Bacillus Calmette-Guérin (BCG) and Preventionof Type 1 and Type 2 Diabetes: Observational Studies
from Bladder Cancer Therapy and Neonatal BCG Vaccination
HF Dias, Y Mochizuki, H Takahashi, H Zheng, WM Kühtreiber, & DL Faustman
MASSACHUSETTS GENERAL HOSPITAL & HARVARD MEDICAL SCHOOL, BOSTON, MA, USA• A double-blind Phase I clinical trial and open-label trial have shown thatadults with type 1 diabetes (T1D) receiving repeat bacillus CalmetteGuérin (BCG) vaccinations experience statistically significantly lowered
HbA1c values over a multi-year time course• BCG has also been used in the United States and Europe for earlystage bladder cancer, administered commonly as 6 intravesical doses
for 6 weeks• We investigated whether BCG therapy for bladder cancer might improveblood sugar levels in those with comorbid T1D or type 2 diabetes (T2D)

Methods

Countries with mandatory neonatal BCG vaccination policies
had a lower T1D incidence in two international databases and alower T2D incidence in one database

20

0GHDx Database
IDF-matched Countries

60

p-value: <0.0001

2060GHDx Database
All GHDx Countries and Territories

40p-value: <0.0001

200Fig. 3. In global population data, BCG neonatal vaccinations consistently correlate with reduced incidence of T1D.
From the IDF list of countries, those with current BCG vaccination programs (blue lines) have significantly reduced
incidence of T1D in children (p = <0.0001). Countries with a mandatory BCG vaccination policy had an average of 65%
reduced incidence. An almost identical trend was observed with a repeat search of T1D incidence for children in the GHDx
database using the same countries as the IDF database. Countries with a childhood BCG program are observed had anaverage of 39% lower incidence for T1D (p = <0.0001). Using all the countries and territories available in the GHDx
dataset, the incidence of T1D in children is reduced by an average of 47% if they earlier received neonatal BCG
vaccinations (p = <0.0001). A Mann-Whitney U test was used to compare the two groups of countries with (blue) andwithout (black) newborn BCG vaccinations based on the country-by-country policies. (N = 33 for BCG- countries, N = 56
for BCG+ for the IDF database.) (N = 45 for BCG-, N = 159 for BCG+ countries and territories in the GHDx database).

1000

GHDx Database
IDF-matched Countries
p-value: 0.0715

750% Change HbA1c after BCG Instillation for Bladder Cancer
Data Source
MGBMSA/Quest

T1DOL

T1D%Δ in A1c

20Combined
201010**00-10-10-20-200120 1 2 3 4 5 6 70 1 2 3 4 5 6 730123Years
T2D20%Δ in A1cT2D1020*1000-10-10-20012-20

0 1 2 3 4 5 6 730 1 2 3 4 5 6 7Years

Years

Years

Average Change HbA1c after BCG Instillation for Bladder Cancer
Data Source
MSA/Quest

T1DOL

MGB

T1D
Average A1c

10.0

7.5

7.8

8.5

7.0

7.6

7.5

6.5

7.4

9.0

*

6.5

8.0

7.2

6.0

0

120 1 2 3 4 5 6 7

3

5.5

7.0

5.5

7.0012Years

T2D
Average A1cCombined

0 1 2 3 4 5 6 73T2D

117.2

7.2

10

7.0

7.0

9

6.8

6.8

6.6

87

6.6

6.466.4

6.2012Years3*6.2
0 1 2 3 4 5 6 7Years

0 1 2 3 4 5 6 7YearsFig 1. Lower HbA1c in T1D,but not T2D, post-BCG
treatment for bladdercancer. In T1D, all threedatasets show a reduction in
HbA1c values (% change in
HbA1c post-BCG instillation)
and a near 10% decrease in
HbA1cs at differing time
points. The combined T1Ddata shows a statisticallysignificant decrease in
HbA1C at Year 1 and Year 5
post-BCG (p = 0.0304,0.0136). In T2D, MSA datashows no change and MGB
data shows an increase inHbA1c values post-BCG
instillation. The combined
T2D data indicates asignificant increase in Year 4
after BCG instillation (p =0.0223). (N for each dataset:
T1D: MGB=4, MSA=9, OL=
6 for T1D; T2D: MGB=97,MSA=9.)

