Single cell transcriptomic analysis of Graves' disease thyroid glands reveals the broad immunoregulatory potential of thyroid follicular and stromal cells and implies a major re-interpretation of the role of aberrant HLA class II expression in autoimmunity.

The study of the immune response in thyroid autoimmunity has been mostly focused on the autoantibodies and lymphocytes, but there are indications that intrinsic features of thyroid tissue cells may play a role in disrupting tolerance that needs further investigation. The overexpression of HLA and adhesion molecules by thyroid follicular cells (TFC) and our recent demonstration that PD-L1 is also moderately expressed by TFCs in autoimmune thyroid indicates that TFCs they may activate but also inhibit the autoimmune response. Intriguingly, we have recently found that in vitro cultured TFCs are able to suppress the proliferation of autologous lymphocyte T in a contact-dependent manner which is independent of the PD-1/PD-L1 signaling pathway. To get a more comprehensive picture of TFC activating and inhibitory molecules/pathways driving the autoimmune response in the thyroid glands, preparations of TFCs and stromal cells from five Graves' disease (GD) and four control thyroid glands were compared by scRNA-seq. The results confirmed the previously described interferon type I and type II signatures in GD TFCs and showed unequivocally that they express the full array of genes that intervene in the processing and presentation of endogenous and exogeneous antigens. GD TFCs lack however expression of costimulatory molecules CD80 and CD86 required for priming T cells. A moderate overexpression of CD40 by TFCs was confirmed. GD Fibroblasts showed widespread upregulation of cytokine genes. The results from this first single transcriptomic profiling of TFC and thyroid stromal cells provides a more granular view of the events occurring in GD. The new data point at an important contribution of stromal cells and prompt a major re-interpretation of the role of MHC over-expression by TFC, from deleterious to protective. Most importantly this re-interpretation could also apply to other tissues, like pancreatic beta cells, where MHC over-expression has been detected in diabetic pancreas.

Thyroid cells from normal and autoimmune thyroid glands suppress T lymphocytes proliferation upon contact revealing a new regulatory inhibitory type of interaction independent of PD1/PDL1.

Immune Checkpoint Receptors include a number of inhibitory receptors that limit tissue damage during immune responses; blocking PD-1/PD-L1 checkpoint receptor axis led to a paradigm shift in cancer immunotherapy but also to autoimmune adverse effects, prominently thyroid autoimmunity. Although PD-L1 is known to be expressed on thyroid follicular cells (TFCs) of autoimmune glands the role on PD-1/PD-L1 in the interaction between T cells and thyroid cells in the tissue has not been investigated. Here we report that autologous primary TFCs, but not transformed TFCs, inhibit CD4 and CD8 T cell proliferation but no cytokine production. This effect is not, however, mediated by PD-1/PD-L1 nor locally produced cytokines. Beta galactosidase analysis excluded culture-induced senescence as an explanation. High resolution flow cytometry demonstrated that autologous TFC/T cells co-culture induced the expansion of several clusters of double negative (DN) T cells characterized by high expression of activation markers and negative immune checkpoints. Single cell transcriptomic profiling demonstrated that dissociated TFC express numerous candidate molecules for mediating this suppressive activity, including CD40, E-Cadherin and TIGIT ligands. These ligands directly or through the generation of a suppressor population of DN T cells, and not the PD-1/PD-L1 axis, are most likely the responsible of TFC immunosuppressive activity. These results contribute to reveal the complex network of inhibitory mechanism that operate at the tissue level to restrain autoimmunity but also point to pathways, other that PD-1/PD-L1, that can contribute to tumor evasion.

Activation of the Monocyte/Macrophage System and Abnormal Blood Levels of Lymphocyte Subpopulations in Individuals with Autism Spectrum Disorder: A Systematic Review and Meta-Analysis.

