Early-onset pulmonary and cutaneous vasculitis driven by constitutively active SRC-family kinase HCK.

BACKGROUND: Inborn errors of immunity are genetic disorders characterized by various degrees of immune dysregulation that can manifest as immune deficiency, autoimmunity, or autoinflammation. The routine use of next-generation sequencing in the clinic has facilitated the identification of an ever-increasing number of inborn errors of immunity, revealing the roles of immunologically important genes in human pathologies. However, despite this progress, treatment is still extremely challenging. OBJECTIVE: We sought to report a new monogenic autoinflammatory disorder caused by a de novo activating mutation, p.Tyr515∗, in hematopoietic cell kinase (HCK). The disease is characterized by cutaneous vasculitis and chronic pulmonary inflammation that progresses to fibrosis. METHODS: Whole-exome sequencing, Sanger sequencing, mass spectrometry, and western blotting were performed to identify and characterize the pathogenic HCK mutation. Dysregulation of mutant HCK was confirmed ex vivo in primary cells and in vitro in transduced cell lines. RESULTS: Mutant HCK lacking the C-terminal inhibitory tyrosine Tyr522 exhibited increased kinase activity and enhanced myeloid cell priming, migration and effector functions, such as production of the inflammatory cytokines IL-1ß, IL-6, IL-8, and TNF-α, and production of reactive oxygen species. These aberrant functions were reflected by inflammatory leukocyte infiltration of the lungs and skin. Moreover, an overview of the clinical course of the disease, including therapies, provides evidence for the therapeutic efficacy of the Janus kinase 1/2 inhibitor ruxolitinib in inflammatory lung disease. CONCLUSIONS: We propose HCK-driven pulmonary and cutaneous vasculitis as a novel autoinflammatory disorder of inborn errors of immunity.

An immunotherapy effect analysis in Rasmussen encephalitis.

BACKGROUND: Immune-mediated mechanisms substantially contribute to the Rasmussen encephalitis (RE) pathology, but for unknown reasons, immunotherapy is generally ineffective in patients who have already developed intractable epilepsy; overall laboratory data regarding the effect of immunotherapy on patients with RE are limited. We analyzed multiple samples from seven differently treated children with RE and evaluated the effects of immunotherapies on neuroinflammation. Immunotherapy was introduced to all patients at the time of intractable epilepsy and they all had to undergo hemispherothomy. METHODS: Immunohistochemistry, flow cytometry, Luminex multiplex bead and enzyme-linked immunosorbent assay techniques were combined to determine: 1) inflammatory changes and lymphocyte subpopulations in 45 brain tissues; 2) lymphocyte subpopulations and the levels of 12 chemokines/cytokines in 24 cerebrospinal fluid (CSF) samples and 30 blood samples; and 3) the dynamics of these parameters in four RE patients from whom multiple samples were collected. RESULTS: Sustained T cell-targeted therapy with cyclophosphamide, natalizumab, alemtuzumab, and intrathecal methotrexate (ITMTX), but not with azathioprine, substantially reduced inflammation in brain tissues. Despite the therapy, the distributions of CD8+ T cells and the levels of C-X-C motif ligand (CXCL) 10, CXCL13, and B cell activating factor (BAFF) in patients' CSF remained increased compared to controls. A therapeutic approach combining alemtuzumab and ITMTX was the most effective in producing simultaneous reductions in histopathological inflammatory findings and in the numbers of activated CD8+ T cells in the brain tissue, as well as in the overall CD8+ T cell population and chemokine/cytokine production in the CSF. CONCLUSIONS: We provide evidence that various T cell-targeted immunotherapies reduced inflammation in the brains of RE patients. The observation that intractable epilepsy persisted in all of the patients suggests a relative independence of seizure activity on the presence of T cells in the brain later in the disease course. Thus, new therapeutic targets must be identified. CXCL10, CXCL13 and BAFF levels were substantially increased in CSF from all patients and their significance in RE pathology remains to be addressed.

Monocytes contribute to DNA sensing through the TBK1 signaling pathway in type 1 diabetes patients.

