Available online at www.sciencedirect.com

ScienceDirect
Bridging the gap between vaccination with BacilleCalmette-Guérin (BCG) and immunological tolerance:
the cases of type 1 diabetes and multiple sclerosis
Giovanni Ristori1,5, Denise Faustman2,5, Giuseppe Matarese3,5,
Silvia Romano1 and Marco Salvetti1,4
At the end of past century, when the prevailing view was thattreatment of autoimmunity required immune suppression,
experimental evidence suggested an approach of immunestimulation such as with the BCG vaccine in type 1 diabetes (T1D)
and multiple sclerosis (MS).Translating these basic studies into
clinical trials, we showed the following: BCG harnessed theimmune system to ‘permanently’ lower blood sugar, even inadvanced T1D; BCG appeared to delay the disease progressionin early MS; the effects were long-lasting (years after vaccination)in both diseases.The recently demonstrated capacity of BCG toboost glycolysis may explain both the improvement of
metabolic indexes in T1D, and the more efficient generation of
inducible regulatory T cells, which counteract the autoimmune
attack and foster repair mechanisms.

the sixties [1]. Throughout the next 30 years we andothers obtained several results that reinforced the rationale, contradicting, at least in part, certain beliefs orparadigms on autoimmune disorders. In particular thefollowing evidence prompted us to translate this approachin human neuroinflammation: the resistance to EAE
induction by the pre-immunization with diverse antigens(both non-self and auto-antigens) [2]; the immune deviation toward a T helper 1 cytokine profile (consideredpathogenic) in multiple sclerosis (MS) patients with asatisfactory response to interferon beta therapy [3]; the‘physiological’ role of molecular mimicry, at least in T cell
responses [4,5]; the safety of influenza and other vaccines
in MS [6,7].

Addresses
1
Center for Experimental Neurological Therapies, Sant’Andrea Hospital,
Department of Neurosciences, Mental Health and Sensory Organs,
Sapienza University of Rome, S. Andrea Hospital, Via di Grottarossa
1035, 00189 Rome, Italy
2
Immunobiology Department, Massachusetts General Hospital andHarvard Medical School, Boston, MA 02129, USA
3
Treg Cell Lab, Dipartimento di Medicina Molecolare e Biotecnologie
Mediche, Università di Napoli ‘Federico II’, IEOS-CNR, Via S. Pansini 5,80131 Napoli, Italy
4
IRCCS Istituto Neurologico Mediterraneo (INM) Neuromed,
86077 Pozzilli, IS, Italy

We studied the effect of BCG vaccine in patients withearly MS. A single cross-over magnetic resonance imaging(MRI)-monitored trial [8] was performed on 14 participants. Comparing monthly MRI scans of pre-vaccinationand post-vaccination period we found that MRI activity,
expressed as gadolinium (Gd)-enhanced lesions plus newand enlarging lesions in T2-weighted (T2W) images,
significantly dropped after BCG. No major adverse eventswere reported [9].

Corresponding authors:
Ristori, Giovanni (giovanni.ristori@uniroma1.it),
Salvetti, Marco (marco.salvetti@uniroma1.it)
5Equal contribution.
Current Opinion in Immunology 2018, 55:89–96
This review comes from a themed issue on Autoimmunity
Edited by Stetson

https://doi.org/10.1016/j.coi.2018.09.016
0952-7915/ã 2018 Published by Elsevier Ltd.

BCG vaccine in neuroinflammation: multiple
sclerosis and beyond
The rationale for the use of adjuvant therapy in Experimental Allergic Encephalomyelitis (EAE) dates back towww.sciencedirect.com

In this same cohort of MS patients we evaluated theeffect of BCG vaccine on the evolution of new Gdenhancing lesions to hypointense lesions on T1weighted MRI images through three new MRI scansover 24 months after vaccination [10]. This allowed toquantify the so-called black holes (BH), expression ofirreversible tissue damage, to evaluate the vaccine influence on axonal loss, that underlies the development ofclinical disability [11]. Once again we compared the twophases of the trial (the run-in versus the post-BCG
percentage of NEL that evolve into BH), and found asignificant result: new enhancing lesions at MRI persisted less and less frequently evolved to black holes
after the BCG vaccine. This post-hoc analysis seemed toindicate that vaccination with BCG might decrease thetissue damage. Moreover, the dynamics of BCG effects
over time prompted us to hypothesize its long-term
benefit and to design subsequent studies on a longer
temporal scale.
The suggestion found confirmation in a third study on
subjects with the first demyelinating episode (usually
Current Opinion in Immunology 2018, 55:89–96

