are pivotal mediators. Here, we test the relationship between copy number variation (CNV) in the CCL4L chemokine gene and rejection risk in LT patients (n=161). Patients with no acute rejection showed a significantly lower mean number of CCL4L copies than patients that showed acute rejection (1.66 vs 1.96, P=0.014), with an even greater number of gene copies seen in patients with more than one episode of acute rejection (1.66 vs 2.30, P=0.001). Additionally, patients with > or =2 CCL4L copies had a significantly higher risk of acute rejection compared with patients that had 0-1 CCL4L copies (odds ratio 2.65; 95% confidence interval, 1.33-5.28; P=0.0046). A combined analysis of CCL4L CNV and the rs4796195 CCL4L single nucleotide polymorphism demonstrated that the effect of CCL4L copy number in acute rejection is mainly because of the number of copies of the CCL4L1 allelic variant. This finding constitutes the first report of CNV as a correlate factor in allograft rejection.

Autoimmune thyroid diseases (AITD) are considered as prototypic organ-specific autoimmune diseases, yet their underlying aetiology remains poorly understood. Among the various pathophysiological mechanisms considered, a failure of central tolerance has received little attention. Here we present evidence in favour of dysregulated thymic function playing a role in AITD. Flow-cytometric analyses conducted in peripheral blood lymphocytes from 58 AITD patients and 48 age- and-sex-matched controls showed that AITD patients have significantly higher blood levels of CD4(+)CD45RA(+), CD4(+)CD31(+) and CD4/CD8 double-positive T lymphocytes, all markers of recent thymic emigrants (RTE). In addition, the alpha-signal joint T cell receptor excision circles (TRECs) content (a molecular marker of RTEs) was higher in the group of AITD patients older than 35 years than in age-matched controls. This was independent from peripheral T cell expansion as assessed by relative telomere length. Comparisons of TREC levels in peripheral blood lymphocytes and intrathyroidal lymphocytes in paired samples showed higher levels within the thyroid during the initial 30 months of the disease, indicating an influx of RTE into the thyroid during the initial stages of AITD. Additionally, a lack of correlation between TREC levels and forkhead box P3 expression suggests that the intrathyroidal RTE are not natural regulatory T cells. These results uncover a hitherto unknown correlation between altered thymic T cell export, the composition of intrathyroidal T cells and autoimmune pathology.

Endocrine epithelial cells, targets of the autoimmune response in thyroid and other organ-specific autoimmune diseases, express HLA class II (HLA-II) molecules that are presumably involved in the maintenance and regulation of the in situ autoimmune response. HLA-II molecules thus expressed by thyroid cells have the "compact" conformation and are therefore expected to stably bind autologous peptides. Using a new approach to study in situ T cell responses without the characterization of self-reactive T cells and their specificity, we have identified natural HLA-DR-associated peptides in autoimmune organs that will allow finding peptide-specific T cells in situ. This study reports a first analysis of HLA-DR natural ligands from ex vivo Graves' disease-affected thyroid tissue. Using mass spectrometry, we identified 162 autologous peptides from HLA-DR-expressing cells, including thyroid follicular cells, with some corresponding to predominant molecules of the thyroid colloid. Most interestingly, eight of the peptides were derived from a major autoantigen, thyroglobulin. In vitro binding identified HLA-DR3 as the allele to which one of these peptides likely associates in vivo. Computer modeling and bioinformatics analysis suggested other HLA-DR alleles for binding of other thyroglobulin peptides. Our data demonstrate that although the HLA-DR-associated peptide pool in autoimmune tissue mostly belongs to abundant ubiquitous proteins, peptides from autoantigens are also associated to HLA-DR in vivo and therefore may well be involved in the maintenance and the regulation of the autoimmune response.

