Underlying T Cell Receptor Methylation Defects in Type 1
Diabetes Are Associated with Density Defects
H Takahashi1, WM Kühtreiber1, S Bien2, D Scheffey2, & DL Faustman1
1MASSACHUSETTS GENERAL HOSPITAL & HARVARD MEDICAL SCHOOL, BOSTON, MA, USA
2ADAPTIVE BIOTECHNOLOGIES, SEATTLE, WA, USA

Introduction• In randomized controlled trials, the bacillus Calmette-Guérin(BCG) vaccine frequently used for tuberculosis prevention has
been shown to gradually improve type 1 diabetes (T1D) andmultiple sclerosis• We investigated whether these autoimmune benefits are due to animpact on the host T cell receptor (TCR) and TCR signal strength
by exploring quantitative defects as a cause for altered TCR
selection at the level of the TCR/CD3 protein complex– TCR is a central regulator of T cell education; efficient structuralorganization and density of TCR/CD3 underlies development andfunction of T cells; T cell maturation defects are associated withautoimmunity, suggesting an underlying defect in TCR selectionMethods• We compared TCR densities on CD4 T cells from subjects with type1 diabetes (T1D, n=80) and non-diabetic controls (NDC, n=37)• We also investigated a quantitative defect in genes in the TCR/CD3
genome for possible underlying overmethylation in T1D• All 13 gene regions of the TCR segments and all 4 versions of CD3
proteins were studied for methylation and protein differences in T1Dvs controls

Results• Significant quantitative defects in TCR and CD3 proteins areobserved in CD4+ T cells from T1D vs controls (Fig. 1)– TCR complex genes and associated CD3 genes wereovermethylated in T1D, resulting in downregulated cell surfaceexpression• Evaluation of TCRαβ expression in CD4+ T cells at the protein level
confirmed methylation patterns– The TCRαβ+ cell population was significantly reduced in T1D vscontrols (p=0.005); MFI density of TCRαβ antibody in T1D was alsosignificantly decreased vs controls (p=0.01)• All CD3 genes except CD3ε had hypermethylated patterns in T1D,
confirmed by RNAseq analysis• Percentage CD3+ T cells in the CD4+ T cell population was reducedin T1D vs controls (p=0.04), as was MFI of CD3+ T cells (p=0.02)

Demethylation of TCR/CD3-related Genes in CD4+
T-cells from T1D Patients after BCG Vaccination
Fig. 2. (A) Top 200 most hyper- andhypomethylated genes in CD4+ T-cells from T1D
treated with BCG vs baseline (prior to vaccination)
at Year 1 (top), Year 2 (middle) and Year 3 (bottom).
Genes were ordered by the difference in β value vsbaseline. TCR-related genes are shown in green(hypermethylated) or red (hypomethylated); CD3related genes are shown in white (hypomethylated);
unrelated genes are shown in black. At Year 1 after
BCG vaccination, 90 and 3 of the 200 mosthypomethylated genes were TCR-related and CD3related, respectively, whereas only 12 TCR-related
genes were hypermethylated. At Year 2, there were74 TCR-related and 3 CD3-related genes among
the top 200 hypomethylated genes and 9 TCRrelated genes among the top 200 hypermethylated
genes. At Year 3, these were 40 (hypomethylated)and 8 (hypermethylated) in TCR-related genes. (B)Histograms show the distribution of β values vsbaseline for CpGs of TCR-related genes at Year 1(top), Year 2 (middle) and Year 3 (bottom). A shift ofβ values toward the left indicates progressive
demethylation over time. (C) Changes in TCRαβexpression after 7-day culture of CD4+ T-cells fromNDC and T1D subjects in the presence of BCG.
CD4+ T-cells were isolated from NDC and T1D
patients and cultured for 7 days in RPMI media +/added BCG, then analyzed by flow cytometry.
Percentage of TCRαβ+ cells for CD4+ T-cells inNDC (n=9) and in T1D (n=8) are shown (average ±SD, *p < 0.05; **p < 0.01; ***p < 0.001, 2-tailed,paired t-test). (D) The MFI of TCRαβ+ cells in NDC(n=9) and T1D (n=8) cultured +/- BCG (average ±SD, *p < 0.05; **p < 0.01; ***p < 0.001, 2-tailed,paired t-test).

