Opinion

BCG Therapy for Type 1 Diabetes:
Restoration of Balanced Immunity andMetabolism
Willem M. Kühtreiber1 and Denise L. Faustman1,*
The bacillus Calmette–Guerin (BCG) vaccine is a microorganism developed as a
vaccine for tuberculosis 100 years ago and used as therapy for bladder cancer
40 years ago. More recently, BCG has shown therapeutic promise for type 1
diabetes (T1D) and several other autoimmune diseases. In T1D, BCG restored
blood sugars to near normal, even in patients with advanced disease of >20
years duration. This clinically important effect may be driven by resetting of theimmune system and the shifting of glucose metabolism from overactive oxidative phosphorylation, a state of minimal sugar utilization, to aerobic glycolysis, astate of high glucose utilization, for energy production. The mechanistic ﬁndings support the Hygiene Hypothesis and reveal the immune and metabolic
synergy of mycobacterial reintroduction in modern humans.

Highlights
The BCG vaccine is an attenuated formof mycobacterium originally developed>100 years ago for tuberculosis prevention. Its safety record is unsurpassed. This vaccine is now being
investigated as a therapy for type 1 diabetes (T1D) and other autoimmune diseases to restore the immune balance.
Repeated BCG vaccinations in long-term
diabetics can restore blood sugars tonear normal by resetting the immune system and by increasing glucose utilization
through a metabolic shift to aerobic glycolysis, a high-glucose-utilization state.

BCG’s Efﬁcacy in Lowering Blood Sugars in T1D Is Based on Immune and
Immunometabolic Effects

BCG-treated subjects given at least
two vaccines do not experience
restoration of blood sugars until about
3 years later, but once the blood
sugars return to normal, the therapeutic effect endures beyond 5 years.

The BCG vaccine (see Glossary), an attenuated Mycobacterium bovis that resembles Mycobacterium tuberculosis, is the most commonly used vaccine in the world. Used globally for over
100 years, it also is heralded as the safest vaccine ever developed. Primarily developed as apreventive vaccine for tuberculosis, high-dose BCG has also been used since the 1970s for earlystage bladder cancer. From an evolutionary viewpoint, humans and Neanderthals evolved with
Mycobacteria in their bone marrow, and it is only in recent times that humans have been without
continuous exposure to these organisms that are commonly found in soil, water, and theenvironment, as well as their common historical interface with humans in less-clean living environments [1–3]. This long evolutionary history explains why global data suggest that Mycobacteria
shape the host immune system, a bacterial–host interaction often referred to as trained immunity,
through epigenetic changes creating beneﬁcial commensalism at the gene level [4–9].
A recently reported Phase I randomized clinical trial ﬁnds that in longstanding T1D, vaccination
with two doses of intradermal BCG achieved, after a delay of 3 years, lowered HbA1c values in
the near-normal range. Once achieved, lower HbA1c values appear to be permanent for the
next 5 years without further BCG vaccinations [10] (Figure 1). Remarkably, reductions in HbA1c
are not associated with hypoglycemic events, a common occurrence with insulin alone andblood sugar control at these near-normal levels. Mechanistic data further reveal that BCG’sreduction of HbA1c appears to be achieved by resetting of the immune system in two ways on
the cellular level: through ‘turning on’ suppressive T regulatory (Treg) cells [10–12]; and by
the killing of pathogenic (cytotoxic) T lymphocytes (T cells) (CTLs) that attack pancreatic islet
cells [13,14] (Figure 2A). Both of these mechanisms rely on BCG’s induction of the cytokine
tumor necrosis factor (TNF). It has been appreciated that these immune regulatory effects of80

Trends in Endocrinology & Metabolism, February 2019, Vol. 30, No. 2© 2018 Elsevier Ltd. All rights reserved.

https://doi.org/10.1016/j.tem.2018.11.006

T1D subjects prior to BCG treatment
have an immune system dominatedby oxidative phosphorylation, a low-glucose-utilization state that predominantly
utilizes the Krebs cycle for energy.
Based on the Hygiene Hypothesis, lifelong underexposure to pathogens could
account for the predominance of oxidative phosphorylation in untreated T1D.
Because the BCG-induced restorationof glucose utilization is through regulated cellular utilization of sugar, episodes of hypoglycemia with nearnormal blood sugars are rarely reported.
1

Department of Medicine,
Immunobiology Laboratories,
Massachusetts General Hospital andHarvard Medical School, Boston, MA,
USA*Correspondence:
faustman@helix.mgh.harvard.edu(D.L. Faustman).

Onset of T1D

10–12 %

Glossary

BCG Rx
7–8 %Standard insulin therapy≈ 20 years

Rx with BCG
Limited hypoglycemia; reduced insulin

Age ≈ 15 years≈ 35 years

HbA1c (%)

Rx with insulin

6.0%≈ 43 years

Figure 1. Bacillus Calmette–Guerin (BCG) Permanently Lowers HbA1c to Near-Normal Levels. The diagram
summarizes the original data in [10] (see Figure 1). Multidosing with BCG, with a delay of approximately 3 years, returns
blood sugars to near-normal levels without hypoglycemia. BCG appears to work even in longstanding adult type 1 diabetic
subjects and persists long term without further BCG treatment.

BCG are also shared by tuberculosis itself: tuberculosis turns on Treg cells and also changes
the host epigenetics of the methylation machinery for Treg expansion [15–18].
Type 1 diabetic adults vaccinated with BCG exhibit within 8 weeks peripheral CD4 T cells with
epigenetic changes in the Treg signature genes (i.e., FoxP3, TNFRSF18, IL2RA, IKZF2, IKZF4, andCTLA4 [10]) (Figure 2B). Prepared DNA shows demethylation changes indicative of increased
gene expression. This was conﬁrmed by de novo host gene expression at the mRNA level of thesesame target Treg genes [10]. It is known that epigenetic modiﬁcations not only drive FoxP3
transcription but also lead to Treg stability [19]. BCG and tuberculosis turn on Tregs by multiple
methods in addition to the direct demethylation of the FoxP3 genes and additional Treg signature
genes. The CREB protein is recognized as an activated protein when phosphorylated and binds toa critical region of the FoxP3 locus to enhance transcription. Mycobacteria – BCG or TB – on
infecting host macrophages produce a burst in cAMP. Mycobacteria are unique among bacteria inthat they have not just one but up to 17 adenylate cyclase genes to phosphorylate host proteinsand change gene expression. Monocytes that are infected with mycobacteria activate criticalproteins such as CREB that in turn can promote FoxP3 expression by enhancing the transcription
machinery of the demethylated locus (Figure 2C) [20–22].