Rearrangement analysis of immunoglobulin genes is an exceptional opportunity to look back at the B lymphocyte differentiation during ontogeny and the subsequent immune response, and thus to study the selective pressures involved in autoimmune disorders. In a recent study to characterize the antigenic specificity of B lymphocytes during T1D progression, we generated hybridomas of islet-infiltrating B lymphocytes from NOD mice and other related strains developing insulitis, but with different degrees of susceptibility to T1D. We found that a sizable proportion of hybridomas produced monoclonal antibodies reactive to peripherin, an intermediate filament protein mainly found in the peripheral nervous system. Moreover, we found that anti-peripherin antibody-producing hybridomas originated from B lymphocytes that had undergone immunoglobulin class switch recombination, a characteristic of secondary immune response. Therefore, in the present study we performed immunoglobulin VL and VH analysis of these hybridomas to ascertain whether they were derived from B lymphocytes that had undergone antigen-driven selection. The results indicated that whereas some anti-peripherin hybridomas showed signs of oligoclonality, somatic hypermutation and/or secondary rearrangements (receptor edition and receptor revision), others seemed to directly derive from the preimmune repertoire. In view of these results, we conclude that anti-peripherin B lymphocytes are positively selected and primed in the course of T1D development in NOD mice, and reinforce the idea that peripherin is a relevant autoantigen targeted during T1D development in this animal model.

The recent description of a large amount of copy number variation (CNV) in the human genome has extended the concept of genome diversity. In this study we integrate the analysis of CNV and single nucleotide polymorphisms (SNPs) in the human CCL4L chemokine gene. CCL4L is a nonallelic copy of CCL4/MIP-1beta chemokine and displays a CNV that also includes the CCL3L gene, a nonallelic copy of CCL3/MIP-1alpha. This CNV and two functionally relevant CCL4L SNPs (rs4796195 and rs3744595) have been recently associated to HIV pathology in three independent studies. We have quantified the CCL4L copy number and genotyped both SNPs in samples from HGDP-CEPH Diversity Panel. A strong correlation between CCL4L CNV and one of the SNPs analyzed is found, whereas no significant linkage disequilibrium is found between the two SNPs despite their close distance (647 bp), suggesting a recent appearance of the second SNP when the diversity in the first one and CNV had already been generated. The present study points out that in genes with CNV, it may be a key issue to combine the assessment of gene copy number with the genotyping of relevant SNPs to understand the phenotypic impact of genome variation in the immune response.

The destruction of beta cells by the islet infiltrating lymphocytes causes type 1 diabetes. Transgenic mice models expressing interferon (IFN)-beta in beta cells, in the non-obese diabetic (NOD) strain and in a diabetes-free, major histocompatibility complex-matched, homologous strain, the non-obese resistant (NOR) mice, developed accelerated type 1 diabetes after 3 weeks of age. Our aim was to determine if natural killer (NK) cells could affect the acceleration of the disease. We determined the amount of NK cells in the pancreas, spleen and lymph nodes from NOD rat insulin promoter (RIP)-IFN-beta mice. Pancreatic cytokines were assessed by quantitative real-time polymerase chain reaction and protein arrays. To confirm the relevance of NK cells in the acceleration of autoimmune diabetes this subset was depleted with anti-asialo GM1 antibodies. An increase of intrapancreatic NK cells characterized the accelerated onset of diabetes both in NOD and NOR RIP-IFN-beta transgenic models. Cytokines involved in NK function and migration were found to be hyperexpressed in the pancreas from accelerated diabetic mice. Interestingly, the depletion of NK cells in vivo abolished completely the acceleration of diabetes. NK cells connect innate to adaptive immunity and might play a role in autoimmunity. We report here that NK cells are required critically in the pancreas for accelerated diabetes. This model links inflammation to acceleration of beta cell-specific autoimmunity mediated by NK cells.

