Updates

from the Faustman Laboratory at Massachusetts General Hospital

Fall 2022

A Note from Dr. Faustman
Another year is almost in thebooks, and we are very excitedabout what we were able to do
and where we are going. As
always, the participants in
our trials have been amazing.
Despite the ongoing challengesof the pandemic, they maketheir appointments. The
five-year Phase II trial isapproaching completion.
This year, we also were able
to launch a multi-center
pediatric trial. Across all of
the trials, we now have almost
200 patients who have receivedat least two doses of BCG.
Like many aspects of our lives,
in-person conferences havestarted to return. At the annual
American Diabetes Association
meeting in New Orleans, wepresented major updates on
the trial as well as new dataon how BCG modifies key
components of the immunesystem.
In August of this year, we
published results from ourCOVID-19 study. When thepandemic hit in early 2020,
the FDA gave us permission toanalyze the ongoing Phase II
trial to see if BCG vaccinations
protected from infection andillness related to COVID-19.
The results, which were coveredby the Wall Street Journal and
New York Times, showed that
BCG was very effective against
COVID-19 as well as a rangeof common infections.
Clinical trials are long andexpensive journeys, but thereward is worth the effort.
We have not lost sight of thegoal to apply to the FDA totreat type 1 diabetes withBCG. This would be thefirst FDA approved immune
intervention to alter theunderlying condition oftype 1 diabetes in people
living with existing disease.
We hope you will help us
move this important workforward. Please contact us
if you have any questions,diabetestrial@partners.org.
Sincerely,Denise L. Faustman, MD, PhD

UpdateFall 2022

BCG Clinical Trials to Reverse Type 1 Diabetes
In addition to the Phase II trial, other trials
are underway. We have now had more than 200
participants with type 1 diabetes vaccinated withmultiple doses of BCG.

The Phase II trial is one year closer to completion.
All 150 BCG-treated and placebo patients in the trial
have been followed for more than four years. The trial
will not read out until the last patient enrolled reaches
five years, which should happen some time in 2023.
TotalNo. Subjects

BCG-Treated(n)Control/
Placebo (n)Double Blinded/
Open-Label
Randomized,
Double-Blinded

Time Followed/Duration

Phase I + Crossover

52943Radiology Study

660Open-Label~3 y/5 yPhase II Study

150

10050

Randomized,
Double-Blinded~3-4 y/5 yTransition Study

29290Open-Label

2 y/5 yPhase II Crossover

6NANA

Phase II Pediatric Study

150

7575

New Pediatric Trial

Randomized,
Double-Blinded
Randomized,
Double-Blinded

2 y/5 y--“BCG is a very old vaccine, but recently has been at the center of an
explosion of clinical trial research showing benefit in multiple

In March, the Lab received approval to test BCG inchildren with type 1 diabetes. The new, 150-subject,multicenter trial, “Repeat BCG Vaccinations for theTreatment of Pediatric Type 1 Diabetes,” is alreadyalmost 50% enrolled. This is a long-anticipated andexciting development for the families who havesupported us for so many years.

autoimmune diseases and diverse infectious diseases. We know thetiming and strain of BCG vaccination is pivotal to outcomes. This is thefirst time multiple doses of a highly efficacious strain of BCG have been
given to adolescents with at least 2 years of type 1 diabetes,” says
Denise Faustman, MD, PhD, director of the Immunobiology Laboratory
at Massachusetts General Hospital and co-investigator on the study.

Research Updates
The 2022 American Diabetes Association (ADA)
Scientific Sessions were held in June in New Orleans.
One of our key findings in 2022, which was presented
at the meeting and also published in the journal
Science Advances, is a new understanding of how BCG
may alter the T cell defects common in people withtype 1 diabetes. We also shared clinical trial updates
and results of a retrospective study of patients withtype 1 diabetes who underwent BCG therapy forbladder cancer. That study showed a reduction inHbA1c values similar to the patients in the Phase I
trials at MGH.

5 or 10 y/10 y1268-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, USAIntroduction•The T cell receptor (TCR) is a central regulatorof 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•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

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••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 cell
population 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 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 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 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 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 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 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

www.faustmanlab.org

BCG and COVID-19

About BCG

Early in the pandemic, researchers began to report seeinga trend of lower infection rates and illness from COVID-19
in countries with BCG vaccination programs. This did notcome as a huge surprise, as researchers have long known
that BCG is protective against respiratory infections inchildren. MGH received permission from the FDA to doa double-blinded analysis of the ongoing type 1 diabetes
trial to see if the participants in the trial benefited from theBCG vaccinations they received before the pandemic hit.
The study, which was covered in the Wall Street Journal andThe New York Times, showed that 12.5% of placebo-treatedindividuals and 1% of BCG-treated individuals met criteria
for confirmed COVID-19, yielding a vaccine effectivenessrate of 92%. BCG’s effectiveness did not appear to be
impacted by differences in COVID-19 variants.
“MGH’s COVID-19 data set is unique and exciting because thepatients were all vaccinated with multiple doses of BCG prior to theonset of the epidemic. Prior to the trial they had no known exposure
to tuberculosis or prior BCG vaccination. This eliminates the major