BACKGROUND: The aberrant recognition of self-nucleic acids by the innate immune system contributes to the pathology of several autoimmune diseases. Although microbial DNA and, in certain instances, self-DNA that is released from damaged cells are primarily recognized by Toll-like receptor 9 (TLR9), recent evidence suggests that other cytosolic sequence-nonspecific DNA sensors contribute to DNA recognition. In this study, we focused on the sensing of microbial and host DNA in type 1 diabetes (T1D) patients. METHODS: Peripheral blood mononuclear cells (PBMCs) and monocytes from pediatric patients with T1D and from healthy donors were stimulated with microbial DNA (CpG) or with self-DNA (DNA contained within neutrophil extracellular traps, NETs). The production of cytokines was measured by flow cytometry and multiplex bead assays. The internalization of microbial DNA and its colocalization with STING was detected by image cytometry. Furthermore, the involvement of the TBK1 kinase was investigated by detecting its phosphorylation with phospho-flow cytometry or by using a TBK1 inhibition assay. RESULTS: We observed a prominent proinflammatory response in T1D PBMCs, especially pDCs and monocytes, to microbial DNA in comparison to that in controls. We further confirmed that monocytes could bind and internalize DNA and respond by releasing proinflammatory cytokines in a more pronounced manner in T1D patients than those in controls. Surprisingly, this cytokine production was not affected by TLR9 blockade, suggesting the involvement of intracellular receptors in DNA recognition. We further identified TBK1 and STING as two crucial molecules in the DNA-sensing pathway that were involved in CpG-DNA sensing by T1D cells. A similar DNA-sensing pathway that was dependent on intracellular DNA sensors and the STING-TBK1 interaction was employed in response to NETs, which were used to model self-DNA. CONCLUSIONS: Here, we show that there were significant differences in DNA sensing in T1D patients compared to that in controls. We demonstrate that monocytes from T1D patients are able to sense microbial- and self-DNA, leading to proinflammatory cytokine secretion through the adaptor protein STING and the TBK1 kinase.

Phase I/II trial of dendritic cell-based active cellular immunotherapy with DCVAC/PCa in patients with rising PSA after primary prostatectomy or salvage radiotherapy for the treatment of prostate cancer.

OBJECTIVE: Immunotherapy of cancer has the potential to be effective mostly in patients with a low tumour burden. Rising PSA (prostate-specific antigen) levels in patients with prostate cancer represents such a situation. We performed the present clinical study with dendritic cell (DC)-based immunotherapy in this patient population. MATERIALS AND METHODS: The single-arm phase I/II trial registered as EudraCT 2009-017259-91 involved 27 patients with rising PSA levels. The study medication consisted of autologous DCs pulsed with the killed LNCaP cell line (DCVAC/PCa). Twelve patients with a favourable PSA response continued with the second cycle of immunotherapy. The primary and secondary objectives of the study were to assess the safety and determine the PSA doubling time (PSADT), respectively. RESULTS: No significant side effects were recorded. The median PSADT in all treated patients increased from 5.67 months prior to immunotherapy to 18.85 months after 12 doses (p < 0.0018). Twelve patients who continued immunotherapy with the second cycle had a median PSADT of 58 months that remained stable after the second cycle. In the peripheral blood, specific PSA-reacting T lymphocytes were increased significantly already after the fourth dose, and a stable frequency was detected throughout the remainder of DCVAC/PCa treatment. Long-term immunotherapy of prostate cancer patients experiencing early signs of PSA recurrence using DCVAC/PCa was safe, induced an immune response and led to the significant prolongation of PSADT. Long-term follow-up may show whether the changes in PSADT might improve the clinical outcome in patients with biochemical recurrence of the prostate cancer.

Anti-N-methyl-D-aspartate receptor encephalitis: the clinical course in light of the chemokine and cytokine levels in cerebrospinal fluid.

