Type 1 diabetes (T1D) results from autoimmune destruction of insulinproducing beta cells in pancreatic islets. Various immune cell populations are involved in disease development and natural course. However, to our knowledge, so far there are no comprehensive comparative investigations of all main immune cell populations and their most important subsets at the onset of disease. Therefore, in the current study, we analyzed 51 peripheral blood immune cell populations in 22 young T1D patients and in 25 age-matched controls using a comprehensive polychromatic flow cytometry panel developed for whole blood by the COST Action no. BM0907 ENTIRE (European Network for Translational Immunology Research and Education: From Immunomonitoring to Personalized Immunotherapy) consortium. We found that in T1D patients, frequencies and absolute counts of natural killer (NK) cells, dendritic cells (DC) and T cells, aswell as their respective subsets, were significantly altered compared to controls. Further, we observed that changes in several cell populations (e.g. CD14 + CD16 + non-classical monocytes, plasmablasts) were dependent on the age of the patient. In addition to age-related changes, we also found that alterations in immune cell patterns were associated with parameters such as the presence of ketoacidosis and C-peptide serum levels. Our study provides a foundation for future studies investigating different cell lineages and their role in T1D and illustrates the value of polychromatic flow cytometry for evaluating all main peripheral immune cells and their subsets in whole blood samples.

Age‐related changes in human T‐cell populations are important contributors to immunosenescence. In particular, terminally differentiated CD8+ effector memory CD45RA+ TEMRA cells and their subsets have characteristics of cellular senescence, accumulate in older individuals, and are increased in age‐related chronic inflammatory diseases. In a detailed T‐cell profiling among individuals over 65 years of age, we found a high interindividual variation among CD8+ TEMRA populations. CD8+ TEMRA proportions correlated positively with cytomegalovirus (CMV) antibody levels, however, not with the chronological age. In the analysis of over 90 inflammation proteins, we identified plasma TRANCE/RANKL levels to associate with several differentiated T‐cell populations, including CD8+ TEMRA and its CD28− subsets. Given the strong potential of CD8+ TEMRA cells as a biomarker for immunosenescence, we used deep‐amplicon bisulfite sequencing to match their frequencies in flow cytometry with CpG site methylation levels and developed a computational model to predict CD8+ TEMRA cell proportions from whole blood genomic DNA. Our findings confirm the association of CD8+ TEMRA and its subsets with CMV infection and provide a novel tool for their high throughput epigenetic quantification as a biomarker of immunosenescence.

Cytokines play a pivotal role in the maintenance of intestinal homeostasis inducing pro- or anti-inflammatory response and mucosal barrier function in celiac disease (CD) and type 1 diabetes (T1D). We aimed to compare the levels of pro- and anti-inflammatory cytokines in CD patients without and with coexisting T1D, as well as to evaluate its association with the presence of enteroviruses (EV), regulatory T cells (Tregs), and dendritic cells (DCs) in small bowel mucosa. Altogether, 72 patients (median age 10.1 years) who had undergone small bowel biopsy were studied. The study group consisted of 24 patients with CD (median age 6.5 years), 9 patients with CD and concomitant T1D (median age 7.0 years), two patients with T1D (median age 8.5 years), and 37 patients (median age 14.0 years) with functional gastrointestinal disorders (FGD) and a normal small bowel mucosa as controls. The levels of 33 cytokines in serum were measured by multiple analysis using the Milliplex® MAP Magnetic Bead assay. The densities of FOXP3+ Tregs, CD11c+ DC, indoleamine 2,3-dioxygenase+ (IDO+) DC, langerin+ (CD207+) DCs, and EV were evaluated by immunohistochemistry as described in our previous studies. Circulating anti-EV IgA and IgG were evaluated using ELISA. The most important finding of the study is the significant increase of the serum levels of IL-5, IL-8, IL-13, IL-15, IL-17F, IL-22, IL-27, IP-10, MIP-1β, sIL-2Rα, sTNFRII, and TNFα in CD patients compared to controls and its correlation with the degree of small bowel mucosa damage graded according to the Marsh classification. The leptin level was higher in females in all study groups. The levels of IL-2, IL-6, IL-12 (P70), IL-15, IP-10, and IFNγ correlated significantly with the density of FOXP3+ Tregs in lamina propria of the small bowel mucosa, which supports the evidence about the signaling role of these cytokines in the peripheral maintenance of FOXP3+ Tregs. At the same time, a significant negative correlation occurred between the level of IL-4 and density of FOXP3+ Tregs in controls. Another important finding of our study was the correlation of IL-17F, IP-10, sTNFRII, MCP-1, and GM-CSF with the density of EV-positive cells in the lamina propria of the small bowel mucosa. Correlation of MIP-1 (CCL-4) with CD103+ DC and langerin+ DC densities may point to their significance in the recruitment of immune cells into the lamina propria and in driving the inflammatory response in CD patients. Our results suggest the predominance of Th1 and Th17 immune responses over EV VP1 protein in CD and T1D patients. The significant elevation of Th2 cytokines, like IL-5 and IL-13, but not IL-4, in CD patients and its correlation with the degree of small bowel mucosa damage could reflect the role of these cytokines in gut defense and inflammation.