BCG is a live, attenuated bacterial
vaccine derived from Mycobacterium
bovis. Discovered over 100 years ago
at the Pasteur Institute in Paris, BCG
has historically been given to protect
against tuberculosis. In the historyof medicine, it is considered to beextremely safe. BCG is on the World
Health Organization’s Model List of
Essential Medicines for adults andchildren and is given to roughly 100
million children per year globally. Overthe last twenty years, a growing groupof researchers and clinicians havebegun to explore the “off-target” effects
of BCG for diseases including type 1
diabetes, multiple sclerosis, allergy,fibromyalgia, Alzheimer’s disease
and respiratory infections such as
COVID-19.

confounding factors that have limited other trials. The results
support the idea that BCG needs time to have a clinical effect, but its
effects may then be very lasting and durable,” says Hazel Dockrell,London School of Hygiene & Tropical Medicine, an infectious
diseases expert who was not officially involved in the study.
REPEAT BCG VACCINATIONS IN ESTABLISHED TYPE 1 DIABETICS• ≥3 BCG (n=96) or placebo injections (n=48) before COVID-19 pandemic
January 2020

BCG VACCINESFOR COVID-19 &INFECTIONPROTECTION

Posterior
Probability

BCG

Placebo

VaccineEfficacy

COVID-19
confirmed by
Point of Careand SARS-CoV-2
Antibodies

1.0%

12.5%

92%

0.99

COVID-19
confirmed byPCR

0.0%

10.4%

100%

0.99(Vaccine Efficacy)

COVID-19 TESTING• COVID Antibodies• PCR

Cumulative COVID-19 Disease (%)≥ 3x

5th International
BCG Working GroupSYMPTOMS• Direct patientreporting• Bi-monthly surveys

BCG
Placebo

05Efficacy: 92%
p = 0.009

10Surveillance Time (months)

15In October, we convened the “Off-Target
Effects of BCG Conference” in Alexandria,
VA. This was our 5th international
conference held since 2013 and the biggest
to date. Over thirty researchers, payers
and policy experts from around the globe
convened to share updates on BCG clinical
trials in type 1 diabetes, multiple sclerosis,
COVID-19 and Alzheimer’s disease. For
more information on the conference andthe exciting research underway, visit
www.bcgworkinggroup.org.

UpdateFall 2022

FUNDRAISINGHow You Can Help

Events Come Back
COVID-19 changed how many events were held, but that did notstop inspired donors from organizing golf tournaments, mountainclimbs and other events. If you are interested in hosting an event,please contact the lab directly.

Please consider making a
tax-deductible donation to thistype 1 diabetes research program.
Every gift makes a difference.
1. To make a secure online donation,
visit www.faustmanlab.org andclick on “Donate.”
2. You may make a gift by check(payable to “Massachusetts
General Hospital”) and mailit to:

Donor Spotlight, Jana Brinkhouse
How has type 1 diabetes impacted
your family?
My daughter Karolina was diagnosed
at the age of 2.5, She comes from afamily with what appears to be a genetic
predisposition toward type 1 diabetes.
Chris, her father, was diagnosed at the ageof 12. Two of his siblings have it as well.
So T1D has had a generational impact onour family.
How did you learn about the BCG clinical trials
and why are you so supportive?
We learned about the trials through a family friend. We were excited by theidea of reversing the disease, not managing the symptoms and that BCG
has a proven safety record and would not require immunosuppression.
Tell us about some of your events.
Our events are inspired by all the children who suffer from T1D. We
wanted to involve children and give them a chance to have fun in theprocess with a type of family-centric process where they could learn aboutT1D as well. Our dance party model was very successful. Our last event,pre-COVID in 2019, was attended by 1000 people.
Any advice to families looking to get involved?
Try to come up with a community-wide event and tell your story, make it
personal, and truly describe a day in the life of a person with T1D. As weknow, there is confusion as to the different types of diabetes and dispellingthese myths is crucial to raising money and ultimately finding a cure.
What’s next for you?
Karolina has many things planned for 2023. Having recently been named
an ambassador for the student-athlete program at Wears Woody, she willtell her story about the challenges of having T1D while being an athlete,
all while raising funds for diabetes. She is also starting a T1D club at herhigh school, with the charity of choice being the Faustman Lab. A possible
return of the successful dance party model is also in the works!

Diabetes Clinical Trial
c/o Dr. Denise Faustman
Immunobiology Laboratory
MGH - East
Building 149, 13th Street, CNY-3601
Charlestown, MA 02129

On the memo line, please write:“Faustman T1D research.”Thank you for joining us in thefight against diabetes!

For more information, visit
www.faustmanlab.org or email
DiabetesTrial@partners.org.
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