Aerobic Glycolysis and Oxidative Phosphorylation, Two Immunometabolic
Steps, Differ in Their Utilization of Serum Glucose
Normally the immune cells produce energy through either oxidative phosphorylation or
aerobic glycolysis based on their functions, their environment, and probably many othercausal factors yet to be identiﬁed (Figure 3A). Oxidative phosphorylation uses the Krebs cycle,
and cells in which this mechanism is predominant transport relatively small amounts of sugar forenergy. In marked contrast, aerobic glycolysis in lymphocytes is a regulated and high-glucose
sugar-transport process that creates purines through the pentose phosphate shunt, andsuch cells underutilize the Krebs cycle for energy. Remarkably, BCG switches immunometabolism from high reliance on oxidative phosphorylation to high reliance on aerobic glycolysis
on a systemic level, monitored in humans by documented effects in the blood (Figure 2B)[10,23,24], thereby lowering glucose levels. It is important to point out that T1D subjects prior to
BCG treatment have overzealous oxidative phosphorylation so the BCG effect on immune

Aerobic glycolysis: a metabolic
pathway used by cells to obtainenergy and to make purines, which
are DNA and RNA building blocks.
Aerobic glycolysis is not overly
dependent on the mitochondria or
the Krebs cycle. This metabolic
pathway utilizes lots of sugar on theexterior of the cell as the energy
course.
Bacillus Calmette–Guerin (BCG)
vaccine: an attenuatedMycobacterium bovis organism
similar to the Mycobacterium
tuberculosis organism. BCG has
been used globally as a preventive
vaccine for tuberculosis for over 100
years, with over 3 billion people
vaccinated.
Cytotoxic T lymphocytes (CTLs):
also known as autoimmune killer
cells; pathogenic cells underlying
autoimmunity that attack and kill selforgans.
Hygiene Hypothesis: anenvironment-based proposal toexplain the increasing incidence of
T1D and other autoimmune
diseases. It asserts that diseases
appear as a result of a change inenvironmental factors such as fewer
childhood exposures tomicroorganisms, increased vaccines
and thus fewer infections, increased
antibiotic usage with fewer infections,cleaner foods without
microorganisms, and a lifestylewithout occupations with closeinteractions with soil and animals.
Ketones: substances produced
during a late step in oxidative
phosphorylation prior to the Krebs
cycle; made from acetyl-CoA.
Krebs cycle: a late metabolic stepduring oxidative phosphorylation thatyields energy in the form of ATP.
Late stages of the Krebs cycle use
the mitochondrial membrane forelectron transport.
NOD mouse: a nonobese diabetic
mouse that is a common murine
model for the study of autoimmunity.
Like humans, NOD micespontaneously develop T1D driven
by too many CTLs and too few
functional Treg cells.
Oxidative phosphorylation: ametabolic pathway used by cells to
obtain energy; dependent on only
small amounts of glucose and utilizes
the mitochondria and Krebs cycle.

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81

metabolism is to restore it to normal by increasing aerobic glycolysis, a state of accelerated
regulated glucose utilization (Figure 2C). It has been appreciated that other forms of mycobacteria like M. tuberculosis also modulate aerobic glycolysis for trained immunity effects [25].
Lowering blood glucose levels in diabetes is a highly investigated topic since the discovery of
insulin. Interventional and pharmacological methods to practically or theoretically lower blood
sugar have relied on diverse biological pathways and processes to achieve euglycemia. Insulinlevels have increased from reintroduction of islets or islet regeneration, decrease in peripheral
insulin resistance, and enhancement of insulin secretion. Glucose-lowering drugs can inhibit
liver glucose production, slow intestinal glucose absorption, or decrease the insulin contraregulatory hormone glucagon. Blood sugar-lowering drugs also include drugs that prevent
glucose uptake in the kidney (SGLT2 inhibitors) or prevent glucose uptake in the intestine(SGLT1 inhibitors). The recent clinical trial on repeat BCG vaccination of T1D identiﬁes a newmechanism for blood sugar lowering that uses immunometabolism.

Can BCG Treatment Regenerate Pancreatic Islets?
BCG treatment with a clinical delay in onset of efﬁcacy appears permanent, without risk ofhypoglycemia since the cellular sugar transport is regulated, and need not be repeated after
initial dosing [10]. Remarkably, the underlying mechanism, immunometabolism, does not needpancreatic regeneration to restore blood sugars, at least in those patients with longstanding
diabetes [10,13]. We have shown previously that BCG treatment of diabetic NOD mice results
in restoration of normoglycemia by means of large-scale regeneration of islets [26,27]. In ourcurrent human T1D studies, however, this appears not to be the primary mechanism. As thedata in our NPJ Vaccines paper show, although HbA1c was stably corrected to the normal
range for over 5 years, stimulated C-peptide levels were almost undetectable and certainly notlarge enough to account for the drop in HbA1c [10]. We therefore conclude that, at least inthese T1D patients with adult disease onset, long duration of disease, and no signiﬁcantresidual C-peptide at the start of the clinical trials, the pancreas after BCG was not playing a rolein controlling blood sugars.
A pediatric trial is planned and we hope it will show whether blood sugar restoration in younger
people can be driven by both pancreatic islet regeneration and regulation of immunometabolism
as observed in adults with longstanding T1D. Regardless of the age or duration of T1D, theimmune imbalance of too few functional Tregs and too many pathologic T effectors is present at
diagnosis and persists for years and BCG appears able to reset these immune defects.