A group of European FOCIS Centers of Excellence adapted panels of the Human Immunophenotyping Consortium (HIPC) for whole blood analysis. Using four core panels [T/regulatory T cell/B/natural killer (T/T reg /B/NK) and myeloid cells] the main leukocyte populations were analyzed in a clinical-diagnostic setting in a harmonized manner across different platforms. As a first step, the consortium presents here the absolute and relative frequencies of the leukocyte subpopulations in the peripheral blood of more than 300 healthy volunteers across six different European centers.

ProblemHealthy pregnancy is associated with a physiologic increase in inflammatory responses. The objective of this study was to assess changes in plasma cytokines associated with uncomplicated pregnancy.Method of studyTo examine these changes, plasma levels of immune response mediators from healthy gravidas (N = 115, gestation weeks 23‐30) were compared with those from healthy non‐pregnant women (N = 42). Comparisons were performed using multiplex analysis for Th1 activity‐related cytokines (IFN‐γ, IL‐2, sIL‐2Rα, IL‐12[P70], and IL‐27), Th2 activity‐related cytokines (IL‐4, IL‐5, and IL‐13), other immune response mediators (GM‐CSF, IL‐1β, sIL‐1RI, IL‐6, IL‐8, IL‐15, IL‐17A, IL‐17F, IL‐21, IL‐22, IL‐23, TGFβ1, TGFβ2, TGFβ3, and TNFα), regulatory T cell–related cytokines (IL‐10 and sTNFRII), adipokines (adiponectin, leptin, PAI‐1, and resistin), chemokines (IP‐10, MCP‐1, and MIP‐1β), and hematopoietic growth factor IL‐7.ResultsMultivariate linear regression models showed increased levels of IL‐7, Th1‐, and Treg activity‐related cytokines and decreased levels of adipokines and chemokines in healthy gravidas compared with healthy non‐pregnant women. Additionally, season of the year, age, pre‐pregnancy body mass index, and HLA‐DR/DQ genotypes for type 1 diabetes risk showed different and sometimes reciprocal influence on cytokine levels.ConclusionOur study stresses the importance of profiling immune response mediators during pregnancy to better understand the effect of healthy pregnancy on cytokine levels.

Aims/Introduction Gestational diabetes (GDM) is characterized by low‐grade systemic inflammation, which manifests as changes in the levels of cytokines in the blood. We aimed to investigate plasma immune mediators during gestational weeks 23–28 in 213 women at risk for GDM, and to find associations between GDM and its complications. Materials and Methods We quantified the levels of adipokines: adiponectin, leptin, plasminogen activator inhibitor‐1 and resistin; chemokines: C‐C motif chemokine ligand 2 (CCL2), CCL4, C‐X‐C motif chemokine ligand 8 (CXCL8) and CXCL10; and cytokines: granulocyte‐macrophage colony‐stimulating factor, interferon‐γ, interleukin (IL)‐1β, soluble (s)IL‐1RI, IL‐2, sIL‐2Ra, IL‐4, IL‐5, IL‐6, IL‐7, IL‐10, IL‐12(p70), IL‐13, IL‐15, IL‐17A, IL‐17F, IL‐21, IL‐22, IL‐23, IL‐27, transforming growth factor (TGF)‐β1, TGF‐β2, TGF‐β3, tumor necrosis factor‐α and soluble tumor necrosis factor receptor 2 using the Milliplex®MAP Magnetic Bead assay on Luminex®200™, and compared the results with clinical data from pregnancy and post‐partum follow up. Results Lower levels of adiponectin and higher levels of CCL2 (Wilcoxon test, P = 3.4E‐03 and P = 0.03, respectively) were found in women with GDM. IL‐27 levels were associated with lower odds of GDM (adjusted logistic regression 0.90, P = 2.4E‐03), and showed a risk association with glutamic acid decarboxylase autoantibody positivity (adjusted odds ratio 1.13, P = 2.8E‐03). Similarly, higher IL‐22 levels increased the odds of glutamic acid decarboxylase autoantibody positivity (adjusted odds ratio 4.23, P = 0.04). TGF‐β1 was associated with post‐partum fasting glucose levels, and CCL4 with post‐partum C‐peptide levels (linear regression, P = 0.04 and P = 0.01, respectively). Women who developed pregnancy complications had higher levels of CXCL10 and CCL4 (linear regression, P = 7.0E‐04 and P = 0.01, respectively). Conclusions Plasma adiponectin and CCL2 concentrations distinguish women with GDM. IL‐27 and IL‐22 levels might select women with an autoimmune reaction, whereas increased TGF‐β1 and CCL4 are associated with post‐partum glucose and insulin metabolism.

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