Abnormally Methylated TCR Genes Observed in T1D
Undergo Gradual De-methylation after BCG Treatment
Fig. 3. (A) Chronological changes in βvalues of TCR-related genes from CD4+ Tcells of T1D (n=13) after BCG vaccinationvs baseline (Year 1 top; Year 2 middle;
Year 3 bottom) are shown (average ± SD,red bars mean significant hypomethylation;p < 0.05, 2-tailed, paired t-test). Thegraphs show progressive demethylation ofthe TCR after BCG vaccination. (B)Chronological change of average changein β values vs baseline in T1D (n=13) atthe TCR-segment level after BCG
vaccination (*p < 0.05; **p < 0.01; ***p <0.001, 2-tailed, paired t-test). (C) Heatmapof change in β values for all TCR-related
genes. The left lane shows difference in βvalue between T1D (n=13) and NDC(n=8), and the three right lanes show
average methylation vs baseline (beforevaccination) at Year 1, 2 and 3 (n=13vaccinated T1D). Shifts to hypomethylation
are displayed in shades of red, and shifts
to hypermethylation are displayed inshades of green.

TCR-Related Genes Are Hypermethylated in CD4+ T
Cells from T1D Patients vs Non-diabetic Controls
Fig. 1. (A) CD4+ T-cells were isolated from blood samplesof 13 subjects with T1D and 8 NDC. β values of all CpGs
per gene were averaged and the genes ordered based onthe difference in average β value between T1D and NDC.
Shown are the 200 most hypermethylated and 200 mosthypomethylated genes of T1D versus NDC among 35184
total genes. 48 of the top 200 hypermethylated genes(24%) were TCR-related (green bars). Among the 200 mosthypomethylated genes, only 9 (4.5%) were TCR-related(red bars). TCR-unrelated genes shown in black. (B)Differences in average β values between 13 T1D atbaseline and 8 NDC (T1D - NDC) are shown for all TCRrelated genes (average ± SD, blue bars indicate significant
hypermethylation; p < 0.05, 2-tailed, unpaired t-test).
Positive bars indicate hypermethylation in T1D compared
to NDC; negative bars indicate hypomethylation. (C)Overall methylation in TCR-segments at baseline; β valuesof the local region genes in C-, J-, V- and D-segments wereaveraged. Differences in β values between 13 T1D atbaseline and 8 NDC are shown at each TCR-segment. (D)Analysis of the expression of TCRαβ on CD4+ T-cells byflow cytometry. Ratio of TCRαβ+ cells among CD4+ T-cellsin NDC (n=11) and T1D (n=10) is shown. (E) Mean
fluorescent intensity (MFI) of APC in TCRαβ+ cells in NDC(n=11) and T1D (n=10) is shown.
*p < 0.05; **p < 0.01; ***p <0.001, 2-tailed, unpaired t-test.

Conclusions• Patients with T1D have quantitative TCR defects, consisting of amarked reduction in receptor density on T cells due tohypermethylation of TCR-related genes– The TCR sequence is not modified through recombination, ruling out aqualitative defect• TCR triggering is affected by intermolecular distance between TCR
proteins; activation diminishes when proximity is increased between
TCR surface proteins, leading to faulty T cell selection• In T1D, altered TCR and CD3 density might be a novel mechanism
for failed T cell selection leading to autoimmunity• BCG corrects this defect gradually over 3 years by demethylating
hypermethylated sites on members of the TCR gene family, leading
to partial correction of densities

No conflicts of interest to declare. Contact: dfaustman@mgh.harvard.edu
www.postersession.com