Dendritic cells (DC) play essential functions in both innate and adaptive immune responses. Peripheral blood DCs are divided into two major subsets, named conventional DC (cDC) and plasmacytoid DC (pDC), which play specific functions in the immune response. The absolute numbers of DCs (and their subsets) in peripheral blood may suffer variations due to physiological or pathological reasons, and therefore the enumeration of DC subsets in blood samples may be of clinical interest. However, to date this enumeration has produced controversial rather than consistent results. Here, using a two-tube platform approach, peripheral blood DCs have been enumerated in samples from healthy blood donors aged 18-65 years old. The results obtained showed a significant age-related decrease in pDC numbers, whilst cDC numbers remained unaltered. The different protocols used to define and enumerate DC subsets from blood samples may account for the controversial results reported before, thus emphasizing the importance of a general consensus to enumerate DCs. Reduced pDC numbers may be related to the higher susceptibility to infection and deficient response to vaccination often observed in aged individuals.

In this second review on chemokines, we focus on the polymorphisms and alternative splicings and on their consequences in disease. Because chemokines are key mediators in the pathogenesis of inflammatory, autoimmune, vascular and neoplastic disorders, a large number of studies attempting to relate particular polymorphisms of chemokines to given diseases have already been conducted, sometimes with contradictory results. Reviewing the published data, it becomes evident that some chemokine genes that are polymorphic have alleles that are found repeatedly, associated with disease of different aetiologies but sharing some aspects of pathogenesis. Among CXC chemokines, single nucleotide polymorphisms (SNPs) in the CXCL8 and CXCL12 genes stand out, as they have alleles associated with many diseases such as asthma and human immunodeficiency virus (HIV), respectively. Of CC chemokines, the stronger associations occur among alleles from SNPs in CCL2 and CCL5 genes and a number of inflammatory conditions. To understand how chemokines contribute to disease it is also necessary to take into account all the isoforms resulting from differential splicing. The first part of this review deals with polymorphisms and the second with the diversity of molecular species derived from each chemokine gene due to alternative splicing phenomena. The number of molecular species and the level of expression of each of them for every chemokine and for each functionally related group of chemokines reaches a complexity that requires new modelling algorithms akin to those proposed in systems biology approaches.

Human and mouse plasmacytoid dendritic cells (PDC) express IL-2 mRNA specifically upon TLR stimulation, but not under CD40L stimulation. Even though the expression of the IL-2R by PDC has been described, the functional implications of this expression remain unknown. Here, we investigated the expression and function of the IL-2R in activated human PDC. The IL-2Ralpha chain, CD25, is expressed in both CpG- and CD40L-activated PDC. CD25 expression is a relatively rapid event, as the receptor was detected 6 h after the initial activation signal. Exogenous IL-2 added to CD40L-activated PDC increased the expression of CD25, enhanced the secretion of pro-inflammatory cytokines and promotes PDC survival. CpG-activated PDC cultured in the presence of IL-2R-blocking monoclonal antibodies showed a reduced expression of pro-inflammatory cytokines, especially TNF-alpha. This reduction was dose and time dependent, suggesting a regulatory role of IL-2 in TNF secretion that might occur at the post-transcriptional level. These results indicate that the expression of the IL-2R is relevant to human PDC activation, and that IL-2 may be an important auto- and/or paracrine factor modulating the activation and survival of PDC. Finally, CD25 expression may be considered as a useful early activation marker for human PDC.

Most of our knowledge of the antigenic repertoire of autoreactive B lymphocytes in type 1 diabetes (T1D) comes from studies on the antigenic specificity of both circulating islet-reactive autoantibodies and peripheral B lymphocyte hybridomas generated from human blood or rodent spleen. In a recent study, we generated hybridoma cell lines of infiltrating B lymphocytes from different mouse strains developing insulitis, but with different degrees of susceptibility to T1D, to characterize the antigenic specificity of islet-infiltrating B lymphocytes during progression of the disease. We found that many hybridomas produced mAbs restricted to the peripheral nervous system (PNS), thus indicating an active B lymphocyte response against PNS elements in the pancreatic islet during disease development. The aim of this study was to identify the autoantigen recognized by these anti-PNS mAbs. Our results showed that peripherin is the autoantigen recognized by all anti-PNS mAbs, and, therefore, a relevant neuroendocrine autoantigen targeted by islet-infiltrating B lymphocytes. Moreover, we discovered that the immune dominant epitope of this B lymphocyte immune response is found at the C-terminal end of Per58 and Per61 isoforms. In conclusion, our study strongly suggests that peripherin is a major autoantigen targeted during T1D development and poses a new question on why peripherin-specific B lymphocytes are mainly attracted to the islet during disease.

