Presence of TRPA1 Modifies CD4+/CD8+ T Lymphocyte Ratio and Activation.

Transient Receptor Potential Ankyrin 1 (TRPA1) has been reported to influence neuroinflammation and lymphocyte function. We analysed the immune phenotype and activation characteristics of TRPA1-deficient mice (knockout-KO) generated by targeted deletion of the pore-loop domain of the ion channel. We compared TRPA1 mRNA and protein expression in monocyte and lymphocyte subpopulations isolated from primary and secondary lymphatic organs of wild type (WT) and KO mice. qRT-PCR and flow cytometric studies indicated a higher level of TRPA1 in monocytes than in lymphocytes, but both were orders of magnitude lower than in sensory neurons. We found lower CD4+/CD8+ thymocyte ratios, diminished CD4/CD8 rates, and B cell numbers in the KO mice. Early activation marker CD69 was lower in CD4+ T cells of KO, while the level of CD8+/CD25+ cells was higher. In vitro TcR-mediated activation did not result in significant differences in CD69 level between WT and KO splenocytes, but lower cytokine (IL-1ß, IL-6, TNF-α, IL-17A, IL-22, and RANTES) secretion was observed in KO splenocytes. Basal intracellular Ca2+ level and TcR-induced Ca2+ signal in T lymphocytes did not differ significantly, but interestingly, imiquimod-induced Ca2+ level in KO thymocytes was higher. Our results support the role of TRPA1 in the regulation of activation, cytokine production, and T and B lymphocytes composition in mice.

Clinical Characteristics and Outcome of Neuronal Surface Antibody-Mediated Autoimmune Encephalitis Patients in a National Cohort.

Background: In our previous single-center study of autoimmune encephalitis (AE) related autoantibody test results we found positivity in 60 patients out of 1,034 with suspected AE from 2012 through 2018 as part of a Hungarian nationwide program. In our current multicenter retrospective study, we analyzed the clinical characteristics and outcome of AE patients with positive neuronal cell surface autoantibody test results. Methods: A standard online questionnaire was used to collect demographic and clinical characteristics, laboratory and imaging data, therapy and prognosis of 30 definitive AE patients in four major clinical centers of the region. Results: In our study, 19 patients were positive for anti-NMDAR (63%), 6 patients (20%) for anti-LGI1, 3 patients for anti-GABABR (10%) and 3 patients for anti-Caspr2 (10%) autoantibodies. Most common prodromal symptoms were fever or flu-like symptoms (10/30, 33%). Main clinical features included psychiatric symptoms (83%), epileptic seizures (73%) and memory loss (50%). 19 patients (63%) presented with signs of central nervous system (CNS) inflammation, which occurred more frequently in elder individuals (p = 0.024), although no significant differences were observed in sex, tumor association, time to diagnosis, prognosis and immunotherapy compared to AE patients without CNS inflammatory markers. Anti-NMDAR encephalitis patients were in more severe condition at the disease onset (p = 0.028), although no significant correlation between mRS score, age, sex and immunotherapy was found. 27% of patients (n = 8) with associated tumors had worse outcome (p = 0.045) than patients without tumor. In most cases, immunotherapy led to clinical improvement of AE patients (80%) who achieved a good outcome (mRS ≤ 2; median follow-up 33 months). Conclusion: Our study confirms previous publications describing characteristics of AE patients, however, differences were observed in anti-NMDAR encephalitis that showed no association with ovarian teratoma and occurred more frequently among young males. One-third of AE patients lacked signs of inflammation in both CSF and brain MRI, which emphasizes the importance of clinical symptoms and autoantibody testing in diagnostic workflow for early introduction of immunotherapy, which can lead to favorable outcome in AE patients.

Regulatory T cells are less sensitive to glucocorticoid hormone induced apoptosis than CD4+ T cells.

