18F-FDG PET/CT Mapping of Functional Microbial Niches to Understand Host GlucoseRegulation after BCG Vaccinations
HF Dias, JF Fu, TG Luck, GE Wolfe, ER Hostetter, NC Ng, H Zheng, WM Kühtreiber, JC Price, C Catana & DL Faustman
MASSACHUSETTS GENERAL HOSPITAL & HARVARD MEDICAL SCHOOL, BOSTON, MA, USA

Introduction• Diverse microbiomes in humans are known to change hostenergy metabolism• When the bacillus Calmette-Guérin (BCG) vaccine (live,avirulent Mycobacterium bovis) is introduced as experimental
therapy into human hosts with underlying type 1 diabetes(T1D), it gradually shifts energy metabolism in host blood
lymphoid cells from oxidative phosphorylation to aerobic
glycolysis, drawing more glucose out of the blood to fuelintracellular metabolism•

This metabolic shift may explain the BCG bacillus’ therapeutic
benefit: Systemic and long-term reduction of excess glucose
from the blood of TID patients• The organ-specific niches where the BCG bacillus alters
metabolism and establishes persistent residence are unknownMethods• In an open-label human clinical trial in participants with T1D(n=6), we mapped organ-specific niches for the BCG-induced
shift to aerobic glycolysis using fluorine-18 fluorodeoxyglucose(18F-FDG) positron emission tomography (PET) and X-ray
computed tomography (CT)•Standardized uptake value ratios were calculated to identify
organs with heightened glucose uptake over a 2-year timeperiod after BCG vaccination and to confirm earlier work that
BCG vaccination gradually lowers blood sugar levels without any
contribution from endogenous insulin• We also tested BALB/c mice (n=17) for the presence of BCG
colonies in particular organ niches before and aftervaccination

Results• Human and murine studies of the BCG microorganism
concurred that the spleen is the major anatomic site offunctional metabolic change and residence• The BCG bacillus also transiently mapped to the bonemarrow, liver, circulating lymphocytes as in the descending
aorta, and lungs

BCG vaccinations affect lymphoid sugar uptake
in humans with T1D
Fig. 1. (A) Percent change in HbA1c values
over 2 years time after 3 BCG vaccinationsin adults with longstanding T1D (n=6).
Mixed-Effects Analysis p-value=0·0402;
Holm-Šídák’s multiple comparisons test vsbaseline provided significant p-values forweek 0 vs week 52 (0·0006), week 0 vsweek 56 (0·0446), and week 0 vs week 78(0·0237). (B) C-peptide, a biomarker ofresidual pancreatic insulin secretion, wasmeasured in all T1Ds receiving in vivo BCG(n=6). The graph shows the sequential
values for the 6 patients at baseline, Year 01
and Year 02 after repeat BCG vaccinations.
The data show negligible C-peptide
production, indicating that residual C-peptide
cannot account for restored glucose
metabolism. (C) Human monocytes
harvested from T1D patients who were(n=11) or were not (n=11) treated with in vivo
BCG vaccination (approx. 2 years’ prior)
were cultured in vitro with or without BCG
exposure. Glucose uptake was analyzed
using a 2-NBDG assay. Both groups (BCGnaïve/unvaccinated and BCG-vaccinated)
had a significant increase in glucose uptake
after in vitro BCG stimulation (p-values of0.0026 and 0.0013 respectively, paired ttest). Even without BCG stimulation inculture, vaccinated T1Ds were observed tohave a significantly higher glucose uptake (pvalue of 0.0062, unpaired t-test).
Significantly increased uptake was also seen
when comparing both unvaccinated vsvaccinated T1Ds after in vitro BCG (p-valueof 0.0032, an unpaired t-test. (D)Characteristics of the 28 T1D subjects
presented in Fig. 1A-C.

18FDG-PET/CT images and gating regions
Fig. 2. (A) F-FDG-PET/CT images ofselect tissues showing an accelerated
glucose uptake signal with the gating
regions used to monitor uptake. Circles
represent the areas where the mean
SUV was measured. The left column is
the CT superimposed onto the 1 8 FFDG-PETimage (red/yellow), with thebrighter the color indicating the greater
the glucose uptake. Right image is the
1 8 F-FDG-PET scan alone, with thedarker the color indicating the greater
the glucose uptake.
18

18F-FDG-PET/CT scans in BCG-treatedparticipants with T1D identify human organs
with accelerated sugar uptake

BCG vaccinations in mice promote long-term
persistence of the BCG organism

Fig 5. (A) BCG culture experiments using tissues from periodically harvested mice (n=17) show that BCG
was significantly detected in the spleen for mice harvested up until week 20 (p-value=<0.0001). There arealso significant signs of BCG being present in the liver for mice harvested until week 12 (p-value=0.0002) andin the bone marrow of mice harvested until week 8 (p-value = 0.0312). (B) Images showing BCG colonies
grown in culture and labelled according to the number of colonies visible; plates with greater than 10 colonies
were just counted as 10. (C) The average number of BCG colonies per mouse is significantly higher in thespleen up until week 20, the liver up until week 12, and the bone marrow up until week 8. (p-value=<0.0001,0.0002 and 0.0312, respectively). One-tailed Wilcoxon ranked test was used to test for significance.
Fig 3. (A) Glucose uptake monitored by 1 8 F-FDG methods at baseline, Year 01 and Year 02 after BCG
vaccinations and then compared to baseline glucose uptake in each BCG-treated subject on an organ-perorgan basis (n=6). The graphs show 5 tissues/organs with augmented glucose uptake (out of 10 studied):spleen, aorta, bone marrow, lungs, and liver. The spleen was the only organ to continue through Year 02 toshow significantly more glucose uptake, according to a linear mixed effects model (p = 0.03). (B) 1 8 F-FDG
PET/CT images of the spleen over 2 years depicting the increase in standardized uptake value ratios(SUVR). (C) Dot plot of the spleen presenting the increase in glucose uptake for open-label patients on anindividual subject basis.

Correlation of organ-specific glucose uptake inhumans and BCG microbe presence in miceFig 6. Comparison of increased glucose uptake inhumans measured by metabolic changes induced
by BCG compared to the physical presence of theBCG microbe in mice. Scoring on a defined scaleof 0-5 (based on the amount of BCG microbepresent in mice tissue and the degree of glucose
uptake observed through the 18-FDG PET scans;
5 reflects the best signal for the presence of BCG
or for the best glucose uptake observed), thespleen, bone marrow, muscle, liver and lungtissues correlate with each other.

Identifying the location and time course of BCG
persistence in BCG-vaccinated miceFig. 4. BCG-treated BALB/c mice wereserially sacrificed to look at the tissue
niches where the BCG organisms might
persist as a live organism. Mice were
harvested at 5 separate times over a 22week time period. Harvested tissues
were tested for the presence of BCG
using the tissue homogenate andculturing in a media specific for BCG
growth. Tissue from the spleen, lungs,
bone marrow, liver, and muscle werecollected and observed for persistence ofthe BCG organism through colonyforming units in culture.

Conclusions••

These human and murine studies support the spleen as theniche for the BCG vaccine’s functional improvement ofmetabolism
The spleen is a lymphoid organ massive enough to explain
BCG’s systemic benefit of lowering blood glucose to nearnormal levels in T1D
Funding: The Iacocca Foundation. No conflicts of interest to declare. Contact:
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dfaustman@mgh.harvard.edu

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