Chemokines are a superfamily of small structurally related cytokines that have evolved to form a complex network of proteins that typically regulate leucocyte traffic but also carry very diverse sets of immune and non-immune functions. Two general features of cytokines, redundancy and promiscuity, are particularly prominent in chemokines. In part, these properties result from repeated processes of gene duplication and diversification, which has led to the present complex genomic map of chemokines, which contains cases of non-allelic isoforms, copy number polymorphisms and classical allelic variation. This genomic complexity is compounded with pre-translational and post-translational mechanisms resulting in a complex network of proteins whose essential functions are maintained, constituting a remarkable case of robustness reminiscent of crucial metabolic pathways. This reflects the adaptation of a system under strong evolutive pressure, supporting the concept that the chemokine system is essential for the coordination, regulation and fine-tuning of the type of immune response. In this first review, we analyse currently available data on the chemokine superfamily, focusing on its complex genomic organization. Genes encoding essential inflammatory chemokines are grouped into defined chromosomal locations as clusters and miniclusters that, from the genetic point of view, can be considered single entities given their overall functions (many ligands of a cluster bind to a few shared receptors). We will try to interpret this genomic organization of chemokines in relation to the main functions acquired by each individual member or by each cluster. In a second review, we shall focus on the relationship of chemokine variability and disease susceptibility.

B-cells participate in the autoimmune response that precedes the onset of type 1 diabetes, but how these cells contribute to disease progression is unclear. In this study, we analyzed the phenotype and functional characteristics of islet-infiltrating B-cells in the diabetes-prone NOD mouse and in the insulitis-prone but diabetes-resistant (NOD x NOR)F1 mouse. The results indicate that B-cells accumulate in the islets of both mice influenced by sex traits. Phenotypically and functionally, these B-cells are highly affected by the islet inflammatory milieu, which may keep them in a silenced status. Moreover, although islet-infiltrating B-cells seem to be antigen experienced, they can only induce islet-infiltrating T-cell proliferation when they act as accessory cells. Thus, these results strongly suggest that islet-infiltrating B-cells do not activate islet-infiltrating T-cells in situ, although they may affect the progression of the disease otherwise.

Background and Objective: The splenic function of patients followed by the Department of General and Digestive Surgery in the Hospital Universitari Germans Trias i Pujol (HUGTiP) from 1985 to 2003 for different degrees of splenic trauma according to the classification of the American Association for the Surgery of Trauma (AAST) 1994 was quantified and related to the treatment received (non surgical, total splenectomy with or without splenosis and splenectomy plus autotransplantation) to detect splenic dysfunction predisposing the development of postsplenectomy sepsis (PSS)., Patients and Method: 43 patients underwent an isotopic study with dynamic splenic gammagraphy and pitted erythrocytes (Normarsky optics) and submembranous vacuoles (transmission electron microscopy) were evaluated., Results: The non surgical group presented normal phagocytic and filtration function with the median speed of splenic enhancement being 3.46 Kcts/s2 (interval: 0.8-6.98). The percentage of pitted erythrocytes was 2% (0-8.8), the number of pits per erythrocyte was 0.03 (0-0.12) and the percentage of erythrocytes with 1, 2, 3 and 4 pits was 1.6%, 0.4%, 0% and 0%, respectively. The percentage of red cells with submembranous vacuoles was 2.55% (0-5.6), the number of vacuoles per red cell was 0.03 (0-0.06) and the percentage of red cells with 1, 2, 3 and 4 vacuoles was 2%, 0.2%, 0% and 0%, respectively. In the operated group, the splenic enhancement speed was 0.08 Kcts/s2 (0-1.75) (p < 0.0001). The percentage of pitted erythrocytes was 38% (0.2-64) (p < 0.0001), the number of pits per erythrocyte was 0.86 (0-1.8) (p < 0.0001) and the percentage of erythrocytes with 1,2,3 and 4 pits was 16.39%, 7.2%, 3.59% and 2.52%, respectively (p < 0.0001). The percentage of red cells with submembranous vacuoles was 11.2% (1.8-31.9) (p = 0.0006); the number of vacuoles per cell was 0.16 (p = 0.0022) and the percentage of red cells with 1, 2, 3 and 4 vacuoles was 6.51%, 1.73%, 0.4% and 0.2%, respectively (p = 0.0246, 0.0010, < 0.0001 and 0.0002, respectively)., Conclusions: Splenic function of patients with a history of splenic trauma receiving conservative treatment is normal, independently of the degree of the lesion, thereby reinforcing the use of this therapeutic approach to avoid the development of postsplenectomy sepsis. In the patients treated with splenectomy, with or without splenosis, splenic function was absent or very altered being partially conserved in cases treated with splenectomy plus autotransplantation.