Earlier we have reported that thymic regulatory T cells (Treg) are resistant to in vivo glucocorticoid hormone (GC)-induced apoptosis, while the most GC-sensitive DP thymocytes died through the activation of mitochondrial apoptotic pathway. Here we analyzed the apoptosis-inducing effect of high dose (10-6 M) in vitro dexamethasone (DX) treatment in mouse thymic- and splenic Tregs and CD4+ T cells. Activation of both extrinsic and intrinsic apoptotic pathways started after 2 h of DX treatment in CD4 SP thymocytes and was 3 × higher than in CD4+ splenocytes, while in Treg cells, weak activation of the extrinsic apoptotic pathway started only after 3 h. We also investigated the expression of 21 apoptosis-related molecules using a protein array and found higher level of both pro-and anti-apoptotic molecules in Tregs compared to CD4+ T cells. 4 h in vitro DX treatment induced upregulation of most apoptosis-related molecules both in Tregs and CD4+ T cells, except for the decrease of Bcl-2 expression in CD4+ T cells. We found high basal cytosolic Ca2+ levels in untreated Treg cells, which further increased after DX treatment, while the specific TCR-induced Ca2+ signal was lower in Tregs than in CD4+ T cells. Our results suggest that in the background of the relative apoptosis resistance of Treg cells to GCs might be their high basal cytosolic Ca2+ level and upregulated Bcl-2 expression. In contrast, downregulation of Bcl-2 expression in CD4+ T cells can explain their higher, DX-induced apoptosis sensitivity.

Toll-Like Receptor Mediated Activation of Natural Autoantibody Producing B Cell Subpopulations in an Autoimmune Disease Model.

Altered expression and function of the Toll-like receptor (TLR) homologue CD180 molecule in B cells have been associated with autoimmune disorders. In this study, we report decreased expression of CD180 at protein and mRNA levels in peripheral blood B cells of diffuse cutaneous systemic sclerosis (dcSSc) patients. To analyze the effect of CD180 stimulation, together with CpG (TLR9 ligand) treatment, on the phenotype defined by CD19/CD27/IgD/CD24/CD38 staining, and function (CD69 and CD180 expression, cytokine and antibody secretion) of B cell subpopulations, we used tonsillar B cells. After stimulation, we found reduced expression of CD180 protein and mRNA in total B cells, and CD180 protein in B cell subpopulations. The frequency of CD180+ cells was the highest in the CD19+CD27+IgD+ non-switched (NS) B cell subset, and they showed the strongest activation after anti-CD180 stimulation. Furthermore, B cell activation via CD180 induced IL-6 and natural autoantibody secretion. Treatment with the combination of anti-CD180 antibody and CpG resulted in increased IL-6 and IL-10 secretion and natural autoantibody production of B cells. Our results support the role of CD180 in the induction of natural autoantibody production, possibly by NS B cells, and suggest an imbalance between the pathologic and natural autoantibody production in SSc patients.

Single-center study of autoimmune encephalitis-related autoantibody testing in Hungary.

OBJECTIVE: Autoantibody detection is crucial for the early diagnosis of autoimmune encephalitis (AIE) since prompt therapy can determine the disease outcome. Here, we report a single-center 6-year retrospective study of autoantibody testing in AIE in the Hungarian population. METHODS: Serum and/or cerebrospinal fluid (CSF) autoantibody tests were performed using cell-based indirect immunofluorescence assay for AIE diagnosis. Samples were provided by neurology clinics as part of a nationwide program. Test results were analyzed for samples received during the period from 2012 to 2018. RESULTS: We tested 1,247 samples from 1,034 patients with suspected AIE. Autoantibodies were present in 60 patients (5.8% of total). The distribution of patients with different autoantibodies by age and sex was as follows: NMDAR (70%), mostly in young females, LGI1 (15%) in middle-aged males, GABAB R (12%) in elderly males, and Caspr2 (7%) in males. Long-term follow-up was conducted in 30 patients with repeated test requests, of which 17 remained positive, and 13 switched to negative. CONCLUSION: We report the most comprehensive clinical laboratory study of autoantibody testing in AIE in the Hungarian population. Our results show that the frequency of different autoantibody types in AIE corresponds to the data described in the literature.

[Difficulties and prospects in the immunotherapy of gliomas]. / A gliómák immunterápiájának nehézségei és reményei.