90 Autoimmunity

referred to as clinically isolated syndrome—CIS), that is
considered, in the presence of a brain and spinal cord MRI
compatible with MS, the onset of clinical disease [12]. In
these parallel groups trials we met several significant end
points after a single vaccination with BCG in people with
CIS: reduced disease activity with MRI monitoring, lesspropensity to develop black holes at 18 months, lower
occurrence of the second demyelinating event (that isconversion to clinically definite MS) at 60 months, without major adverse event due to vaccine (Figure 1) [13].

The mechanisms of action of BCG vaccine in MS are
not clear. However, clinical observations along withgenetic and functional studies suggest a net beneficial
role of TNF in human neuroinflammation, that may be
deficient in MS patients, and seems to be at variancewith more complex effects seen in EAE [15]. In fact,diseases worsening occurred when therapies antagonizing tumor necrosis factor (TNF) were tried in MS [16],contrary to other autoimmune disorders where antiTNF is beneficial, but bears risks of demyelinating
episodes. Moreover, an MS-associated genetic variant
directs increased expression of a soluble protein thatacts as an analogous of anti-TNF agents [17]; notably,
this variant is not associated with autoimmune disorders
where TNF antagonism results beneficial. A potent
TNF inducer, such as BCG vaccination, may be usefulto rescue TNF deficit in MS, and this is in keeping withevidences coming from type 1 diabetes (see next
section).

Underway currently is our trial with the BCG vaccine inpeople with ‘radiologically isolated syndromes’ (RIS): a
new subclinical entity, that occurs when an MRI of thecentral nervous system shows results compatible withMS, but without any symptom or sign of disease [14].
The trial stemmed from reasoning that safe and manageable approaches, that can be used with the (biological)
onset of the disease, are important unmet needs in MS. In
fact, the last-decade therapeutic progress for the overt
relapsing-remitting form was reached at the cost of safety,
quality of life and overtreatment. Being safe, cheap andavailable, the BCG vaccine seemed appropriate as a frontline immune-modulatory approach for people with RIS.

Another promising field for adjuvant approach seems tobe that of more common neurological disorders, such as
Alzheimer’s and Parkinson disease, classically considered
of degenerative nature. Recent evidences consistently

Figure 1

(a)

(b)

2

1,0

Placeboplacebo + DMT
BCG + DMT

Cumulative survival of free relapse

Mean changes (±SEM) in total T-1 hypontense
lesions including ‘black holes’

BCG

1

0

0,8

0,6

0,4

0
Baseline-month 6

Baseline-month 12

Baseline-month 18

10

20

30

40

50

60

Follow-up (months)
Current Opinion in Immunology

Long-term effects of a single BCG vaccination in people with first demyelinating episode. (a) Mean change in the total T1-hypointense lesions,
including ‘black holes’ that express irreversible tissue damage, over 18 months. (b) Proportion of people free from the second clinical event (thatis not converting to clinically definite multiple sclerosis) over 60 months.

Current Opinion in Immunology 2018, 55:89–96

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Bridging the gap between vaccination with Bacille Calmette-Guérin (BCG) and immunological tolerance Ristori et al. 91

showed a neuroinflammatory component in these diseases, with a prevalent role for glial cells and innate
immune system [18]. In this context a neuroprotective
action of BCG vaccine emerged in experimental modelsof neurodegenerative diseases. Two works in models of
PD suggested a BCG-mediated immune stimulation, that
may limit deleterious microglial response to a neuronal
insult [19], may promote T regulatory protective
responses [20]. Other two works in models of AD elucidated additional mechanisms of neuroprotection: activation of tumor necrosis factor receptor 2 (TNFR2), thatblock neuroinflammation and promote neuronal survival[21], as well as recruitment of inflammation-resolving
monocyte (producing anti-inflammatory cytokines andneurotrophic factors) at the choroid plexus and perivascular spaces of plaque pathology [22]. Along the same lineof research several works showed a beneficial effect of
neonatal BCG vaccination on neurodevelopment and on
counteracting neurobehavioral impairment and neuroinflammation due to secondary immune challenge in adultmice [23,24].
BCG and type 1 diabetes