Tacrolimus is a widely used immunosuppressive agent. Although T cells are the main targets of these pharmacological drugs, antigen presentation may also be affected. Among antigen-presenting cells, plasmacytoid dendritic cells (PDCs) are the main source of type I interferons upon microbial challenge, and are involved in several diseases and autoimmune disorders. The aim of this study was to evaluate whether tacrolimus can modulate the function of PDCs in vitro. Maturation and function of PDCs was determined using flow cytometry, enzyme-linked immunosorbent assay and cytometry bead arrays. The effect of tacrolimus on PDCs was observed mainly when the cells were pretreated with the immunosuppressive agent before activation. Upon dinucleotide-oligodeoxynucleotide (CpG-ODN) activation, tacrolimus pretreated PDCs showed a significant reduction in the surface expression of co-stimulatory molecules and human leucocyte antigen D-related (HLA-DR) and secreted reduced levels of tumour necrosis factor (TNF)-alpha. These results show that tacrolimus treatment of PDCs impairs CpG-induced activation, which could affect the outcome of the immune response.

Celiac disease is a gluten-induced T-cell mediated autoimmune process that results in the destruction of the intestinal mucosa and is associated with an expansion of CD8(+) CD103(+) TCRalphabeta intraepithelial lymphocytes (IELs) in the damaged epithelium. The role of this IEL population in the pathology is unknown. The aim of this work was to compare the cytokine profile and the cytotoxicity pattern from CD8(+) IEL clones isolated from celiac (CD) and non-celiac (NCD) biopsies. We report that the number of IL-10 producing CD clones was significantly lower (26%) than that obtained from the NCD sample (62%). Instead, IL-2 was produced by more CD (44%) than NCD clones (26%). Cytotoxicity patterns against intestinal epithelial cell lines suggest different functional subsets of CD8(+) IELs. CD clones capable of high cytotoxicity produced IL-2 whereas most cytotoxic NCD IELs produced IL-10. This clonal analysis indicates that an impaired immune regulation in celiac mucosa may be partially attributed to the low generation of regulatory CD8(+) IELs that produce IL-10.

Aims/hypothesis: The expression of IFNbeta in beta cells results in accelerated type 1 diabetes. The REG family of beta cell proliferation factors have been described as autoantigens in autoimmune diabetes. The aim of this study was to determine the effect of IFNbeta on Reg expression, and the implications of this in terms of autoimmunity., Methods: Reg gene expression was determined in islets from non-obese diabetic (NOD) RIP-HuIFNbeta mice by cDNA microarray, quantitative real-time PCR and immunohistochemistry. The effect of IFNbeta on Reg1 and Reg2 expression was assessed in the NOD insulinoma cell line NIT-1. IL-6, known to induce Reg expression, was measured in the insulitis microenvironment. Morphological studies were carried out to determine islet enlargement in this model., Results: Reg2 was upregulated in islets from the NOD RIP-HuIFNbeta mice at the onset of the autoimmune attack. IFNbeta upregulates Reg1 and Reg2 genes in NIT-1 cells. The expression of Il6 was increased in islets from transgenic mice and in NIT-1 cells exposed to HuIFNbeta. Moreover, islets from transgenic mice were enlarged compared with those from wild-type mice., Conclusions/interpretation: Reg overexpression correlates well with the acceleration of diabetes in this model. The upregulation of Reg suggests that islets try to improve hyperglycaemia by regenerating the cells lost in the autoimmune attack. Reg expression is regulated by several factors such as inflammation. Therefore, the overexpression of an IFNbeta-induced autoantigen (REG) in the islets during inflammation might contribute to the premature onset of diabetes.

