1268-P

Underlying T Cell Receptor Methylation Defects in Type 1 Diabetes Associated with Quantitative Defects
H. Takahashi1, W.M. Kühtreiber1, A.H. Lee1, A. Aristarkhova1, H.F. Dias1, N. Ng1, S. Bien2, D. Scheffey2, & D.L Faustman1
1MASSACHUSETTS GENERAL HOSPITAL & HARVARD MEDICAL SCHOOL, BOSTON, MA, USA; 2ADAPTIVE BIOTECHNOLOGIES, SEATTLE, WA, USA

Introduction•The T cell receptor (TCR) is a central regulator
of T cell education•Efficient structural organization and density ofTCR/CD3 underlies the development andfunction of T cells•

T cell maturation defects are associated withautoimmunity, suggesting an underlying defectin TCR selection•

Here we explore quantitative defects as acause for altered TCR selection at the level ofthe TCR/CD3 protein complex

Demethylation of TCR/CD3-related Genes in CD4+
T-cells from T1D Patients after BCG Vaccination

TCR-related Genes Are Hypermethylated in CD4+ TCells from T1D Patients vs Non-diabetic Controls

Abnormally Methylated TCR Genes Observed inT1D Undergo Gradual De-methylation after BCG
Vaccine TreatmentMethods•••We compared TCR densities on CD4 T cells
from subjects with type 1 diabetes (T1D, n=80)
and non-diabetic controls (NDC, n=37)
We also investigated a quantitative defect ingenes in the TCR/CD3 genome for possible
underlying overmethylation in T1D
All 13 gene regions of the TCR segments andall 4 versions of CD3 proteins were studied formethylation and protein differences in T1D vscontrols

Results• Significant quantitative defects in TCR andCD3 proteins are observed in CD4+ T cells ofT1D vs controls (Fig. 1)•Over-methylation of TCR complex genes and CD3genes resulted in a functional defect:
downregulated cell surface expression• Evaluation of TCRαβ expression in CD4+ T cells
at the protein level confirmed methylation patterns• The TCRαβ+ cell population was significantly
reduced in T1D vs controls (p=0.005); MFI densityof TCRαβ antibody in T1D was also significantly
decreased vs controls (p=0.01)• In T1D, all CD3 genes except CD3ε had
hypermethylated patterns, confirmed by RNAseqanalysis• The percentage CD3+ T cells in the CD4+ T cellpopulation was reduced in T1D vs controls(p=0.04), as was the MFI of CD3+ T cells(p=0.02)

Fig. 1. (A) CD4+ T-cells were isolated from blood samples of 13 T1D and 8 NDC patients. The βvalues of all CpGs per gene were averaged and the genes ordered based on the difference inaverage β value between T1D and NDC. Shown are the top 200 most hypermethylated and top
200 most hypomethylated genes of T1D versus NDC among 35184 total genes. Forty-eight ofthe 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 areshown in black. (B) Differences in average β values between T1D at baseline (n=13) and NDC(n=8) (T1D - NDC) are shown for all TCR-related genes (average ± SD, blue bars indicate
significant hypermethylation; p < 0.05, 2-tailed, unpaired t-test). Positive bars indicate
hypermethylation in T1D as compared to NDC, whereas negative bars indicate hypomethylation.
(C) Overall methylation in TCR-segments at baseline; β values of the local region genes in C-, J, V- and D-segments were averaged. The differences in β values between NDC (n=8) and T1D(n=13) at baseline (T1D-NDC) are shown at each TCR-segment (average, *p < 0.05; **p < 0.01;***p < 0.001, 2-tailed, unpaired t-test). (D) Analysis of the expression of TCRαβ on CD4+ T-cellsby flow cytometry. The ratio of TCRαβ+ cells among CD4+ T-cells in NDC (n=11) and T1D (n=10)
is shown (average ± SD, *p < 0.05; **p < 0.01; ***p < 0.001, 2-tailed, unpaired t-test). (E) The
mean fluorescent intensity (MFI) of APC in TCRαβ+ cells in NDC (n=11) and T1D (n=10) isshown (average ± SD, *p < 0.05; **p < 0.01; ***p < 0.001, 2-tailed, unpaired t-test).

Fig. 2. (A) Top 200 most hyper- and hypomethylated 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 versus baseline. TCR-related genes are shown in green(hypermethylated) or red (hypomethylated); CD3-related genes are shown in white(hypomethylated); unrelated genes are shown in black. At Year 1 after BCG vaccination, 90 and 3of the top 200 most hypomethylated genes were TCR- and CD3-related respectively, whereas
only 12 TCR-related genes were hypermethylated. At Year 2, there were 74 TCR- and 3 CD3related genes among the top 200 hypomethylated genes and 9 TCR-related genes among the top200 hypermethylated genes. At Year 3, these were 40 (hypomethylated) and 8 (hypermethylated)
in TCR-related genes. (B) Histograms show the distribution of β values vs baseline for CpGs ofTCR-related genes at Year 1 (top), Year 2 (middle) and Year 3 (bottom). A shift of β values toward
the left indicates progressive demethylation over time. (C) Changes in TCRαβ expression after 7day culture of CD4+ T-cells from NDC 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. The percentage of TCRαβ+ cells for CD4+ T-cells in NDC (n=9)
and in T1D (n=8) are shown (average ± SD, *p < 0.05; **p < 0.01; ***p < 0.001, 2-tailed, paired ttest). (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).

Fig. 3. (A) Chronological changes in β values of TCR-related genes from CD4+ T-cells of T1D(n=13) after BCG vaccination vs 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 of the TCR after BCG vaccination. (B) Chronologicalchange of average change in β values vs baseline in T1D (n=13) at the TCR-segment level after
BCG vaccination (*p < 0.05; **p < 0.01; ***p < 0.001, 2-tailed, paired t-test). (C) Heatmap ofchange in β values for all TCR-related genes. The left lane shows difference in β value between
T1D (n=13) and NDC (n=8), and the three right lanes show average methylation vs baseline(before vaccination) at Year 1, 2 and 3 (n=13 vaccinated T1D). Shifts to hypomethylation aredisplayed in shades of red, and shifts to hypermethylation are displayed in shades of green.

Conclusions•Patients with T1D have quantitative defects in the TCR/CD3 protein complex, resulting in decreased expression•TCR triggering is affected by intermolecular distance between TCR proteins; activation diminishes when proximity isincreased 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 therapy causes a gradual de-methylation of the TCR and CD4 genes, leading to partial correction of densities
No conflicts of interest to declare. Contact: www.postersession.com
dfaustman@mgh.harvard.edu