Methylation Analysis in Distinct Immune Cell Subsets in Type 1 Diabetes

Abstract

Epigenetics provides a mechanism in which the environment can interact with the genotype to produce a variety of phenotypes. These epigenetic modifications have been associated with altered gene expression and silencing of repetitive elements, and these modifications can be inherited mitotically. DNA methylation is the best characterized epigenetic mark and earlier studies have examined DNA methylation profiles in peripheral blood mononuclear cells in disease. However, any disease-related signatures identified would just display differences in the relative abundance of individual cell types as each cell subset generates a unique methylation profile. Therefore is it important to identify cell- or tissue-specific changes in DNA methylation, particularly in autoimmune diseases such as type 1 diabetes.

Decreased HLA-DQ expression on peripheral blood cells in children with varying number of beta cell autoantibodies

“…The peripheral blood cell extraction protocol was adapted from Ref. [ 26 ], a protocol followed as cells were intended for bulk epigenetics analysis [ 27 ]. Briefly, the five 10 mL blood samples were pooled at room temperature at equal volumes into two 50 mL Falcon tubes and diluted 1:1 in RPMI 1640 Medium (GlutaMAX TM Supplement, HEPES, Invitrogen) supplemented with 1% (v/v) penicillin-streptomycin and 0.2% (v/v) Bip-Pure Human Serum Albumin (catalog number 05-720-1B, Biological Industries, Cromwell, Connecticut, USA).…”

“…Isolated cell subsets were stained with titrated monoclonad antibodies ( Table 1 A), adapted from Ref. [ 26 ]. The HLA-DQ antibody clone REA303 recognizes the same epitopes as the pan DQ-specific SPV-L3.…”

“…Gating strategy for identifying cell populations and HLA-DQ cell surface MFI on the isolated cell subsets was adapted from Ref. [ 26 ] and illustrated in Supplemental Fig. 1 .…”

Possible Relationship between the HLA-DRA1 Intron Haplotype of Three Single-Nucleotide Polymorphisms in Intron 1 of the HLA-DRA1 Gene and Autoantibodies in Children at Increased Genetic Risk for Autoimmune Type 1 Diabetes

“…In brief, a peripheral blood sample (3050 ml) was obtained to isolate CD19 1 B cells, CD16 1 cells, CD14 1 CD16 À monocytes, CD4 1 T cells, CD8 1 T cells, and CD16 1 CD66 1 neutrophils from PBMCs and RBCs, respectively, using magnetic-associated cell sorting (Miltenyi Biotec, Bergisch Gladbach, Germany). HLA-DQ MFI was identified with flow cytometry using a gating strategy for identifying cell populations and HLA-DQ MFI on the isolated cell subsets adapted from Dang et al (29).…”

“…The titrated mAbs were used to stain cell subsets in a gating strategy (Supplemental Fig. 2) adapted from Dang et al (29). First, duplicate events were removed using forward scatter height plotted against forward scatter area.…”