Autoimmune thyroid diseases are characterized by lymphocytic infiltration of the thyroid gland. Chemokines are crucial in the recruitment of lymphocytes and might play an important role in the pathogenesis of autoimmune thyroid disease. The aim of this study was to test the feasibility of analysing by one-tube reverse-transcriptase polymerase chain reaction (RT-PCR) technique CC chemokine profiles in samples obtained by fine needle aspiration biopsy (FNAB). In 27 out of 35 (77%) samples, the material was sufficient for analysis and in 16 (59%) chemokines were detected, thus demonstrating the potential of this technique. Moreover, even in this small group, a statistically significant increase of CCL3 and CCL4 was found in samples from patients with autoimmune thyroid disease as compared to those with multinodular goiter. Chemokine profile measured by improved multiamplification techniques in FNAB thyroid samples may become a useful complementary tool for the management of thyroid autoimmune disease as it constitutes a source of data for research of their pathogenesis.

HLA genotyping by polymerase chain reaction (PCR) has some inherent labor-intensive and effort-demanding limitations. To overcome them, we have developed a real-time PCR with hybridization probes approach able to obtain a medium-low resolution HLA-B genotyping with fewer tubes and probes and with a shorter time requirement. Our strategy used 18 simultaneous reactions amplifying HLA-B alleles and an internal control. Monitorization of both amplifications in each tube is performed by the simultaneous application of two fluorescent resonance emission transfer probes: the first probe, different for each tube, is specific for the HLA-B locus and the second probe detects the control gene. A medium-low resolution (300 HLA-B allelic groups) typing is obtained for each sample by analyzing the melting curve patterns. Because some alleles may be determined without using the complete set of reactions, we present an alternative strategy: a first round of seven reactions and, according to the result, a second (or third) round of PCRs to solve the ambiguities. This method was validated in pretyped clinical samples and the results were completely concordant. Moreover, fewer ambiguous results were obtained. In summary, we present a new, faster, and more accurate method than currently used PCR techniques to type HLA-B alleles.

The frequent observation of organized lymphoid structures that resemble secondary lymphoid organs in tissues that are targeted by chronic inflammatory processes, such as autoimmunity and infection, has indicated that lymphoid neogenesis might have a role in maintaining immune responses against persistent antigens. In this Review, we discuss recent progress in several aspects of lymphoid neogenesis, focusing on the similarities with lymphoid tissue development, the mechanisms of induction, functional competence and pathophysiological significance. As more information on these issues becomes available, a better understanding of the role of lymphoid neogenesis in promoting chronic inflammation might eventually lead to new strategies to target immunopathological processes.

It is well established that the polymorphisms at the 5' of the insulin gene (IDDM2) confers susceptibility to type 1 diabetes, probably by modifying the level of insulin expression in the thymus that in turn influences immunological tolerance to insulin as self-antigen. AIRE is a transcription regulator which controls the expression of many peripheral antigens within the thymus, among them insulin. Results presented here confirm that insulin gene copies from both parental chromosomes are expressed in human thymus and that IDDM2 class III protective alleles are indeed associated with a higher level of insulin message expression. However, differences in insulin mRNA expression among different thymi were far wider than those determined by the class I and class III insulin gene alleles and maintained a clear correlation with AIRE expression. These results confirm the effect of IDDM2 alleles on insulin expression in the thymus, but suggest that the levels of AIRE may exert a stronger influence than IDDM2 alleles themselves.