BACKGROUND: Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is an autoimmune disorder of the central nervous system (CNS). Its immunopathogenesis has been proposed to include early cerebrospinal fluid (CSF) lymphocytosis, subsequent CNS disease restriction and B cell mechanism predominance. There are limited data regarding T cell involvement in the disease. To contribute to the current knowledge, we investigated the complex system of chemokines and cytokines related to B and T cell functions in CSF and sera samples from anti-NMDAR encephalitis patients at different time-points of the disease. One patient in our study group had a long-persisting coma and underwent extraordinary immunosuppressive therapy. METHODS: Twenty-seven paired CSF/serum samples were collected from nine patients during the follow-up period (median 12 months, range 1-26 months). The patient samples were stratified into three periods after the onset of the first disease symptom and compared with the controls. Modified Rankin score (mRS) defined the clinical status. The concentrations of the chemokines (C-X-C motif ligand (CXCL)10, CXCL8 and C-C motif ligand 2 (CCL2)) and the cytokines (interferon (IFN)γ, interleukin (IL)4, IL7, IL15, IL17A and tumour necrosis factor (TNF)α) were measured with Luminex multiple bead technology. The B cell-activating factor (BAFF) and CXCL13 concentrations were determined via enzyme-linked immunosorbent assay. We correlated the disease period with the mRS, pleocytosis and the levels of all of the investigated chemokines and cytokines. Non-parametric tests were used, a P value <0.05 was considered to be significant. RESULTS: The increased CXCL10 and CXCL13 CSF levels accompanied early-stage disease progression and pleocytosis. The CSF CXCL10 and CXCL13 levels were the highest in the most complicated patient. The CSF BAFF levels remained unchanged through the periods. In contrast, the CSF levels of T cell-related cytokines (INFγ, TNFα and IL17A) and IL15 were slightly increased at all of the periods examined. No dynamic changes in chemokine and cytokine levels were observed in the peripheral blood. CONCLUSIONS: Our data support the hypothesis that anti-NMDAR encephalitis is restricted to the CNS and that chemoattraction of immune cells dominates at its early stage. Furthermore, our findings raise the question of whether T cells are involved in this disease.

Low marginal zone-like B lymphocytes and natural antibodies characterize skewed B-lymphocyte subpopulations in del22q11 DiGeorge patients.

PURPOSE: Patients with DiGeorge syndrome suffer from T-lymphopenia. T-cells are important for the maturation and regulation of B-cell function. Our aim was to characterize the B-cell compartment in DiGeorge syndrome patients. METHODS: B-cell subset phenotypization using flow cytometry. Serum BAFF (B-cell activating factor) and serum anti-alpha-galactosyl IgM measurement using ELISA. Serum IgG measurement using nephelometry. RESULTS: We observed a significantly increased number of naïve B-cells and decreased number of switched memory B-cells in DiGeorge patients. Furthermore, we observed increased BAFF levels and a trend toward hypergammaglobulinemia later in life. Surprisingly, we detected a decrease in marginal zone-like (MZ-like) B-cells and natural antibodies in DiGeorge patients. CONCLUSION: The maturation of B-cells is impaired in DiGeorge patients, with high naïve and low switched memory B-cell numbers being observed. There is a clear trend toward hypergammaglobulinemia later in life, coupled with increased serum BAFF levels. Surprisingly, the T-independent humoral response is also impaired, with low numbers of MZ-like B-cell and low levels of anti-alpha-galactosyl IgM natural antibodies being detected.

Negativity for Specific Autoantibodies in Patients with Type 1 Diabetes That Developed on a Background of Common Variable Immunodeficiency.

Common variable immunodeficiency (CVID) is a heterogeneous group of disorders characterized by disturbed antibody production and a dysregulated immune system. Aside from recurrent infections, the most common complications of CVID are autoimmune complications, particularly autoimmune cytopenias. To date, type 1 diabetes mellitus (T1D) in combination with CVID has only been described as an unusual complication in several reports, but the true incidence of T1D with CVID remains unknown. We describe 2 patients with a combination of T1D and CVID with serious impairment of antibody production. We also provide a review of the available literature. T1D-specific insulin autoantibodies and autoantibodies to glutamic acid decarboxylase and tyrosine phosphatase IA2 were not detected in either of our patients at the time of diagnosis or during the course of the disease. In both cases, T1D manifestation and diagnosis preceded the discovery of CVID by several years. Following the diagnosis of immunodeficiency and the start of immunoglobulin substitution therapy, their clinical status improved, manifesting as a lower frequency of infections and improved T1D control, with decreased glycosylated hemoglobin A1c values. Based on these reported cases, we assume that T1D might be more frequent than previously reported in patients with CVID. To verify the actual incidence of T1D among CVID patients, we searched the European Society for Immunodeficiencies Registry database, and found 25 cases of T1D in 1,671 listed CVID patients, suggesting a higher occurrence of T1D among CVID patients than previously thought. Early diagnosis and treatment of immunodeficiency improve both the prognosis and the course of CVID, reduce the frequency and severity of infections and may contribute to better management of T1D.