Autism spectrum disorder (ASD) is a neurodevelopmental condition with a so far unknown etiology. Increasing evidence suggests that a state of systemic low-grade inflammation may be involved in the pathophysiology of this condition. However, studies investigating peripheral blood levels of immune cells, and/or of immune cell activation markers such as neopterin are lacking and have provided mixed findings. We performed a systematic review and meta-analysis of studies comparing total and differential white blood cell (WBC) counts, blood levels of lymphocyte subpopulations and of neopterin between individuals with ASD and typically developing (TD) controls (PROSPERO registration number: CRD CRD42019146472). Online searches covered publications from 1 January 1994 until 1 March 2022. Out of 1170 publication records identified, 25 studies were finally included. Random-effects meta-analyses were carried out, and sensitivity analyses were performed to control for potential moderators. Results: Individuals with ASD showed a significantly higher WBC count (k = 10, g = 0.29, p = 0.001, I2 = 34%), significantly higher levels of neutrophils (k = 6, g = 0.29, p = 0.005, I2 = 31%), monocytes (k = 11, g = 0.35, p < 0.001, I2 = 54%), NK cells (k = 7, g = 0.36, p = 0.037, I2 = 67%), Tc cells (k = 4, g = 0.73, p = 0.021, I2 = 82%), and a significantly lower Th/Tc cells ratio (k = 3, g = −0.42, p = 0.008, I2 = 0%), compared to TD controls. Subjects with ASD were also characterized by a significantly higher neutrophil-to-lymphocyte ratio (NLR) (k = 4, g = 0.69, p = 0.040, I2 = 90%), and significantly higher neopterin levels (k = 3, g = 1.16, p = 0.001, I2 = 97%) compared to TD controls. No significant differences were found with respect to the levels of lymphocytes, B cells, Th cells, Treg cells, and Th17 cells. Sensitivity analysis suggested that the findings for monocyte and neutrophil levels were robust, and independent of other factors, such as medication status, diagnostic criteria applied, and/or the difference in age or sex between subjects with ASD and TD controls. Taken together, our findings suggest the existence of a chronically (and systemically) activated inflammatory response system in, at least, a subgroup of individuals with ASD. This might have not only diagnostic, but also, therapeutic implications. However, larger longitudinal studies including more homogeneous samples and laboratory assessment methods and recording potential confounding factors such as body mass index, or the presence of comorbid psychiatric and/or medical conditions are urgently needed to confirm the findings.

Analysis of the PD-1/PD-L1 axis in human autoimmune thyroid disease: Insights into pathogenesis and clues to immunotherapy associated thyroid autoimmunity.

Autoimmune thyroid diseases (AITDs), i.e., Graves' disease (GD) and Hashimoto thyroiditis (HT), are the most prevalent organ-specific autoimmune diseases, but their pathogenesis is still incompletely understood. The PD-1/PD-L1 pathway is an important mechanism of peripheral tolerance that has not been investigated in AITDs. Here, we report the analysis of the expression of PD-1, PD-L1 and PD-L2 in PBMCs, infiltrating thyroid lymphocytes (ITLs) and in thyroid follicular cells (TFCs) in GD, HT and multinodular goiter (MNG) patients and healthy controls PBMCs (HC). By combining flow cytometry, tissue immunofluorescence and induction experiments on primary and thyroid cell line cultures, we show that: 1) while PD-1+ T cells are moderately expanded in PBMCs from GD vs HC, approximately half of T cells in the infiltrate are PD-1+ including some PD-1hi; 2) PD-L1, but not PD-L2, is expressed by 81% of GD glands and in 25% of non-autoimmune glands; 3) PD-L1, was expressed by TFCs in areas that also contain abundant PD-1 positive T cells but; 4) co-localization in TFCs indicated only partial overlap between the smaller areas of the PD-L1+ and the larger areas of HLA class II+ expression; 5) IFNγ is capable of inducing PD-L1 in >90% of TFCs in primary cultures and cell lines. Collectively these results indicate that the PD-1/PD-L1 axis is operative in AITD glands and may restrain the autoimmune response. Yet the discrepancy between easy induction in vitro and the limited expression in vivo (compared to HLA) suggests that PD-L1 expression in vivo is partially inhibited in GD and HT glands. In conclusions 1) the PD-1/PD-L1 pathway is activated in AITD glands but probably not to the extent to inhibit disease progression and 2) Thyroid autoimmunity arising after PD-1/PD-L1 blocking therapies in cancer patients may result from interfering PD-1/PD-L1 tolerance mechanism in thyroid with minimal (focal) thyroiditis. Finally acting on the PD-1/PD-L1 pathway could be a new approach to treat AITD and other organ-specific autoimmunity in the future.