The role played by dendritic cell (DC) subsets in the immune response to alloantigens is not well defined. In vitro experiments have extensively shown that freshly isolated myeloid (M)DCs induce a strong T lymphocyte proliferation whereas plasmacytoid (P)DCs do not, unless activated by CD40 ligation. The aim of these studies was to explore whether the interplay among PDCs, MDCs and T cells modulates alloresponse. Freshly isolated MDCs and PDCs were merged in different proportions and used as antigen presenting cells (APCs) in mixed lymphocyte cultures (MLC). As described, isolated PDCs only induced a mild alloresponse, while MDCs were potent inducers of alloproliferation. Unexpectedly, when PDCs were merged with even low numbers of MDCs (down to 100 cells) and used as APCs, a potent Th1 cell proliferation was detected. Survival and maturation of PDCs was increased in these MLC conditions, which could partially explain the magnitude of the T-cell response. Interestingly, the proportion of IFNgamma-producing cells generated in such cultures was higher compared to MDC-stimulated cultures. These data suggest that the interaction between both DC subsets is determinant to generate a potent Th1 response, at least in an allogeneic situation, and may be relevant to the outcome of allogeneic stem cell transplantation.

Human CCL4/macrophage inflammatory protein (MIP)-1beta and CCL3/MIP-1alpha are two highly related molecules that belong to a cluster of inflammatory CC chemokines located in chromosome 17. CCL4 and CCL3 were formed by duplication of a common ancestral gene, generating the SCYA4 and SCYA3 genes which, in turn, present a variable number of additional non-allelic copies (SCYA4L and SCYA3L1). In this study, we show that both CCL4 loci (SCYA4 and SCYA4L) are expressed and alternatively generate spliced variants lacking the second exon. In addition, we found that the SCYA4L locus is polymorphic and displays a second allelic variant (hereinafter SCYA4L2) with a nucleotide change in the intron 2 acceptor splice site compared with the one described originally (hereinafter SCYA4L1). Therefore, the pattern of SCYA4L2 transcripts is completely different from that of SCYA4L1, since SCYA4L2 uses several new acceptor splice sites and generates nine new mRNAs. Furthermore, we analyzed the contribution of each locus (SCYA4 and SCYA4L1/L2) to total CCL4 expression in human CD8 T cells by RT-amplified fragment length polymorphism and real-time PCR, and we found that L2 homozygous individuals (L2L2) only express half the levels of CCL4 compared with L1L1 individuals. The analysis of transcripts from the SCYA4L locus showed a lower level in L2 homozygous compared with L1 homozygous individuals (12% vs 52% of total CCL4 transcripts). A possible clinical relevance of these CCL4 allelic variants was suggested by the higher frequency of the L2 allele in a group of HIV(+) individuals (n = 175) when compared with controls (n = 220, 28.6% vs 16.6% (p = 0.00016)).

Polymerase chain reaction (PCR)-based human leukocyte antigen (HLA) typing methods currently used in most histocompatibility laboratories, such as PCR-sequence-specific primers (PCR-SSP) and PCR-sequence-specific oligonucleotide probes (PCR-SSO), are time-consuming and are at risk of contamination during the post-PCR process. The aim of this study was to develop a real-time PCR (rtPCR)-based HLA-DRB1 and -DRB3/4/5 low-medium resolution typing method to avoid these problems. This new method combined the use of specific primers and probes for HLA-DRB alleles. One pair of DRB gene primers and two DRB-specific probes (FAM and VIC) were used per reaction in each of a set of 16 PCR reaction tubes. To provide an internal positive control, each tube also contained a pair of primers and a TET probe for glyceraldehyde phosphate dehydrogenase. This allowed a very significant reduction in the number of reactions and the processing time, whereas typing resolution increased. After successful testing on 100 samples, the technique was validated in 200 clinical samples that had previously been typed for HLA-DRB using a standard PCR-based method. Identical results were obtained with all samples. This new method also reduced ambiguous results and was faster and less cumbersome than currently used PCR-SSP or PCR-SSO techniques.