BCG’s Immune and Immunometabolic Effects Support the Hygiene
Hypothesis
It is commonly appreciated that microorganisms frequently utilize aerobic glycolysis as themechanism for energy production when they reside in infected cells, using simple sugars as asource of energy. Therefore, if autoimmunity is driven by the Hygiene Hypothesis theobservation of a high baseline of oxidative phosphorylation ﬁts well. The Hygiene Hypothesis
contends that the rise in allergies and autoimmunity in modern societies is due to the lack ofinteraction between microorganisms and the immune system. Microorganisms, including BCG,
convert the host immune system to aerobic glycolysis at least at the site of infection. Our data
support the Hygiene Hypothesis by showing that the reintroduction of a bacterium (BCG) into
T1D subjects ameliorates autoimmunity. We also have shown that improper T cell training bypoor Treg function and pathogenic cytotoxic T cells is also corrected and that this correction
appears to be driven by the host production of TNF, a natural cytokine that is induced bypathogens as the ﬁrst line of defense.
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Pentose phosphate shunt: themetabolic pathway for cellularsynthesis of purines and pyrimidines;
very active in aerobic glycolysis but
underactive in oxidative
phosphorylation.
Purines: building blocks of thenucleic acids DNA and RNA.
TB: M. tuberculosis, the pathologic
version of mycobacteria that causes
disease in humans.
Treg-speciﬁc demethylation
region (TSDR): the DNA region of
the FoxP3 gene critical for Treg
generation and stability. This DNA
region must to demethylated for thecritical CREB protein to bind; CREB
must additionally be phosphorylated
to bind to the demethylated region.
T regulatory (Treg) cells:
suppressive cells that help to quiet
the immune response. In
autoimmune disease, they aredeﬁcient either in numbers or in theirsuppressive capabilities.
Tumor necrosis factor (TNF): anaturally made cytokine that helps tomold the immune response. TNF is
known to be the ligand for Treg
expansion and also the ligand forselective death of cytotoxic
lymphocytes.
Type 1 diabetes (T1D): an
autoimmune disease wherein the
immune system abnormally attacks
the insulin-secreting cells of thepancreas and thus causes lifethreatening hyperglycemia.
Type 2 diabetes (T2D): a disorderwhere early in the disease insulin isstill made by the pancreas but the
peripheral tissues where insulin actsare resistant to insulin action. It is
generally believed that this insulin
resistance eventually drives themassive amounts of pancreatic
insulin production to decline.

(A)

CTL

Treg

CTL

BCG
Treg

CTL

TNF

Treg

CTL

CTL

Treg
Treg

Treg

CTL

Treated autoimmune environment

Autoimmune environment(B)

Methylated Treg signature genes
FoxP3

TNFRSF18

IL2RA

IKZF2

IKZF4

CTLA4

IKZF4

CTLA4

De-methylated genes aer BCG treatment

FoxP3

TNFRSF18

IKZF2

IL2RA

(C)

Phosphorylaon

BCG or TB infected
monocytes

TB (RV0386 gene)cAMP
BCG (Mb 0393 gene)

CREB

CREB

Foxp3 TSDR

Figure 2. Immune effects of Bacillus Calmette–Guerin (BCG) in Type 1 Diabetic Subjects. (A) The autoimmune environment comprises too few suppressive
T regulatory (Treg) cells and too many cytotoxic T lymphocytes (CTLs). With BCG treatment and its associated tumor necrosis factor (TNF) induction, Treg cell expansion
and augmented function occurs, and CTLs die thus restoring the immune balance towards normal at the autoimmune site. Black circles are CTLs, green circles are
Tregs, and blue circles are the insulin-secreting islets. (B) At the DNA level, BCG in vivo causes direct demethylation of the six signature genes of Treg expression and(Figure legend continued on the bottom of the next page.)
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Figure 3C compares the balance of oxidative phosphorylation and aerobic glycolysis bylymphoid cells from normal subjects, from unvaccinated T1D subjects, and from BCG-treated
T1D subjects. It summarizes our mechanistic ﬁndings from full-genome mRNA sequencing ofseparated cell populations as well as comprehensive metabolomic and epigenetic studies [10].
Untreated T1D lymphocytes display an abnormally high reliance on oxidative phosphorylation,
which is a state of low glucose utilization. BCG-treated lymphocytes use more glucose forenergy and thus lower serum glucose in a regulated fashion. The high oxidative phosphorylation
state of unvaccinated T1D subjects is associated with brisk Krebs cycle utilization, theoverproduction of ketones, and underutilization of glucose transport to fuel energy production.
With BCG therapy not only does accelerated and regulated glucose transport occur at the cell
membrane, but the pentose phosphate shunts turns on, and fewer metabolites are funneledthrough the Krebs cycle. This suggests that BCG-treated T1D subjects are less likely to
produce ketones. Since ketones have long been associated with only T1D and not type 2
diabetes (T2D), the high oxidative phosphorylation state in T1D may explain why moreketones are produced with equivalent amounts of elevated blood sugars and no ketones inT2D. Whether looking at purine synthesis or at the pentose phosphate shunt mechanism,
untreated T1D has the hallmarks of predominant oxidative phosphorylation driving immunometabolism. It is also important to point out that the measurement of purine metabolites inunvaccinated T1D compared with age-matched controls consistently shows diminished levels,a clear marker of insufﬁcient baseline aerobic glycolysis (Figure 4). This suggests that T1D has
an underlying defect in metabolic balance, which BCG partially corrects. The data as a whole
do not suggest that BCG shifts normal immune metabolism in an overzealous fashion to ahighly upregulated abnormal state of aerobic glycolysis, but that it is merely resetting thebalance. Since many microorganisms including tuberculosis mycobacteria and BCG utilize
aerobic glycolysis, the data in total support that immune-metabolic imbalance in T1D could
stem from too few environmental exposures, which have been eliminated as a result of moresterile environments.
It could be argued that although it is observed that immune metabolism after repeat BCG
therapy ﬂips glucose utilization, the metabolic marker shifts might indicate that near-normal
HbA1c, compared with elevated HbA1c, drives immunometabolism to predominantly corrected aerobic glycolysis. However, this is not the case, because there is no difference in thepattern of metabolites associated with aerobic glycolysis and oxidative phosphorylation whencomparing unvaccinated T1D with high HbA1c vs low HbA1c values. Instead, an entirely
different group of metabolites, unrelated to carbohydrate metabolism, appears to be correlatedwith glucose levels in T1D (Figure 5).