Decreased dendritic cell numbers but increased TLR9-mediated interferon-alpha production in first degree relatives of type 1 diabetes patients.

OBJECTIVE: Dendritic cells (DCs) play an important role in pathogenesis of autoimmunity, including type 1 diabetes (T1D). In this study, we investigated DC subpopulations and their responses to TLR stimulation in T1D patients and their relatives. METHODS: We analyzed the frequency of myeloid (mDCs) and plasmacytoid DCs (pDCs) in 97 T1D patients (69 onset, 28 long-term), 67 first-degree relatives, and 64 controls. We additionally tested the IFN-alpha production by pDCs upon stimulation with TLR 7, 8 and 9 agonists. RESULTS: A lower number of mDCs and pDCs were found in T1D patients and their relatives. Of all the tested TLR ligands, only stimulation with CpG 2216 induced IFN-alpha production that was the highest in T1D relatives, except of autoantibody-negative relatives bearing the protective haplotypes. CONCLUSION: Our data demonstrate disturbances in DC number and function expressed most significantly in T1D relatives and point to a potential role of TLR9-induced IFN-alpha production in T1D development.

Helios expression in T-regulatory cells in patients with di George Syndrome.

PURPOSE: Syndrome diGeorge is associated amongst other clinical signs with various degrees of thymic dysplasia, related immunodeficiency and autoimmune disorders. Helios, a transcription factor from Ikaros family, has been proposed as a marker for thymus derived Tregs. We therefore examined Helios + Tregs in a cohort of patients with genetically proven diGeorge syndrome with typical T cell lymphopenia due to the thymic pathology. METHODS: T cells, FoxP3+ Tregs and Helios + FoxP3+ Tregs were examined in 52 samples from 37 patients. One patient with diGeorge/CHARGE syndrome with total thymic aplasia was also included. Statistical analysis was performed using a linear regression comparison. RESULTS: Total absolute Tregs were significantly lower in diGeorge patients as compared to controls in all age groups (0-20 years) (p = 0.0016). The difference was more expressed in the first four years of age. Relative Treg numbers expressed as the percentage of Tregs in CD4+ T-cells, however, were not different in patients and controls in all age groups (p = 0.661), neither could we find any significant difference in the percentage of Helios + Tregs between patients and controls (p = 0.238). Helios + Tregs were still present in a patient with diGeorge/CHARGE syndrome with complete athymia 7 years after partially matched unrelated repeated T lymphocytes infusions. CONCLUSION: Our findings show that while there was a significant decrease in absolute numbers of Tregs in patients with diGeorge syndrome, the relative percentage of this population did not differ between patients and controls. Low absolute Tregs thus reflected typical T cells lymphopenia in patients. Helios expression was not affected in diGeorge syndrome.

Changes in innate and adaptive immunity over the first year after the onset of type 1 diabetes.