Fig 2. Decreasing trends
in average HbA1c for T1D,
but not T2D. In T1D, lower
HbA1cs are seen in all threedatabases for subjects
treated with BCG forbladder cancer. When all
three T1D datasets werecombined, a Student’s
paired t-test showedsignificance whencomparing year 0 to year 5(p = 0.0133). In T2D, thetrend is in an upwards
direction post BCG
instillation, a significant
increase when comparingyear 0 to year 4 (p =0.0460). (N for eachdataset: T1D: MGB=4,MSA=9, OL=6; T2D:
MGB=97, MSA=9)

500

250

Type 2 Diabetes
Incidence per 100,000

BCG therapy in adults with comorbid diabetes and bladdercancer was associated with multi-year and stable lowering of
HbA1c in T1D, but not in T2D

BCG BCG +400• Databases were Management Sciences Associates/Quest Diagnostics(MSA, N=263 million adults), Massachusetts General Brigham (MGB,N=6.5 million) and Optum Labs (OL, N=45 million)– Global Health Data Exchange (GHDx) and International DiabetesFederation (IDF) datasets; children ages 0 to 14 years; countries divided
into those with and without a policy of mandatory neonatal BCG
vaccination

p-value: <0.000140• We investigated whether BCG therapy for bladder cancer via
intravesical instillation improves blood sugar levels in patients withcomorbid T1D or T2D by retrospectively analyzing three large patient
databases in the United States, identifying subjects with documented
T1D (N=19) or T2D (N=106) who underwent BCG therapy for bladdercancer and assessing BCG’s subsequent year-by-year impact on HbA1c• We also performed an ecological study to determine whether BCG
exposure reduces onset of T1D and T2D by examining country-bycountry associations between mandatory neonatal BCG vaccination
programs and T1D and T2D incidence

IDF Database60

Type 1 Diabetes
Incidence per 100,000

Introduction

0BCG BCG +1200

GHDx DatabaseAll GHDx Countries and Territories
p-value: <0.0001

8004000Fig. 4. In global population data, neonatal BCG vaccinations might confer some protection from T2D onset. Using
IDF-matched countries in the GHDx database, the T2D incidence was not significantly different between countries with achildhood BCG program (in blue) and those without neonatal BCG vaccinations (in black) (p = 0.0715). However, using allcountries and territories available in the GHDx dataset, the T2D incidence was reduced by an average of 28% in countrieswith neonatal vaccine programs (p <0.0001). A Mann-Whitney U test was used to compare the two groups of countries withand without newborn BCG vaccinations policies. (N = 33 for BCG- countries, N = 55 for BCG+ for the IDF database.) (N =45 for BCG-, N = 159 for BCG + countries and territories in the GHDx database).

Conclusions• Elderly patients with longstanding T1D who received BCG bladdercancer treatment show a gradual lowering of HbA1c, but this is notseen in those with T2D• The bladder cancer data suggests that high-dose intravesical BCGtherapy may contribute towards a drop in HbA1c values in T1D, butnot T2D (likely due to metformin interference in BCG’s ability tochange glycolysis pathways)• Ecological analysis of global data suggests a role for neonatal BCG
in the prevention of T1D and, to a lesser extent, T2D• The epidemiological evidence analyzed here suggests that BCG
may play a role in the prevention and treatment of T1D and in thepossible prevention, but not treatment, of T2D
No conflicts of interest to declare. Contact: dfaustman@mgh.harvard.edu
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Underlying T Cell Receptor Methylation Defects in Type 1Diabetes 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 hasbeen 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 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 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 β
values of TCR-related genes from CD4+ Tcells 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)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+ TCells 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 on
the 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-cells
in NDC (n=11) and T1D (n=10) is shown. (E) Meanfluorescent 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
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