Phenotypic Analysis of Human Lymph Nodes in Subjects With New-Onset Type 1 Diabetes and Healthy Individuals by Flow Cytometry.

Background: Ultrasound guided sampling of human lymph node (LN) combined with advanced flow cytometry allows phenotypic analysis of multiple immune cell subsets. These may provide insights into immune processes and responses to immunotherapies not apparent from analysis of the blood. Methods: Ultrasound guided inguinal LN samples were obtained by both fine needle aspiration (FNA) and core needle biopsy in 10 adults within 8 weeks of diagnosis of type 1 diabetes (T1D) and 12 age-matched healthy controls at two study centers. Peripheral blood mononuclear cells (PBMC) were obtained on the same occasion. Samples were transported same day to the central laboratory and analyzed by multicolour flow cytometry. Results: LN sampling was well-tolerated and yielded sufficient cells for analysis in 95% of cases. We confirmed the segregation of CD69+ cells into LN and the predominance of CD8+ Temra cells in blood previously reported. In addition, we demonstrated clear enrichment of CD8+ naïve, FOXP3+ Treg, class-switched B cells, CD56bright NK cells and plasmacytoid dendritic cells (DC) in LNs as well as CD4+ T cells of the Th2 phenotype and those expressing Helios and Ki67. Conventional NK cells were virtually absent from LNs as were Th22 and Th1Th17 cells. Paired correlation analysis of blood and LN in the same individuals indicated that for many cell subsets, especially those associated with activation: such as CD25+ and proliferating (Ki67+) T cells, activated follicular helper T cells and class-switched B cells, levels in the LN compartment could not be predicted by analysis of blood. We also observed an increase in Th1-like Treg and less proliferating (Ki67+) CD4+ T cells in LN from T1D compared to control LNs, changes which were not reflected in the blood. Conclusions: LN sampling in humans is well-tolerated. We provide the first detailed "roadmap" comparing immune subsets in LN vs. blood emphasizing a role for differentiated effector T cells in the blood and T cell regulation, B cell activation and memory in the LN. For many subsets, frequencies in blood, did not correlate with LN, suggesting that LN sampling would be valuable for monitoring immuno-therapies where these subsets may be impacted

Activation of the G(i) heterotrimeric G protein by ANCA IgG F(ab')2 fragments is necessary but not sufficient to stimulate the recruitment of those downstream mediators used by intact ANCA IgG.

Anti-neutrophil cytoplasm autoantibodies (ANCA) are implicated in the pathogenesis of systemic vasculitis. Intact ANCA IgG activate superoxide generation in cytokine-primed neutrophils after binding their antigens and co-engaging Fcgamma receptors (FcgammaR). The contribution of antigen binding via ANCA F(ab')(2) fragments to signaling has been unclear. This study shows that both ANCA IgG and F(ab')(2) fragments of ANCA IgG induce significant GTPase activity, which could be blocked with pertussis toxin and anti-G(i) protein antibodies. Pertussis toxin inhibited ANCA IgG-induced superoxide generation but was without effect on superoxide production after conventional FcgammaR ligation. ANCA F(ab')(2) fragments did not induce superoxide generation. ANCA IgG activated PI 3-kinase-generating PIP(3), activated protein kinase B (PKB), and p21(ras); activation of each mediator was inhibited with pertussis toxin, but PI3K and PKB were not activated by ANCA IgG F(ab')(2) fragments. Intact ANCA IgG induced tyrosine phosphorylation, whereas F(ab')(2) fragments did not, and ANCA IgG-mediated superoxide generation was inhibited with genistein. Both genistein and pertussis toxin together completely abrogated the ANCA-induced oxidative burst. Genistein also inhibited ANCA IgG-induced PIP(3) generation and p21(ras) activation. These data implicate a novel ANCA IgG stimulated signaling pathway that involves both F(ab')(2)-mediated antigen binding and Fc-mediated FcgammaR ligation in cooperative interactions between G(i) proteins and tyrosine kinases that facilitates activation of downstream mediators

Multiple independent variants at the TERT locus are associated with telomere length and risks of breast and ovarian cancer

TERT-locus single nucleotide polymorphisms (SNPs) and leucocyte telomere measures are reportedly associated with risks of multiple cancers. Using the iCOGs chip, we analysed ~480 TERT-locus SNPs in breast (n=103,991), ovarian (n=39,774) and BRCA1 mutation carrier (11,705) cancer cases and controls. 53,724 participants have leucocyte telomere measures. Most associations cluster into three independent peaks. Peak 1 SNP rs2736108 minor allele associates with longer telomeres (P=5.8×10−7), reduced estrogen receptor negative (ER-negative) (P=1.0×10−8) and BRCA1 mutation carrier (P=1.1×10−5) breast cancer risks, and altered promoter-assay signal. Peak 2 SNP rs7705526 minor allele associates with longer telomeres (P=2.3×10−14), increased low malignant potential ovarian cancer risk (P=1.3×10−15) and increased promoter activity. Peak 3 SNPs rs10069690 and rs2242652 minor alleles increase ER-negative (P=1.2×10−12) and BRCA1 mutation carrier (P=1.6×10−14) breast and invasive ovarian (P=1.3×10−11) cancer risks, but not via altered telomere length. The cancer-risk alleles of rs2242652 and rs10069690 respectively increase silencing and generate a truncated TERT splice-variant

Multiple independent variants at the TERT locus are associated with telomere length and risks of breast and ovarian cancer

<p>TERT-locus SNPs and leukocyte telomere measures are reportedly associated with risks of multiple cancers. Using the Illumina custom genotyping array iCOG, we analyzed similar to 480 SNPs at the TERT locus in breast (n = 103,991), ovarian (n = 39,774) and BRCA1 mutation carrier (n = 11,705) cancer cases and controls. Leukocyte telomere measurements were also available for 53,724 participants. Most associations cluster into three independent peaks. The minor allele at the peak 1 SNP rs2736108 associates with longer telomeres (P = 5.8 x 10(-7)), lower risks for estrogen receptor (ER)-negative (P = 1.0 x 10(-8)) and BRCA1 mutation carrier (P = 1.1 x 10(-5)) breast cancers and altered promoter assay signal. The minor allele at the peak 2 SNP rs7705526 associates with longer telomeres (P = 2.3 x 10(-14)), higher risk of low-malignant-potential ovarian cancer (P = 1.3 x 10(-15)) and greater promoter activity. The minor alleles at the peak 3 SNPs rs10069690 and rs2242652 increase ER-negative (P = 1.2 x 10(-12)) and BRCA1 mutation carrier (P = 1.6 x 10-14) breast and invasive ovarian (P = 1.3 x 10(-11)) cancer risks but not via altered telomere length. The cancer risk alleles of rs2242652 and rs10069690, respectively, increase silencing and generate a truncated TERT splice variant.</p>