Adult Vaccination with BCG Is Efﬁcacious in Diverse Human Diseases
Research over the past 10 years has investigated the therapeutic beneﬁts of BCG for an array of
autoimmune, allergic, and induced adaptive immune responses to childhood infections[4,13,28–34]. In multiple sclerosis, BCG delivered in a double-blind randomized controlled
trial halted new onset disease, but the clinical effect was most striking after a delay of nearly 5
years [29]. Three BCG vaccines administered in childhood were associated with lower incidence of T1D by age 12 years [33]. In a Phase I trial, two doses of BCG in long-term T1D
subjects elicited favorable biomarker responses, such as increased beneﬁcial Treg cells, killing

subsequent augmented mRNA expression of the corresponding genes (i.e., FoxP3, TNFRSF18, IL2RA, IKZF2, IKZF4, and CTLA4). (C) Ancient organisms likemycobacteria use genome-encoded bacterial adenylyl cyclases, the enzymes that generate cAMP, as second messengers to regulate host genes. For proper Treg
stability, phosphorylated CREB binds the FoxP3 Treg-speciﬁc demethylation region (TSDR).

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Trends in Endocrinology & Metabolism, February 2019, Vol. 30, No. 2

Aerobic glycolysis

OxidaƟve phosphorylaƟon
(A)

GlucoseGlucosePurinesPentose shunt

Krebs cycle
Accelerated sugar uƟlizaƟon

Poor sugar uƟlizaƟon

(B)

NormalUntreated T1D

OxidaƟve phosphorylaƟon
Aerobic glycolysis(C)

BCG-treated T1D

Poor sugar uƟlizaƟon

Restore sugar uƟlizaƟon

Normal subject

T1D

T1D aŌer BCG Rx

GlucoseGlucoseGlucoseGlucoseGlucoseGlucoseGlucose 6P

Pentoseshunt

Purines

Fructose 6P

Pentoseshunt

Purines

Glucose6P
Fructose 6P

Glyceraldehyde 3P

Glyceraldehyde 3P

Pyruvate

Pyruvate

Acetyl-CoA

Ketones

Acetyl-CoA

Krebs cycle

Krebs cycle

Balanced oxidaƟve
phosphorylaƟon andaerobic glycolysis

Dominant oxidaƟve
phosphorylaƟon

Pentoseshunt
Pentoseshunt

BCG

Glucose 6P

Pentoseshunt

Purines

Fructose 6P

Pentoseshunt

Purines

Glyceraldehyde 3P

Pyruvate

Ketones

Acetyl-CoA

Krebs cycle

Restored balance

Figure 3. The Balance between Oxidative Phosphorylation and Aerobic Glycolysis in Lymphoid Cells. (A) Glucose utilization by lymphocytes is dictated bytheir metabolic state. With predominant oxidative phosphorylation, there is little glucose utilization but high Krebs cycle utilization for energy metabolism. With high(Figure legend continued on the bottom of the next page.)
Trends in Endocrinology & Metabolism, February 2019, Vol. 30, No. 285

of pathogenic (cytotoxic) T cells, and temporary restoration of pancreatic insulin, but, by the endof the 20-week trial, failed to lower HbA1c [13]. Three more years had to elapse before HbA1c
was lower, according to the Phase I extension study [10]. In low-birthweight infants, BCG
vaccination in a case-cohort study conferred a survival advantage in a diversity of infections
unrelated to tuberculosis, and in healthy populations conferred a lifelong and long-term survival
advantage [35–38].

Lessons about the Therapeutic Beneﬁts of BCG
The evidence base offers important lessons on the timing of BCG administration relative to disease
onset, the number of BCG doses, and strain differences in BCG efﬁcacy. If NOD mice are given BCG
after they display early signs of diabetes (prediabetes), new-onset diabetes, or full-blown diabetes, itpermanently reverses diabetes [26,27,39,40]. However, giving BCG at birth in diabetes-prone
humans or NOD mice as a single injection has no beneﬁt, so the disease must be apparent for BCG tobe effective in mice and humans [41,42]. In humans, a single dose of BCG is not associated with
reduced incidence of T1D by age 12 years, but at least two doses are beneﬁcial [33,43].
Human and mouse studies reveal variable efﬁcacy of different BCG strains. A single dose of theMoreau BCG substrain appears to decrease the progression of new-onset diabetes in humans,
but three subsequent human studies using less potent BCG strains, such as TICE, demonstrated no clinical beneﬁt in humans, at least with limited follow-up time [44,45]. The TICE strainof BCG, for example, is known to have poor immunoregulatory properties in both NOD miceand humans and when studied in vitro has lower efﬁcacy for the induction of TNF and thetranscription factor NFkB [46].
The age of subjects treated with BCG may drive mechanisms underlying blood sugar restoration. In young NOD mice, repeat administration of BCG causes brisk pancreatic regeneration[26,27]. In adult NOD mice and humans with longstanding diabetes, BCG repeat administration
leads to restored blood sugars through immune effects and immunometabolism, not throughpancreatic regeneration. Pediatric trials have not yet been conducted to determine the relative
contribution of pancreatic regeneration as a cause of restored blood sugars versus accelerated
aerobic metabolism as the primary driver of restoration of blood sugars to normal levels.