Despite the spectacular development of clinical immunotherapy (IT) in the last decade, the regular treatment approaches for the most common central nervous system (CNS) tumors, the malignant gliomas (MGs) has not changed yet. The most important pitfalls of the routine application of immunotherapy can be imputed to the special and originally immunosuppressed microenvironment and the extreme heterogeneity of MGs, however the defensive role of the blood-brain barrier, the general usage of steroids and the difficulties in the evaluation of brain images can also play a role in these types of difficulties. Additionally, in the case of MGs, well-accepted IT biomarker assays (PDL1 positivity, mismatch repair deficiencies, tumor mutation burden, etc.) generally reveal only minimal levels of immunogen activities. Nevertheless, there are some promising results with the utilization of checkpoint inhibitors and other IT modalities (such as virus-based therapies, tumor vaccines, adoptive T cell therapies) and with the combination of conventional oncotherapy methods in case of CNS malignancies, as well. In conclusion, although the CNS is not any more considered as an "immunological sanctuary" and despite some encouraging experimental and clinical results in CNS oncotherapy, the routine application of IT in case of MGs is still awaited.

Glucocorticoid hormone differentially modulates the in vitro expansion and cytokine profile of thymic and splenic Treg cells.

OBJECTIVE: Functional disturbances in regulatory T cells (Treg) have been described in autoimmune diseases, and their potential therapeutic use is intensively studied. Our goal was to investigate the influence of glucocorticoid hormone on the in vitro differentiation of Treg cells from thymic and splenic CD4+ T cells under different conditions to establish methods for generating stable and functionally suppressive iTregs for future use in adoptive transfer experiments. METHODS: Thymic and splenic CD4+ T lymphocytes were isolated from 3 to 4 week-old control and in vivo dexamethasone (DX) pretreated BALB/c mice using magnetic bead negative selection, followed by CD25 positive selection. The cells were cultured with anti-CD3/CD28 beads and IL-2 in the presence or absence of TGFß and/or DX for 3-6 days. Multiparametric flow cytometry was performed using CD4, CD25, CD8, TGFß (LAP) cell surface and Foxp3, IL-4, IL-10, IL-17 and IFNγ intracellular staining. Quantitative RT-PCR was performed to measure IL-10, TGFß cytokine and Foxp3 mRNA levels. RESULTS: Differentiation of thymus-derived CD4+ cells in vitro into iTreg cells was most effective (24-25%) when anti-CD3/CD28 beads, IL-2, and TGFß were present. Splenic CD4+ T cell expansion under same conditions resulted in a higher (44-45%) iTreg cell ratio that further increased (up to 50% Treg) in the presence of DX. Elevated immunosuppressive cytokine (IL-10 and TGFß) production by iTregs could be measured both at protein and mRNA levels without elevation of Th1/Th2 or Th17 cytokine production. We got the highest iTreg ratio (74%) and TGFß production when CD4+CD25+ splenic T cells were stimulated in the presence of TGFß. In vivo 4 days DX pretreatment resulted in enhanced in vitro expansion and Foxp3 expression of thymus-derived iTregs and decreased differentiation of spleen-derived iTreg cells. In these Tregs the relative expression of IL-10 mRNA significantly decreased under all in vitro stimulation conditions, while TGFß mRNA level did not change. CONCLUSION: DX promotes the expansion of thymic and splenic Treg cells, and enhances Foxp3+ expression and the production of immunosuppressive cytokines IL-10 and TGFß in vitro. In vivo pretreatment of mice with DX inhibited the immunosuppressive cytokine production of in vitro differentiated Treg cells. We hypothesize that patients receiving GC therapy may need special attention prior to in vitro expansion and transplantation of Treg cells.

Development of a robust and standardized immunoserological assay for detection of anti-measles IgG antibodies in human sera.