Our 20-year journey towards using BCG to treat type1 diabetes (T1D) started off in a paradoxical direction.
Studying both human and mouse models, we discovered Tlymphocyte (T cell) imbalances, both an overabundance of
pathogenic cytotoxic T cells (CD8 CTLs), and a paucity ofsuppressive T-regulatory (CD4 Treg) cells. Importantly
these immune cell imbalances could be corrected withtumor necrosis factor (TNF), a well-known pro-inflammatory cytokine with immune-stimulatory properties. At thetime, however, the prevailing view was that treatment of
T1D and other forms of autoimmunity required immunosuppression, not immunostimulation, the same belief that
also dominated the multiple sclerosis field.
Through basic science, we discovered that the antigenpresenting cells of T1D patients had interruptions in CD8
T cell education due to a paucity of self-peptides being
generated for the HLA class I groove, a process controlled
by intracellular processing in antigen-presenting cells [25
,26,27]. This was some of early T1D data implementing
CD8 T cells in the disease; prior studies thought CD4 T
cells were the central cells in T1D. The intracellularantigen presenting process normally utilized the TAP
and proteasome genes and this presentation of self antigens
was altered in T1D. Interestingly these HLA class I presenting genes were located in the high-risk HLA region[28–33]. Our research suggested that HLA class I presentation of self-proteins was necessary for tolerance [30]. With
proteasome dysfunction, additional intracellular processing
defects were also detected in T1D. NFkB activation, aproteasome-dependent process for T cell education pathways, was also dysfunctional [34,35]. TNF is the cytokine
that normally stimulates the NFkB signaling pathway. This
TNF-stimulated pathway promotes antigen presentation,www.sciencedirect.com

T cell maturation and proper education of T cells. Interruptof this pathway would be predicted to cause autoimmunity.
If there was perhaps too little TNF or at least sluggish
TNF-mediated signaling in the immune system, could
something as simple of adding back TNF be therapeuticfor T1D? Indeed, TNF selectively killed autoreactive T
cells causing T1D and multiple sclerosis and also in otherautoimmune diseases such a multiple sclerosis antigen
presenting defects in HLA class I have been reported[36–39]. The data started to accumulate that this inflammatory cytokine, TNF might be therapeutic. Animal models of autoimmunity also demonstrated the therapeutic
benefit of TNF. Disease causing cytotoxic T cells, harvested from the spleens of diabetes affect mice, normallytransfer disease to naı̈ve animals. If these splenocytes werefirst in vitro treated with TNF, disease transfer was prevented [36]. TNF itself or TNF induction with the BCG
vaccine was found to prevent onset as well as reverse evenend-stage disease in the NOD mouse model when administered in vivo [40,41]. This suggested that autoimmune
diseases needed TNF, not immunosuppression, as a therapeutic approach (Figure 2). As further refinements ofthese experiments continued, TNF or TNF stimulation
through the TNFR2 receptor was the central pathway for
disease modification [42,43].
Translating these basic studies into clinical trials of T1D
could be accomplished by two routes: manufacture andstart a TNF drug program (TNFR2 agonistic antibodies
were not available at the time) or develop and recycle asafe 100 year-old drug, BCG, an inactive version of
Mycobacteria bovis originally developed to protect fromtuberculosis and a known inducer of TNF. Because ofmonetary limitations, we decided to launch a T1D trial
with BCG as a method to induce and restore TNF.
In 2012, we published the Phase I results from multidosing BCG in long-term T1Ds (>10 years disease duration) [44]. This was a safety trial that also studied
important biomarkers of efficacy. We established that
BCG was safe in this patient population, and showed
consistent trends with the proposed mechanism of actionof BCG: death of autoreactive T cells, induction of Tregs,
and minor increase of C-peptide secretion (a marker of
insulin production by the pancreas), suggesting some
restored activity [44]. In support of BCG in T1D, anepidemiology study from Turkey has since showed thatmulti-dosing BCG may not only be beneficial in T1D
treatment, but also in its diabetes prevention. Participants
who received greater than 2 childhood vaccines of BCG
had diminished lifelong risk for developing T1D [45].
Our 12-week-long Phase 1 trial showed that C-peptide
production was not increased in a clinically meaningful
way, although seeing any restored C-peptide in T1D withthis long standing disease was unusual. We asked, had wefollowed our patients long enough? We subsequently
Current Opinion in Immunology 2018, 55:89–96