Overactive Oxidative Phosphorylation in Unvaccinated T1D May Explain the
Well-Known Propensity to Ketosis
The discovery of overactive oxidative phosphorylation in unvaccinated T1D may explain the clinicalobservation of heightened ketosis. It has been known for years that T1D versus T2D subjects havedifferent susceptibilities to ketosis. Even in well-controlled studies, the same glucose dysregulation consistently reveals the ketosis speciﬁc to the type 1 diabetic etiology [47].

aerobic glycolysis, lymphocytes use much more glucose, utilize the pentose shunt with augmented purine metabolites, and produce less energy from the Krebs cycle.
(B) In normal cells from nondiabetic controls, lymphocytes exhibit a balance between oxidative phosphorylation and aerobic glycolysis. At baseline, type 1 diabetes(T1D) subjects have underactive aerobic glycolysis and overactive oxidative phosphorylation, resulting in poorly regulated glucose utilization. With bacillus Calmette–
Guerin (BCG) treatment of T1D, aerobic glycolysis is restored towards normal and the lymphoid cells utilize more glucose in a regulated metabolic fashion avoiding
hypoglycemia. (C) Normal lymphoid cells exhibit a balance in energy production between oxidative phosphorylation that utilizes the Krebs cycle and aerobic glycolysis
that utilizes the pentose phosphate shunt to make purines. T1D lymphoid cells overutilize oxidative phosphorylation and the Krebs cycle for energy metabolism; thisstate is associated with minimal serum glucose utilization since the Krebs cycle is very efﬁcient in generating a lot of ATP while using only a little glucose. It should be
noted that overutilization of oxidative phosphorylation might result in a tendency for overproduction of ketones since too much acetyl-CoA is produced prior to the Krebs
cycle. As a result, too much acetyl-CoA can be shunted to ketones. With BCG treatment of T1D, the immune metabolism of lymphocytes is restored in large part back to
the normal balance. T1D subjects have lowered purine synthesis and pentose phosphate shunt; regulated sugar transport is restored and the tendency of T1D subjects
to make ketones is likely to be minimized.

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Trends in Endocrinology & Metabolism, February 2019, Vol. 30, No. 2

Adenine

Scaled intensity

2.0

Pentose phosphate
shunt

1.5
1.0
0.5

D
T1

Co

nt
ro
l

0.0

N6-carbamoylthreonyladenosine

1.0
0.5

D

Co

T1

l

0.0

nt
ro

Ribulose 5phosphate

1.5

Scaled intensity

6-Phospho
gluconate

7-methylguanine

1.0
0.5

D
T1

nt
ro

l

0.0

Co

Purines
Adenine,N6-carbamoylthreonyladenosine,
7-methylguanine,
N2,N2-dimethylguanosine

N2,N2-dimethylguanosine
1.5

Scaled intensity

Ribose 5phosphate

Scaled intensity

1.5

1.0
0.5

T1
D

Co
nt
ro

l

0.0

Figure 4. Unvaccinated Type 1 Diabetes (T1D) Subjects Have a Suppressed Pentose Phosphate Shunt as
Demonstrated by Reduced Purine Metabolites. The pentose phosphate shunt leads to the production of various
purines. Metabolomic analysis shows that several key purines are signiﬁcantly reduced in the serum of unvaccinated T1D(Figure legend continued on the bottom of the next page.)
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87

HbA1c top 50%
HbA1c boƩom 50%

ADSGEGDFXAEGGGVR
N6-Methyladenosine
2-Linoleoyl-GPE (18:2)
1-Linoleoyl-GPE (18:2)
N-Linoleoylglycine
Pro-hydroxy-pro
3-Phenylpropionate
03
1
2
Scaled intensity

4

Figure 5. Excellent or Poor HbA1c Control Does Not Affect Metabolites of Aerobic Glycolysis and Oxidative
Phosphorylation. Since BCG treatment lowers blood sugars leading to lowered HbA1c values, it was important to ruleout metabolically that blood sugar control was the consequence, not the cause, of the shift in type 1 diabetes (T1D)
lymphocytes from high oxidative phosphorylation to aerobic glycolysis. We therefore divided a group of 100 T1D subjects
into the highest 50% and the lowest 50% based on HbA1c and compared the metabolomic proﬁles of these two groups.
These data showed that the altered metabolites of high blood sugars versus low blood sugars in unvaccinated T1D wereunrelated to the metabolic shifts secondary to bacillus Calmette–Guerin (BCG). Thus, average blood sugar levels had no
impact on carbohydrate metabolism. The graph shows only those metabolites that had statistical signiﬁcance as it relates
to HbA1c levels. The metabolites shown are in the peptide pathway (ADSGEGDFXAEGGGVR, P = 0.000 2, Q = 0.012),
the nucleotide pathway (N6-methyladenosine, P = 0.001, Q = 0.041), the lipid pathway [N-linoleoylglycine, P = 0.000 6,
0.028; 1-linoleoyl-GPE (18:2), P = 0.000 1, Q = 0.01; 2-linoleoyl-GPE (18:2), P = 0.000 3, Q = 0.016 8), and the aminoacid pathway (3-phenylpropionate, P = 0.001 1, Q = 0.041; pro-hydroxy-pro, P = 2.5E–5, Q = 0.041). Thus, none of thesemetabolites relates directly to metabolic control of blood sugar. The data in this ﬁgure were published at the 2018 ADA 78th
Scientiﬁc Sessions meeting in Orlando, Florida [50]. For a description of the metabolomic methods and statistics, pleaserefer to the legend of Figure 4.

Figure 3 shows that cells with highly active oxidative phosphorylation push metabolites down
towards the Krebs cycle, which is a very active pathway for energy. If the downward drive ofoxidative phosphorylation is strong, acetyl CoA will generate ketones. Therefore, high oxidative
phosphorylation is expected to be associated with higher ketone production. By contrast, theBCG-induced shift to aerobic glycolysis is expected to diminish ketone production and could
be an added beneﬁt of BCG therapy in T1D.

patients (n = 100) compared with nondiabetic controls (n = 50). This indicates that the pentose phosphate shunt in T1D isdownregulated. P and Q values, respectively, are: adenine (2.3E 5, 0.000 2); N6-carbamoylthreonyladenosine (0.003,
0.000 5); 7-methylguanine (9E 8, 1.5E 5), and N2,N2-dimethylguanlosine (0.008, 0.039). Underutilization of thepentose phosphate shunt is a symptom of underactive aerobic glycolysis. The data in this ﬁgure are taken from [10] (seeFigure 5b). Please refer to this paper for a description of the methods and statistics that were used. A description of thedemographics for the patient groups is given in [10] (see Online Supplementary Table S1c). P and Q statistics are in [10](see Online Supplementary Table S4). Brieﬂy, the patients had an average disease duration of 8  1 years. Serum samples
from 100 unvaccinated T1D patients and 50 nondiabetic controls were used in this study. The samples were sent for
metabolic proﬁling and statistical analysis to Metabolon, Inc. (Durham, NC). Samples were analyzed on Metabolon’s
integrated discovery platform comprising gas and liquid chromatography for separation and mass spectrometry for
detection and identiﬁcation. The metabolomic platform distinguished a total of 690 metabolites.