Because of measles outbreaks there is a need for continuous monitoring of immunological protection against infection at population level. For such monitoring to be feasible, a cost-effective, reliable and high-throughput assay is necessary. Herein we describe an ELISA protocol for assessment of anti-measles antibody levels in human serum samples that fulfills the above criteria and is easily adaptable by various laboratories. A serum bank of anonymous patient sera was established (N > 3000 samples). Sera were grouped based on measles immunization schedules and/or changes in vaccine components since the introduction of the first measles vaccine in Hungary in 1969. Newly designed ELISA was performed by using Siemens BEP 2000 Advance System and data were confirmed using commercially available kits. Our indirect ELISA was compared to indirect immunfluoresence and to anti-measles nucleocapsid (N) monoclonal antibody-based sandwich ELISA. The results obtained are in high agreement with the confirmatory methods, and reflect measles vaccination history in Hungary ranging from pre-vaccination era, through the initial period of measles vaccination, to present. Based on measurement of 1985 sera, the highest ratio of low/questionable antibody level samples was detected in cluster '1978-1987' (~25.4%), followed by cluster '1969-1977' (~15.4%).Our assay is suitable for assessment of anti-measles immunity in a large cohort of subjects. The assay is cost-effective, allows high-throughput screening and has superior signal-to-noise ratio. This assay can serve as a first step in assessment of the effectiveness of all three components of the MMR vaccine.

Glucocorticoid hormone treatment enhances the cytokine production of regulatory T cells by upregulation of Foxp3 expression.

OBJECTIVE: Despite the fact that glucocorticoids (GC) are important therapeutic tools, their effects on regulatory T cells (Treg) are not well defined. The aim of our work was to investigate how GCs influence in vivo the thymic (tTreg) and peripheral Treg (pTreg) differentiation, survival and cytokine production. METHODS: Tregs were detected with flow cytometry in lymphatic organs of 4-6 weeks old BALB/c mice after repeated (2-4days), high-dose in vivo GC treatment using CD4/CD25 cell surface and Foxp3/IL-10/TGFß/glucocorticoid receptor (GR) intracellular staining. Cytokine, Foxp3, and GR mRNA levels of sorted CD4+CD25high T cells were analyzed using RT-PCR. Foxp3 and GR localization in Treg cells was investigated with confocal microscopy. RESULTS: GC treatment of mice resulted in increased relative tTreg frequency in the thymus, which was due to decreased total thymocyte numbers with unchanged absolute tTreg cell count. In contrast the relative pTreg cell ratio in secondary lymphatic organs decreased or showed no changes after GC treatment, while the absolute number of pTregs decreased. Elevated intracellular IL-10+ and TGFß+ tTreg and pTreg ratios were measured in GC-treated animals, accompanied with elevated Foxp3 mRNA expression. In addition, GC treatment caused increased TGFß and IL-35 mRNA expression in CD4+CD25high+ splenic and elevated IL-10 mRNA level in thymic tTregs. GR expression of thymic tTreg cells was lower than in pTregs. GC treatment caused an opposite change in GR levels, elevating GR in tTregs but decreasing it in pTregs. We observed a nuclear localization of GR in both tTregs and pTregs, which showed high colocalization (∼60%) with Foxp3 transcription factor. These data suggest an interaction of these two transcription factors with further increase due to GC treatment in splenic pTregs. CONCLUSION: Our data show selective survival of tTregs and elevated production of immunosuppressive cytokines by Treg cells after GC treatment, which may contribute to the immunosuppressive effects of GCs.

Increased proportions of functionally impaired regulatory T cell subsets in systemic sclerosis.

Treg abnormalities have been implicated in the pathogenesis of systemic sclerosis (SSc). Treg subpopulations and their cytokines, IL-10 and TGF-ß in the peripheral blood of early stage SSc patients were investigated. We hypothesized that epigenetically regulated methylation of the FOXP3 promoter and enhancer regions are altered in Tregs of SSc patients, which might be involved in the T cell imbalance. CD4+CD25+Foxp3+CD127- Treg cells were significantly elevated in patients with diffuse cutaneous SSc and in patients with anti-Scl-70/RNA-Pol-III autoantibody positivity and with lung fibrosis. Increased CD62L+ Treg cells were present in all SSc subgroups. The production of immunosuppressive cytokines by both CD127- and CD62L+ Tregs was diminished. We observed reduced methylation of Treg specific FOXP3 enhancer regions, and elevated FOXP3 gene expression in active SSc cases with negative correlation in the frequency of CD62L+IL-10+ Tregs. Our data indicate an inappropriate distribution and cytokine production of Treg cells in early form SSc.