AIMS: The development of the immune phenotype in patients with type 1 diabetes (T1D) during the first year following disease onset remains poorly described, and studies analysing the longitudinal development of a complex set of immunological and metabolic parameters are missing. Thus, we aim to provide such complex view in a cohort of 38 children with new onset T1D who were prospectively followed for 1 year. METHODS: All subjects were tested for a set of immunological parameters (complete blood count; serum immunoglobulins; and T, B and dendritic cells), HbA1c and daily insulin dose at baseline and at 6 and 12 months after T1D diagnosis. A mixed meal tolerance test was administered to each of the subjects 12 months after diagnosis, and the C-peptide area under the curve (AUC) was noted and was then tested for association with all immunological parameters. RESULTS: A gradual decrease in leukocytes (adjusted p = 0.0012) was reflected in a significant decrease in neutrophils (adjusted p = 0.0061) over the post-onset period, whereas Tregs (adjusted p = 0.0205) and originally low pDCs (adjusted p < 0.0001) increased. The expression of the receptor for BAFF (BAFFR) on B lymphocytes (adjusted p = 0.0127) markedly increased after onset. No immunological parameters were associated with C-peptide AUC; however, we observed a linear increase in C-peptide AUC with the age of the patients (p < 0.0001). CONCLUSIONS: Our study documents substantial changes in the innate and adaptive immune system over the first year after disease diagnosis but shows no association between immunological parameters and residual beta-cell activity. The age of patients remains the best predictor of C-peptide AUC, whereas the role of the immune system remains unresolved.

Kinetics of dendritic cells reconstitution and costimulatory molecules expression after myeloablative allogeneic haematopoetic stem cell transplantation: implications for the development of acute graft-versus host disease.

Allogeneic hematopoetic stem cell transplantation (HSCT) represents a unique opportunity to monitor the kinetics of reconstitution of dendritic cells (DCs) and their dynamics in distinct pathologies. We analyzed DCs reconstitution after myeloablative HSCT. We separately analyzed patients with acute GVHD. DCs were monitored from the earliest phase of hematopoetic reconstitution until day +365. Both myeloid DCs and plasmacytoid DCs appeared at earliest stages after engraftment and relative numbers within white blood cells compartment peaked between days 19-25 after HSCT. Their proportion then gradually declined and absolute numbers of both DC subsets remained lower than in controls during the whole follow-up. Patients with acute GVHD had significantly lower numbers of circulating DCs. Decrease in DC counts preceded onset of clinical symptoms by at least 24 h and was independent of corticosteroids administration. This study reveals quantification of plasmacytoid and myeloid DCs as a potential biomarker for the prediction of acute GVHD development.

Utility of chemokines CCL2, CXCL8, 10 and 13 and interleukin 6 in the pediatric cohort for the recognition of neuroinflammation and in the context of traditional cerebrospinal fluid neuroinflammatory biomarkers.

BACKGROUND: The recognition of active inflammation in the central nervous system (CNS) in the absence of infectious agents is challenging. The present study aimed to determine the diagnostic relevance of five selected chemo/cytokines in the recognition of CNS inflammation and in the context of traditional cerebrospinal fluid (CSF) biomarkers (white blood cell [WBC] counts, oligoclonal bands, protein levels, CSF/serum albumin ratios) and clinical diagnoses. METHODS: C-C and C-X-C motif ligands (CCL2, CXCL8, 10 and 13) and interleukin (IL) 6 levels in the CSF and serum from 37 control and 87 symptomatic children with ten different (mostly noninfectious) inflammatory CNS disorders (16 of which had follow-up samples after recovery) were determined using Luminex multiple bead technology and software. Nonparametric tests were used; p < 0.05 was considered statistically significant. Receiver operating characteristic curves were constructed to analyze controls and 1) all symptomatic samples or 2) symptomatic samples without CSF pleocytosis. RESULTS: Compared with the control CSF samples, levels of all investigated chemo/cytokines were increased in symptomatic CSF samples, and only IL-6 remained elevated in recovery samples (p ≤ 0.001). CSF CXCL-13 levels (> 10.9 pg/mL) were the best individual discriminatory criterion to differentiate neuroinflammation (specificity/sensitivity: 97/72% and 97/61% for samples without pleocytosis), followed by CSF WBC counts (specificity/sensitivity: 97/62%). The clinical utility of the remaining CSF chemo/cytokine levels was determined in descending order of sensitivities corresponding to thresholds that ensured 97% specificity for neuroinflammation in samples without pleocytosis (pg/mL; sensitivity %): IL-6 (3.8; 34), CXCL8 (32; 26), CXCL10 (317; 24) and CCL2 (387; 10). Different diagnosis-related patterns of CSF chemo/cytokines were observed. CONCLUSIONS: The increased CSF level of CXCL13 was the marker with the greatest predictive utility for the general recognition of neuroinflammation among all of the individually investigated biomarkers. The potential clinical utility of chemo/cytokines in the differential diagnosis of neuroinflammatory diseases was identified.