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Non-treated T1DAB

A Electron transfer inhibitors

Proton eﬄux rate

B Glucose transport compeƟƟon

Basal glycolysis
Compensatory glycolysis
with 2-deoxyglucose (aerobic)

Time

BCG-treated T1D
BProton eﬄux rate

A

Time

Proton produc on rate (pmol/min)

BCG-treated and non-treated T1D
50
40
30
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Time (min)Figure 6. Principles of the Seahorse Glycolytic Rate Assay (GRA) as a Method to Measure In Vitro theSuppressed Aerobic Glycolysis or Compensated Accelerated Glucose Transport in Type 1 Diabetes (T1D)
Monocytes Before and After Bacillus Calmette–Guerin (BCG) Treatment. The GRA assay measures both theoxygen consumption rate (OCR) and the extracellular acidiﬁcation rate (ECAR) of cells in vitro. Aerobic glycolysis
contributes to extracellular acidiﬁcation, resulting in a glycolytic proton efﬂux rate. This metric mirrors the amount oflactate acid produced during aerobic glycolysis. After collecting three baseline data points, the analyzer injects the
mitochondrial electron transport chain inhibitors rotenone and antimycin A, effectively inhibiting all oxidative phosphorylation and thus the Krebs cycle. The cells respond by increasing aerobic glycolysis to compensate. The increased glycolysis
causes an increase in lactate production, which acidiﬁes the medium. After collecting three more data points, the analyzer
injects 2-deoxyglucose, which competes with glucose uptake into the cells but cannot be metabolized by the cells. This
(Figure legend continued on the bottom of the next page.)
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Minimal Hypoglycemia with BCG-Induced Lowered HbA1c Levels Is
Possibly Explained by Upregulated Aerobic Glycolysis
Long-term T1D subjects with near-normal blood sugar corrections in our Phase I clinical trial
continue to take insulin, although in reduced amounts. Remarkably, our 5-year hypoglycemic
surveys reveal that HbA1c corrected to the 5.7–6.0% range is not associated with increased
hypoglycemic episodes. For standard insulin therapy with insulin pumps or with continuous
glucose monitors, hypoglycemia usually worsens with increased insulin use, thus precludingtight blood sugar regulation. BCG-induced blood sugar lowering is due to altered expression ofgenes that regulate glucose transport and glucose utilization through oxidative phosphorylation
and aerobic glycolysis. The hypoglycemic risk is minimized after BCG treatment because mostcellular glucose transporters are regulated (turned on or off) by exterior extracellular glucose
levels; that is, if the blood sugar is high the lymphocytes continue to transport glucose but if theblood glucose is low glucose transport into the cell is stopped. Insulin, of course, is a fabulous
way to lower blood sugars but has the well-known limitation of not reducing glucose transport if
the ambient glucose concentration is lowered. This is why insulin therapy cannot be usedaggressively since insulin is not ‘smart’ and does not know when to stop lowering serum
glucose levels.

New Tests Under Development for Measuring Metabolism and Predicting
BCG ResponsivenessThe overactive state of oxidative phosphorylation in T1D subjects was determined by fullgenome mRNA sequencing of separated cell populations, comprehensive metabolomics, andgenomic epigenetic studies. These are powerful research tools, but the additional goal is todevelop a standardized blood-sampling method that would deﬁne a priori the oxidative/aerobic
state of lymphocytes. This not only might predict, if quantitative, the rate of BCG responsiveness but could also be used to proﬁle T2D subjects for possible BCG responsiveness.
Will BCG also lower blood sugars in T2D subjects? Although diverse obese animal model datasuggest that BCG could similarly control blood sugars and perhaps even impact metabolic
syndrome, studies are under way to develop a diagnostic [48,49]. One in vitro test, still underdevelopment, is a Seahorse Glycolytic Rate Assay (GRA) as a method to measure in vitro the
suppressed aerobic glycolysis or compensated accelerated glucose transport in T1D monocytes after BCG treatment (Figure 6). GRA is based on the measurements of both the oxygen
consumption rate (OCR) and the extracellular acidiﬁcation rate (ECAR) of cells in vitro with andwithout BCG added to the wells as a prototype method of analysis. After coculture of
monocytes with or without BCG, this glycolytic rate assay treats the mitochondrial electrontransport chain with the inhibitors rotenone and antimycin A. They effectively inhibit oxidative
phosphorylation and thus the Krebs cycle. The cells respond by increasing glycolysis tomaximum levels if aerobic glycolysis is already primed. The increased glycolysis causes anincrease in lactate production, which acidiﬁes the medium. As is shown in Figure 6, T1D cells bythemselves have almost no compensated glycolysis. The analyzer then injects 2-deoxyglucose, which competes with glucose uptake into the cells but cannot be metabolized by the
shuts down glucose metabolism. Actual data for the GRA assay show T1D monocytes cultured for 24 h in the presence of
BCG (red line) to have a robust increase in glycolysis compared with control monocytes (blue line); that is, they can respondand convert to a high-glucose-utilization status if provided with BCG to augment this change in cellular metabolism that is
not present at baseline. Monocytes from a single T1D patient were isolated by magnetic separation from whole blood usinga Stem Cell Technologies Monocyte Direct Isolation kit. The cells were cultured for 24 h at 37  C and 5% CO2 in the‘miniplates’ that are designed to be used on a Seahorse XFp instrument. Wells contained either 200 000 monocytes alone
or 200 000 monocytes and 200 000 colony-forming units (CFU) BCG.