B-cells accumulate in pancreatic islets during the autoimmune response that precedes the onset of type 1 diabetes. However, the role and antigenic specificity of these cells remain a mystery. To elucidate the antigenic repertoire of islet-infiltrating B-cells in type 1 diabetes, we generated hybridoma cell lines of islet-infiltrating B-cells from nonobese diabetic (NOD) mice and NOD mice expressing a diabetogenic T-cell receptor (8.3-NOD). Surprisingly, characterization of the tissue specificity of the antibodies secreted by these cells revealed that a predominant fraction of these hybridomas produce antibodies specific for the pancreatic nervous system. Similar results were obtained with B-cell hybridomas derived from mild insulinic lesions of diabetes-resistant (NOD x NOR)F1 and 8.3-(NOD x NOR)F1 mice. Immunoglobulin class analyses further indicated that most islet-derived hybridomas had arisen from B-cells that had undergone immunoglobulin class switch recombination, suggesting that islet-associated B-cells are involved in active, T-helper-driven immune responses against local antigenic targets. This is the first evidence showing the existence of a predominant active B-cell response in situ against pancreatic nervous system elements in diabetogenesis. Our data are consistent with the idea that this B-cell response precedes the progression of insulitis to overt diabetes, thus strongly supporting the idea that pancreatic nervous system elements are early targets in type 1 diabetes.

Genetic and environmental factors are decisive in the etiology of type 1 diabetes. Viruses have been proposed as a triggering environmental event and some evidences have been reported: type I IFNs exist in the pancreata of diabetic patients and transgenic mice expressing these cytokines in beta cells develop diabetes. To determine the role of IFNbeta in diabetes, we studied transgenic mice expressing human IFNbeta in the beta cells. Autoimmune features were found: MHC class I islet hyperexpression, T and B cells infiltrating the islets and transfer of the disease by lymphocytes. Moreover, the expression of beta(2)-microglobulin, preproinsulin, and glucagon in the thymus was not altered by IFNbeta, thus suggesting that the disease is caused by a local effect of IFNbeta, strong enough to break the peripheral tolerance to beta cells. This is the first report of the generation of NOD (a model of spontaneous autoimmune diabetes) and nonobese-resistant (its homologous resistant) transgenic mice expressing a type I IFN in the islets: transgenic NOD and nonobese-resistant mice developed accelerated autoimmune diabetes with a high incidence of the disease. These results indicate that the antiviral cytokine IFNbeta breaks peripheral tolerance to beta cells, influences the insulitis progression and contributes to autoimmunity in diabetes and nondiabetes- prone mice.

Several polymerase chain reaction (PCR)-based human leukocyte antigen (HLA) genotyping methods are in use, but none is fully satisfactory. The introduction of real-time PCR (rt-PCR) with fluorescence resonance energy transfer (FRET) probes provides a powerful tool to overcome the drawbacks of current methods such as the long processing time and the requirement for post-PCR manual procedures. Here we present evidence that the FRET-fluorotyping principle may resolve HLA-B27 variants, providing a higher resolution in less time than the techniques currently in use. The protocol uses between one and three consecutive amplification reactions depending on the resolution required. The first reaction, aimed at detecting HLA-B27-positive samples, uses beta-globin coamplification as control. The second reaction, aimed at resolving most frequent B27 alleles, uses two hybridization probes whose melting temperatures curves allow the classification of HLA-B27 alleles into eight groups. By adding a third reaction, even the rarest alleles associated and not associated to ankylosing spondylitis (AS) may be discriminated. The technique was blindly tested on 60 samples from individuals previously typed and confirmed by standard PCR sequence-specific oligoprobes-PCR sequence and PCR-based typing PCR-SBT (30 B27+, 30 non-B27). There was a complete concordance rate, thus confirming the potential of this new technique for clinical HLA-B27 typing and for HLA typing in general.