Data on microbial DNA-induced IL-1ß production in monocytes of type 1 diabetes patients.

Inflammasomes are large protein complexes involved in the maturation of IL-1ß, a cytokine associated with the pathophysiology of type 1 diabetes (T1D). The data presented in this article focused on the role of inflammasomes in DNA recognition in T1D patients. This data extend knowledge on DNA sensing in T1D patients and relate to our research paper "Monocytes contribute to DNA sensing through the TBK1 signaling pathway in type 1 diabetes patients" Zentsova et al., 2009. To examine inflammasome involvement, we blocked the known mechanism of inflammasome activation - potassium efflux via various approaches: 1) high concentration of KCl; 2) Glybenclamide, which selectively blocks the ATP sensitive K+ channel; 3) KN-62, an inhibitor of P2X7 receptor, which activates K+ channel after ATP binding. Moreover, we used an inhibitor which blocks Nod-like receptor family containing pyrin domain 3 (NLRP3) inflammasome. In T1D patients, we show that secretion of cytokines IL-1ß, TNFα, IL-6 and IFNα after microbial DNA stimulation is promoted by potassium efflux and is not dependent on P2X7 receptor signaling. Surprisingly, the microbial DNA induced IL-1ß release was independent of NLRP3.

Alteration of B cell subsets and the receptor for B cell activating factor (BAFF) in paediatric patients with type 1 diabetes.

BACKGROUND: Lately, mounting evidence has shown that B cells play an important role in the pathogenesis of type 1 diabetes (T1D). Here, we present alterations in B cell subsets including BAFF receptor (BAFFR) expression in cohorts of patients with type 1 diabetes (T1D) and their relatives. PATIENTS AND METHODS: B cells were studied in 438 patients with T1D (158 at disease onset and 280 with long-term disease), 136 first-degree relatives and 53 healthy controls. The B cell panel included transitional, naïve, MZ-like, switched memory B cells and plasmablasts. We also measured serum BAFF levels as well as BAFFR expression on both B and T cells. Moreover, the effect of BAFF on T and B lymphocytes was analysed in vitro. RESULTS: We observed a significant decrease in the proportion of transitional B cells in the patients with T1D, accompanied by an increased proportion of plasmablasts, especially in recent-onset patients and their relatives. While the BAFF serum levels did not differ in the patients with T1D, BAFFR-expressing B and especially T cell numbers were reduced in the T1D cohort, with the exception of patients with recent-onset disease who exhibited a significant increase in the number of BAFFR-expressing T cells. T cell activation and B cell proliferation were more pronounced after activation with BAFF in the T1D cohort compared to controls. CONCLUSION: The B cell panel in patients with T1D is characterized by significantly reduced populations of B cells in their early stages of development with a shift towards plasma cells. The dynamics of BAFFR-expressing B and T cells and the more pronounced responsiveness of the T1D T cells to BAFF point to the role of BAFF and T and B cell cooperation in the development of T1D.