Th1-skewed profile and excessive production of proinflammatory cytokines in a NFKB1-deficient patient with CVID and severe gastrointestinal manifestations.

Monoallelic loss-of-function mutations in NFKB1 were recently recognized as the most common monogenic cause of common variable immunodeficiency (CVID). The prototypic clinical phenotype of NFKB1-deficient patients includes common CVID features, such as hypogammaglobulinaemia and sinopulmonary infections, plus other highly variable individual manifestations. Here, we describe a patient with a profound CVID phenotype and severe gastrointestinal manifestations, including chronic and recurrent diarrhoea. Using an NGS customized panel of 323 genes related to primary immunodeficiencies, we identified a novel monoallelic loss-of-function mutation in NFKB1 leading to a truncated protein (c.1149delT/p.Gly384Glu ∗ 48). Interestingly, we also found a rare variant in NOD2 previously associated with Crohn's disease (p.His352Arg). Our patient had hypogammaglobulinaemia with a small number of B cells, most of which were naïve. The most noteworthy findings included marked skewing towards a Th1 phenotype in peripheral blood T cells and excessive production of proinflammatory cytokines (IL-1ß, TNFα). The patient's 6-year-old daughter, a carrier of the NFKB1 mutation, is clinically asymptomatic, but has started to show cellular and molecular changes. This case of NFKB1 deficiency appears to be a combination of immunodeficiency and a hyperinflammatory state. The current situation of the patient's daughter provides a glimpse of the preclinical phase of the condition.

Central Tolerance Mechanisms to TSHR in Graves' Disease: Contributions to Understand the Genetic Association.

In the last 3 years, the association of thyrotropin receptor gene (TSHR) variations to Graves' disease (GD) has been confirmed. It is now well established that a 30 Kb region of intron 1 of the TSHR gene is linked to GD predisposition. Elucidating the mechanism(s) by which these polymorphisms confer susceptibility is difficult but would constitute an important advance in endocrine autoimmunity in general. Two hypotheses, both postulating TSHR gene regulatory mechanisms, are discussed. One postulates differential level of expression in the thymus, involving central tolerance. The other postulates a shift in TSHR differential splicing leading to the production of soluble proteins that will have easy access to antigen presenting cells, so it is focused in peripheral tolerance. A combination of the 2 hypothesis is feasible, especially under the light of recent evidence that have identified epigenetic factors acting on TSHR intron 1.

Graves' disease TSHR-stimulating antibodies (TSAbs) induce the activation of immature thymocytes: a clue to the riddle of TSAbs generation?

Graves' disease (GD) is an autoimmune thyroid disease defined by the production of stimulating autoantibodies to the thyroid-stimulating hormone receptor (TSHR) (TSAbs) that induce a sustained state of hyperthyroidism in patients. We previously demonstrated that TSHR, the target of this autoimmune response, is also a key susceptibility gene for GD, probably acting through thymic-dependent central tolerance. We also showed that TSHR is, unexpectedly, expressed in thymocytes. In this report, we confirm the expression of TSHR in thymocytes by protein immunoblotting and quantitative PCR, and show that expression is confined to maturing thymocytes. Using functional assays, we show that thymic TSHR is functional and that TSAbs can stimulate thymocytes through this receptor. This new activity of TSAbs on thymocytes may: 1) explain GD-associated thymic enlargement (hyperplasia), and 2) suggest the provocative hypothesis that the continuous stimulation of thymocytes by TSAbs could lead to a vicious cycle of iterative improvement of the affinity and stimulating capability of initially low-affinity antibacterial (e.g., Yersinia) Abs cross-reactive with TSHR, eventually leading to TSAbs. This may help to fill one of the gaps in our present understanding of unusual characteristics of TSAbs.

Lymphoid neogenesis in chronic inflammatory diseases.

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.

Clinical and structural impact of mutations affecting the residue Phe367 of FOXP3 in patients with IPEX syndrome.

Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a monogenic autoimmune disease characterized by early-onset life-threatening multisystemic autoimmunity. This rare hereditary disorder is caused by loss-of-function mutations in the gene encoding the forkhead box P3 (FOXP3) transcription factor, which plays a key role in the differentiation and function of CD4(+)CD25(+) natural regulatory T cells (Tregs), essential for the establishment and maintenance of natural tolerance. We identified a novel mutation in the FOXP3 gene affecting the Phe367 residue of the protein (F367V) in a family with three male siblings affected by IPEX. Two other mutations affecting the FOXP3 Phe367 residue (F367L and F367C) have been described previously. This unique situation of three mutations affecting the same residue in FOXP3 led us to study the molecular impact of these mutations on the structure of FOXP3 protein. Structure analysis showed that Phe367 is involved in a rich interaction network related to both monomer and dimer structure stabilization, and is crucial for FOXP3 regulatory activity. The relevance of this location is confirmed by the results of SIFT and PolyPhen-2 pathogenicity predictions for F367V mutation. In summary, as assessment of the pathogenicity of a novel mutation is crucial to achieve a proper molecular diagnosis, we analysed the impact of mutations affecting the Phe367 residue using a combined approach that provides a mechanistic view of their pathogenic effect.

Novel Mutations Causing C5 Deficiency in Three North-African Families.

The complement system plays a central role in defense to encapsulated bacteria through opsonization and membrane attack complex (MAC) dependent lysis. The three activation pathways (classical, lectin, and alternative) converge in the cleavage of C5, which initiates MAC formation and target lysis. C5 deficiency is associated to recurrent infections by Neisseria spp. In the present study, complement deficiency was suspected in three families of North-African origin after one episode of invasive meningitis due to a non-groupable and two uncommon Meningococcal serotypes (E29, Y). Activity of alternative and classical pathways of complement were markedly reduced and the measurement of terminal complement components revealed total C5 absence. C5 gene analysis revealed two novel mutations as causative of the deficiency: Family A propositus carried a homozygous deletion of two adenines in the exon 21 of C5 gene, resulting in a frameshift and a truncated protein (c.2607_2608del/p.Ser870ProfsX3 mutation). Families B and C probands carried the same homozygous deletion of three consecutive nucleotides (CAA) in exon 9 of the C5 gene, leading to the deletion of asparagine 320 (c.960_962del/p.Asn320del mutation). Family studies confirmed an autosomal recessive inheritance pattern. Although sharing the same geographical origin, families B and C were unrelated. This prompted us to investigate this mutation prevalence in a cohort of 768 North-African healthy individuals. We identified one heterozygous carrier of the p.Asn320del mutation (allelic frequency = 0.065 %), indicating that this mutation is present at low frequency in North-African population.

Autoimmune predisposition in Down syndrome may result from a partial central tolerance failure due to insufficient intrathymic expression of AIRE and peripheral antigens.

Down syndrome (DS), or trisomy of chromosome 21, is the most common genetic disorder associated with autoimmune diseases. Autoimmune regulator protein (AIRE), a transcription factor located on chromosome 21, plays a crucial role in autoimmunity by regulating promiscuous gene expression (pGE). To investigate if autoimmunity in DS is promoted by the reduction of pGE owing to dysregulation of AIRE, we assessed the expression of AIRE and of several peripheral tissue-restricted Ag genes by quantitative PCR in thymus samples from 19 DS subjects and 21 euploid controls. Strikingly, despite the 21 trisomy, AIRE expression was significantly reduced by 2-fold in DS thymuses compared with controls, which was also confirmed by fluorescent microscopy. Allele-specific quantification of intrathymic AIRE showed that despite its lower expression, the three copies are expressed. More importantly, decreased expression of AIRE was accompanied by a reduction of pGE because expression of tissue-restricted Ags, CHRNA1, GAD1, PLP1, KLK3, SAG, TG, and TSHR, was reduced. Of interest, thyroid dysfunction (10 cases of hypothyroidism and 1 of Graves disease) developed in 11 of 19 (57.9%) of the DS individuals and in none of the 21 controls. The thymuses of these DS individuals contained significantly lower levels of AIRE and thyroglobulin, to which tolerance is typically lost in autoimmune thyroiditis leading to hypothyroidism. Our findings provide strong evidence for the fundamental role of AIRE and pGE, namely, central tolerance, in the predisposition to autoimmunity of DS individuals.