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cells. This completely shuts down glucose metabolism. Actual data from the GRA assay showthat T1D monocytes cultured for 24 h in the presence of BCG show a much more robust
increase in aerobic glycolysis than untreated monocytes. This additionally shows that although
T1D monocytes from fresh blood are deﬁcient in aerobic glycolysis, they respond even after
short-term culture in the presence of BCG by restoring and augmenting aerobic glycolysis andsugar utilization from the medium. Future use of these in vitro sugar-utilization assays will help toanswer the question regarding BCG as a method to lower blood sugars in various diabetic
subjects. It is not known whether, like T1D subjects, T2D patients have at baseline suppressed
aerobic glycolysis or whether BCG administration in T2D will need to augment normal aerobic
glycolysis to heightened levels for improved blood sugar control.

Concluding Remarks
BCG is emerging as an efﬁcacious therapy for autoimmune diseases. In T1D, subjects treatedwith two BCG vaccines, after a delayed time, exhibited lowering of blood sugars to near-normal
levels in an 8-year clinical trial. With such a marked beneﬁt, it is important to understand theimmune and metabolic effects of BCG. This Opinion article summarizes the literature showingthat BCG resets the immune system by restoring Tregs and selectively killing pathogenic T
cells. The fact that even people with advanced T1D beneﬁt from lowered stable blood sugars
without severe hypoglycemia warrants attention to BCG’s immune metabolic effects. T1Dsubjects have overactive oxidative phosphorylation, a state of low glucose utilization, highketone production, and high Krebs cycle utilization. After BCG vaccinations, the metabolism of
the immune system gradually shifts to high and regulated glucose transport through aerobic
glycolysis. These ﬁndings support of the Hygiene Hypothesis and show the magnitude of therestoration of the human host immune system by bacteria.
References
1. Comas, I. et al. (2013) Out-of-Africa migration and Neolithic
coexpansion of Mycobacterium tuberculosis with modern
humans. Nat. Genet. 45, 1176–1182

14. Ban, L. et al. (2008) Selective death of autoreactive T cells inhuman diabetes by TNF or TNF receptor 2 agonism. Proc. Natl.
Acad. Sci. U. S. A. 105, 13644–13649

2. Barnes, I. et al. (2011) Ancient urbanization predicts genetic
resistance to tuberculosis. Evolution 65, 842–848

15. Morikawa, H. et al. (2014) Differential roles of epigenetic
changes and Foxp3 expression in regulatory T cell-speciﬁctranscriptional regulation. Proc. Natl. Acad. Sci. U. S. A. 111,
5289–5294

3. Strachan, D.P. (1989) Hay-fever, hygiene, and household size.
BMJ 299, 1259–1260
4. Goodridge, H.S. et al. (2016) Harnessing the beneﬁcial heterologous effects of vaccination. Nat. Rev. Immunol. 16, 392–400
5. Jensen, K.J. et al. (2015) Heterologous immunological effects ofearly BCG vaccination in low-birth-weight infants in Guinea-Bissau: a randomized-controlled trial. J. Infect. Dis. 211, 956–967
6. Netea, M.G. and van Crevel, R. (2014) BCG-induced protection:effects on innate immune memory. Semin. Immunol. 26, 512–517
7. Arts, R.J. et al. (2016) Immunometabolic pathways in BCGinduced trained immunity. Cell Rep. 17, 2562–2571
8. Kleinijenhuis, J. et al. (2014) Long-lasting effects of BCG vaccination on both heterologous Th1/Th17 responses and innate
trained immunity. J. Innate Immun. 6, 152–158
9. Kleinnijenhuis, J. et al. (2012) Bacille Calmette–Guerin induces
NOD2-dependent nonspeciﬁc protection from reinfection via epigenetic reprogramming of monocytes. Proc. Natl. Acad. Sci. U.
S. A. 109, 17537–17542
10. Kuhtreiber, W.M. et al. (2018) Long-term reduction in hyperglycemia in advanced type 1 diabetes: the value of induced aerobic
glycolysis with BCG vaccinations. NPJ Vaccines 3, 23
11. Okubo, Y. et al. (2013) Homogeneous expansion of human T-regulatory cells via tumor necrosis factor receptor 2. Sci. Rep. 3, 3153
12. Okubo, Y. et al. (2016) Treg activation defect in type 1 diabetes:
correction with TNFR2 agonism. Clin. Transl. Immunol. 5, e56
13. Faustman, D.L. et al. (2012) Proof-of-concept, randomized, controlled clinical trial of Bacillus–Calmette–Guerin for treatment oflong-term type 1 diabetes. PLoS One 7, e41756

Outstanding Questions
Will BCG’s ability to regulate blood
sugar in T1D apply to T2D? Although
global animal model studies suggest
this might be the case, no clinical data
exist yet addressing BCG introduction
into humans with T2D.
Why are BCG’s beneﬁts for T1D, multiple sclerosis, and protective immunity
against tuberculosis delayed by anumber of years, although revaccinations appear unnecessary and the
beneﬁts appear to be permanent?
Why do type 1 diabetics have a propensity to ketosis but not type 2 diabetics with the same levels of blood
sugar control? Is this ketotic tendency
corrected with BCG therapy?
Currently all subjects treated with BCG
have reduced insulin needs. Will some
sort of therapy be added to BCG toeliminate the need for insulin? Options
include oral agents used predominantly in T2D.
BCG’s important blood sugar-lowering effect in T1D is in part magniﬁedby these patients having lowered aerobic glycolysis at baseline. Can BCG in
diseases without this underlying balance defect in energy usage – like,perhaps, T2D – also be of beneﬁt?