Short Stature in a Boy with Multiple Early-Onset Autoimmune Conditions due to a STAT3 Activating Mutation: Could Intracellular Growth Hormone Signalling Be Compromised?
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BACKGROUND: Germline STAT3 gain-of-function (GOF) mutations cause multiple endocrine and haematologic autoimmune disorders, lymphoproliferation, and growth impairment. As the JAK-STAT pathway is known to transduce the growth hormone (GH) signalling, and STAT3 interacts with STAT5 in growth regulation, we hypothesised that short stature in STAT3 GOF mutations results mostly from GH insensitivity via involving activation of STAT5. CASE REPORT: A boy with a novel STAT3 c.2144C>T (p.Pro715Leu) mutation presented with short stature (-2.60 SD at 5.5 years). He developed diabetes mellitus at 11 months, generalised lympho-proliferation, autoimmune thyroid disease, and immune bicytopenia in the subsequent years. At 5.5 years, his insulin-like growth factor-1 (IGF-I) was 37 µg/L (-2.22 SD) but stimulated GH was 27.7 µg/L. Both a standard IGF-I generation test (GH 0.033 mg/kg/day sc; 4 days) and a high-dose prolonged IGF-I generation test (GH 0.067 mg/kg/day sc; 14 days) failed to significantly increase IGF-I levels (37-46 and 72-87 µg/L, respectively). The boy underwent haematopoietic stem cell transplantation at 6 years due to severe neutropenia and massive lymphoproliferation, but unfortunately deceased 42 days after transplantation from reactivated generalised adenoviral infection. CONCLUSIONS: Our findings confirm the effect of STAT3 GOF mutation on the downstream activation of STAT5 resulting in partial GH insensitivity. 
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T regulatory lymphocytes in type 1 diabetes: Impaired CD25 expression and IL-2 induced STAT5 phosphorylation in pediatric patients.

T regulatory cells (Tregs) are essential for maintaining tolerance and preventing autoimmune diseases, such as type 1 diabetes (T1D). In our study, we investigated CD25 + FoxP3 + Tregs and thymic FoxP3 + Helios + Tregs in large cohorts of children with T1D at onset and with long-term T1D, and further in their relatives and healthy controls. We observed significantly decreased numbers of CD25 + FoxP3 + Tregs, but not FoxP3 + Helios + Tregs, in long-term patients compared with the control group and T1D onset. Furthermore, long-term T1D patients exhibited highly significant decrease of CD25 expression on both CD25 + FoxP3 + Tregs and FoxP3 + Helios + Tregs, independently on age or the duration of diabetes. A similar reduction of CD25 expression was also found in T1D relatives, more significant in those with positive autoantibodies. Low CD25 expression was associated with impaired signal transducer and activator of transcription 5 (STAT5) phosphorylation after IL-2 exposure. Our results show that the frequency of Tregs is altered in a large cohort of long-term T1D patients, a profound decrease in CD25 expression and altered IL-2 signaling are typical features of Tregs populations in long-term diabetic patients and their relatives.

NF-κB, p38 MAPK, ERK1/2, mTOR, STAT3 and increased glycolysis regulate stability of paricalcitol/dexamethasone-generated tolerogenic dendritic cells in the inflammatory environment.

Tolerogenic dendritic cells (tDCs) may offer an intervention therapy in autoimmune diseases or transplantation. Stable immaturity and tolerogenic function of tDCs after encountering inflammatory environment are prerequisite for positive outcome of immunotherapy. However, the signaling pathways regulating their stable tolerogenic properties are largely unknown. In this study, we demonstrated that human monocyte-derived tDCs established by using paricalcitol (analogue of vitamin D2), dexamethasone and monophosphoryl lipid A exposed for 24h to LPS, cytokine cocktail, polyI:C or CD40L preserved reduced expression of co-stimulatory molecules, increased levels of inhibitory molecules ILT-3, PDL-1 and TIM-3, increased TLR-2, increased secretion of IL-10 and TGF-ß, reduced IL-12 and TNF-α secretion and reduced T cell stimulatory capacity. tDCs further induced IL-10-producing T regulatory cells that suppressed the proliferation of responder T cells. In the inflammatory environment, tDCs maintained up-regulated indoleamine 2, 3 dioxygenase but abrogated IκB-α phosphorylation and reduced transcriptional activity of p65/RelA, RelB and c-Rel NF-κB subunits except p50. Mechanistically, p38 MAPK, ERK1/2, mTOR, STAT3 and mTOR-dependent glycolysis regulated expression of ILT-3, PDL-1 and CD86, secretion of IL-10 and T cell stimulatory capacity of tDCs in the inflammatory environment. Stability of tDCs in the inflammatory environment is thus regulated by multiple signaling pathways.

Phase I/II clinical trial of dendritic-cell based immunotherapy (DCVAC/PCa) combined with chemotherapy in patients with metastatic, castration-resistant prostate cancer.