Central T cell tolerance: Identification of tissue-restricted autoantigens in the thymus HLA-DR peptidome.

Promiscuous gene expression (pGE) of tissue-restricted self-antigens (TRA) in medullary thymic epithelial cells (mTECs) is in part driven by the Autoimmune Regulator gene (AIRE) and essential for self-tolerance. The link between AIRE functional mutations and multi-organ autoimmunity in human and mouse supports the role of pGE. Deep sequencing of the transcriptome revealed that mouse mTECs potentially transcribe an unprecedented range of >90% of all genes. Yet, it remains unclear to which extent these low-level transcripts are actually translated into proteins, processed and presented by thymic APCs to induce tolerance. To address this, we analyzed the HLA-DR-associated thymus peptidome. Within a large panel of peptides from abundant proteins, two TRA peptides were identified: prostate-specific semenogelin-1 (an autoantigen in autoimmune chronic prostatitis/chronic pelvic pain syndrome) and central nervous system-specific contactin-2 (an autoantigen in multiple sclerosis). Thymus expression of both genes was restricted to mTECs. SEMG1 expression was confined to mature HLA-DR(hi) mTECs of male and female donors and was AIRE-dependent, whereas CNTN2 was apparently AIRE-independent and was expressed by both populations of mTECs. Our findings establish a link between pGE, MHC-II peptide presentation and autoimmunity for bona fide human TRAs.

Identification and characterization of a novel splice site mutation in the SERPING1 gene in a family with hereditary angioedema.

Hereditary angioedema due to C1-inhibitor deficiency (HAE-C1INH) is a rare autosomal-dominant disease caused by mutations in SERPING1 gene. The main clinical feature of C1INH deficiency is the spontaneous edema of the subcutaneous and submucosal layers. More than 280 different mutations scattering the entire SERPING1 gene have been reported. We identified and characterized a new mutation in SERPING1 gene in a Spanish family with hereditary angioedema. The mutation (c.685 + 2 T > A) disrupts the donor splice site of intron 4 leading to the loss of exon 4 in mutant mRNA. We demonstrated that mutant mRNA is mostly degraded, probably by the surveillance pathway no-go mRNA decay. Bioinformatic analysis showed that the mutant protein, if produced, would be non-functional since the protein lacks a stretch of 45 amino acids affecting the functional RCL loop. Finally, we found a reduction of the wild-type mRNA expression in c.685 + 2 T > A carriers.

A novel splice site mutation in the SERPING1 gene leads to haploinsufficiency by complete degradation of the mutant allele mRNA in a case of familial hereditary angioedema.

Hereditary angioedema due to C1-inhibitor deficiency (HAE-C1INH) is a rare autosomal-dominant and life-threatening disorder caused by mutations in SERPING1 gene. It is characterized by attacks of angioedema involving the skin and/or the mucosa of the upper airways, as well as the intestinal mucosa. Here we report the case of a patient with HAE-C1INH without family history of angioedema. By sequencing the SERPING1 gene we detected a novel mutation (c.1249 + 5G > A) affecting the 5' donor splice site in intron 7. We analyzed the SERPING1 cDNA expecting a defect in splicing process but only the wild type allele was detected. SNP analysis of the cDNA sequence demonstrated that only one of the two alleles was present, indicating that the mRNA from the mutated allele was completely degraded. This study reinforces the concept of incomplete penetrance of this disorder since the patients' mother never presented any sign of angioedema despite carrying the same mutation.

Composition of the HLA-DR-associated human thymus peptidome.

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.

Association of an SNP with intrathymic transcription of TSHR and Graves' disease: a role for defective thymic tolerance.

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.

Stable antigen-specific T-cell hyporesponsiveness induced by tolerogenic dendritic cells from multiple sclerosis patients.

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.

Regulatory T cells and other lymphocyte subpopulations in patients with melanoma developing interferon-induced thyroiditis during high-dose interferon-α2b treatment.

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.

Ligation of Notch receptors in human conventional and plasmacytoid dendritic cells differentially regulates cytokine and chemokine secretion and modulates Th cell polarization.

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.