16. Scott-Browne, J.P. et al. (2007) Expansion and function of
Foxp3-expressing T regulatory cells during tuberculosis. J.
Exp. Med. 204, 2159–2169
17. Yadav, V. et al. (2015) Understanding the host epigenetics in Mycobacterium tuberculos infection. J. Genet. Genome Res. 2, 016
18. Yaseen, I. et al. (2015) Mycobacteria modulate host epigenetic
machinery by Rv1988 methylation of a non-tail arginine of histone
H3. Nat. Commun. 6, 8922
19. Bettini, M.L. et al. (2012) Loss of epigenetic modiﬁcation driven by
the Foxp3 transcription factor leads to regulatory T cell insufﬁciency. Immunity 36, 717–730
20. Liu, Y. et al. (2014) Two histone/protein acetyltransferases, CBP
and p300, are indispensable for Foxp3+ T-regulatory cell development and function. Mol. Cell. Biol. 34, 3993–4007
21. Bai, G. et al. (2011) Cyclic AMP signalling in mycobacteria:
redirecting the conversation with a common currency. Cell.
Microbiol. 13, 349–358
22. Rehren, G. et al. (2007) Differential gene expression between
Mycobacterium bovis and Mycobacterium tuberculosis. Tuberculosis (Edinb.) 87, 347–359
23. Shi, L. et al. (2016) Immunometabolism in tuberculosis. Front.
Immunol. 7, 150
24. Gleeson, L.E. et al. (2016) Cutting edge: Mycobacterium tuberculosis induces aerobic glycolysis in human alveolar macrophages that is required for control of intracellular bacillary
replication. J. Immunol. 196, 2444–2449

Trends in Endocrinology & Metabolism, February 2019, Vol. 30, No. 2

91

25. Cheng, S.C. et al. (2014) mTOR- and HIF-1a-mediated aerobic
glycolysis as metabolic basis for trained immunity. Science 345,
1250684

38. Pollard, A.J. et al. (2017) Non-speciﬁc effects of vaccines: plausible and potentially important, but implications uncertain. Arch.
Dis. Child. 102, 1077–1081

26. Ryu, S. et al. (2001) Reversal of established autoimmune diabetes
by restoration of endogenous beta cell function. J. Clin. Invest.
108, 63–72

39. McInerney, M.F. et al. (1991) Prevention of insulitis and diabetes
onset by treatment with complete Freund’s adjuvant in NOD
mice. Diabetes 40, 715–725

27. Kodama, S. et al. (2003) Islet regeneration during the reversal of
autoimmune diabetes in NOD mice. Science 302, 1223–1227

40. Sadelain, M.W.J. et al. (1990) Prevention of type I diabetes in
NOD mice by adjuvant immunotherapy. Diabetes 39, 583–589

28. Faustman, D.L. (2014) TNF, BCG, and the proteasome in autoimmunity: an overview of the pathways & results of a Phase I study
in type 1 diabetes. In The Value of BCG and TNF in Autoimmunity(1st edn) (Faustman, D., ed.), pp. 81–104, Academic Press

41. McDevitt, H. et al. (2002) Multiple roles for tumor necrosis factoralpha and lymphotoxin alpha/beta in immunity and autoimmunity.
Arthritis Res. 4, S141–S152

29. Ristori, G. et al. (2014) Effects of bacille Calmette–Guerin after theﬁrst demyelinating event in the CNS. Neurology 82, 41–48
30. Arnoldussen, D.L. et al. (2011) BCG vaccination and allergy: asystematic review and meta-analysis. J. Allergy Clin. Immunol.
127, 246–253.e21
31. Shann, F. (2011) The nonspeciﬁc effects of vaccines and the
expanded program on immunization. J. Infect. Dis. 204, 182–184

42. Huppmann, M. et al. (2005) Neonatal bacille Calmette–Guerin
vaccination and type 1 diabetes. Diabetes Care 28, 1204–1206
43. Karaci, M. and Aydin, M. (2012) The effect of BCG vaccine fromprotection of type 1 diabetes mellitus. J. Contemp. Med. 2, 1–8
44. Shehadeh, N. et al. (1994) Effect of adjuvant therapy on development of diabetes in mouse and man. Lancet 343, 706–707(see comments)

32. Kristensen, I. et al. (2000) Routine vaccinations and child survival:
follow up study in Guinea-Bissau, West Africa. BMJ 321, 1435–1438

45. Allen, H.F. et al. (1999) Effect of bacillus Calmette–Guerin vaccination on new-onset type 1 diabetes. A randomized clinical study.
Diabetes Care 22, 1703–1707

33. Karaci, M. (2014) The protective effect of the BCG vaccine on thedevelopment of type 1 diabetes in humans. In The Value of BCG
and TNF in Autoimmunity (1st edn) (Faustman, D., ed.), pp. 52–
62, Academic Press

46. Hayashi, D. et al. (2009) Comparable studies of immunostimulating activities in vitro among Mycobacterium bovis Bacillus Calmette-Guérin (BCG) substrains. FEMS Immunology and Medical
Microbiology 56, 116–128

34. Kiraly, N. et al. (2015) BCG for the prevention of food allergy –exploring a new use for an old vaccine. Med. J. Aust. 202, 565–566

47. Drouin, P. et al. (2009) Diagnosis and classiﬁcation of diabetes
mellitus. Diabetes Care 32, S62–S67

35. Higgins, J.P. et al. (2016) Association of BCG, DTP, and measles
containing vaccines with childhood mortality: systematic review.
BMJ 355, i5170

48. van Dam, A.D. et al. (2016) BCG lowers plasma cholesterol levels
and delays atherosclerotic lesion progression in mice. Atherosclerosis 251, 6–14

36. Biering-Sørensen, S. et al. (2017) Early BCG-Denmark and neonatal mortality among infants weighing <2500 g: a randomized
controlled trial. Clin. Infect. Dis. 65, 1183–1190

49. Inafuku, M. et al. (2015) Intravenous Mycobacterium bovis bacillus Calmette–Guerin ameliorates nonalcoholic fatty liver disease
in obese, diabetic ob/ob mice. PLoS One 10, e0128676

37. Rieckmann, A. et al. (2017) Vaccinations against smallpox andtuberculosis are associated with better long-term survival: a Danish
case-cohort study 1971–2010. Int. J. Epidemiol. 46, 695–705

50. Kuhtreiber, W.M. et al. (2018) Impact of blood sugar control on
metabolites and carbohydrate metabolism in type 1 diabetes.
Diabetes 67, A473

92

Trends in Endocrinology & Metabolism, February 2019, Vol. 30, No. 2