PURPOSE: We conducted an open-label, single-arm Phase I/II clinical trial in metastatic CRPC (mCRPC) patients eligible for docetaxel combined with treatment with autologous mature dendritic cells (DCs) pulsed with killed LNCaP prostate cancer cells (DCVAC/PCa). The primary and secondary endpoints were safety and immune responses, respectively. Overall survival (OS), followed as a part of the safety evaluation, was compared to the predicted OS according to the Halabi and MSKCC nomograms. EXPERIMENTAL DESIGN: Twenty-five patients with progressive mCRPC were enrolled. Treatment comprised of initial 7 days administration of metronomic cyclophosphamide 50 mg p.o. DCVAC/PCa treatment consisted of a median twelve doses of 1 × 107 dendritic cells per dose injected s.c. (Aldara creme was applied at the site of injection) during a one-year period. The initial 2 doses of DCVAC/PCa were administered at a 2-week interval, followed by the administration of docetaxel (75 mg/m2) and prednisone (5 mg twice daily) given every 3 weeks until toxicity or intolerance was observed. The DCVAC/PCa was then injected every 6 weeks up to the maximum number of doses manufactured from one leukapheresis. RESULTS: No serious DCVAC/PCa-related adverse events have been reported. The median OS was 19 months, whereas the predicted median OS was 11.8 months with the Halabi nomogram and 13 months with the MSKCC nomogram. Kaplan-Meier analyses showed that patients had a lower risk of death compared with both MSKCC (Hazard Ratio 0.26, 95% CI: 0.13-0.51) and Halabi (Hazard Ratio 0.33, 95% CI: 0.17-0.63) predictions. We observed a significant decrease in Tregs in the peripheral blood. The long-term administration of DCVAC/PCa led to the induction and maintenance of PSA specific T cells. We did not identify any immunological parameter that significantly correlated with better OS. CONCLUSIONS: In patients with mCRPC, the combined chemoimmunotherapy with DCVAC/PCa and docetaxel was safe and resulted in longer than expected survival. Concomitant chemotherapy did not preclude the induction of specific anti-tumor cytotoxic T cells.

The TREC/KREC assay for the diagnosis and monitoring of patients with DiGeorge syndrome.

UNLABELLED: DiGeorge syndrome (DGS) presents with a wide spectrum of thymic pathologies. Nationwide neonatal screening programs of lymphocyte production using T-cell recombination excision circles (TREC) have repeatedly identified patients with DGS. We tested what proportion of DGS patients could be identified at birth by combined TREC and kappa-deleting element recombination circle (KREC) screening. Furthermore, we followed TREC/KREC levels in peripheral blood (PB) to monitor postnatal changes in lymphocyte production. METHODS: TREC/KREC copies were assessed by quantitative PCR (qPCR) and were related to the albumin control gene in dry blood spots (DBSs) from control (n = 56), severe immunodeficiency syndrome (SCID, n = 10) and DGS (n = 13) newborns. PB was evaluated in DGS children (n = 32), in diagnostic samples from SCID babies (n = 5) and in 91 controls. RESULTS: All but one DGS patient had TREC levels in the normal range at birth, albeit quantitative TREC values were significantly lower in the DGS cohort. One patient had slightly reduced KREC at birth. Postnatal DGS samples revealed reduced TREC numbers in 5 of 32 (16%) patients, whereas KREC copy numbers were similar to controls. Both TREC and KREC levels showed a more pronounced decrease with age in DGS patients than in controls (p < 0.0001 for both in a linear model). DGS patients had higher percentages of NK cells at the expense of T cells (p < 0.0001). The patients with reduced TREC levels had repeated infections in infancy and developed allergy and/or autoimmunity, but they were not strikingly different from other patients. In 12 DGS patients with paired DBS and blood samples, the TREC/KREC levels were mostly stable or increased and showed similar kinetics in respective patients. CONCLUSIONS: The combined TREC/KREC approach with correction via control gene identified 1 of 13 (8%) of DiGeorge syndrome patients at birth in our cohort. The majority of patients had TREC/KREC levels in the normal range.