92 Autoimmunity

Figure 2

BCG
Treg

CTL

TL
Treg
CTL
CTL

TNF

CTL
CTL
CTL

Treg

CTL

CTL

Treg
Treg
Treg

Treg

UnhealthyTreg
Autoimmune target tissue

CTL

Cytotoxic T cell

CTL

Cytotoxic T cells

Treg

Activated TNFR2 Treg

CTL
Current Opinion in Immunology

Immune mechanisms for the action of TNF induction through BCG. BCG through direct TNF induction or TNFR2 agonism on infected antigen
presenting cells can directly and selectively kill cytotoxic T cells and proliferate Treg cells, thus re-establish immune tolerance.

learned from the multiple sclerosis trials by Ristori et al. that
BCG’s benefit, while slow to start, showed measurable
disease improvement over the next 2–3 years. With thisin mind, we re-opened our Phase I trial to follow our T1D
patients for another 8 years. Similar to the timeframe withmultiple sclerosis, we found that, by year 3, blood sugar
began to drop and hemoglobin A1c (HbA1c) gradually
returned to near normal levels. This slow-to-materialize,
but remarkable, correction of blood sugars towards normal
by BCG persisted for 5 more years [46]. Repeat BCG
vaccines were a powerful way to harness the immune
system and alter metabolism to ‘permanently’ lower blood
sugars, even in advanced TID. In 2018, we showed that themechanism for better blood sugar control by BCG was due
to a systemic shift in metabolism from oxidative phosphorylation to aerobic glycolysis, a metabolic state with accelerated transport and utilization of serum sugar for energy.
BCG therapy in human autoimmunity thus opened up theexciting possibility of a safe and an effective vaccine forresetting the immune system even in existing T1D diseasestates. The BCG vaccine confirmed and verified an entirely
novel way to regulate blood sugars. Fully enrolled andongoing human clinical trials continue to test the validityof BCG lowering of blood sugars in large patient populations and unlike most immune intervention trials is T1D,
the BCG vaccine was potent enough to have these
Current Opinion in Immunology 2018, 55:89–96

beneficial clinical effects in patients with long standing
diabetes. Before this time, immune interventions were
exclusively tried in only new onset T1D, a clinical setting
thought easier to reverse.

Immunometabolic effects of BCG in
autoimmunity
BCG, as therapeutic opportunity to induce immunological tolerance, is a very attractive one. At mechanistic level,
despite compelling experimental evidence suggests thecapacity of BCG to affect immune responses, the cellularand molecular determinants controlling this process arestill not fully understood. Originally, BCG was considereda powerful tool to stimulate immunity against Mycobacterium tuberculosis (MTB); indeed BCG infection is characterized by a classical strong induction of Th1/Th17
responses in immunized individuals, able to exert protection against MTB infection, particularly in very young
individuals. The induction of this type of response is in‘apparent contrast’ with the capacity of BCG to protectfrom induction of mouse EAE and the development of
T1D in the NOD mouse, both models of disease caused
by pro-inflammatory Th1/Th17 switches [47,48].
A possible explanation for this ‘apparent paradox’ comesfrom recent studies linking immunometabolic pathways
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Bridging the gap between vaccination with Bacille Calmette-Guérin (BCG) and immunological tolerance Ristori et al. 93

to the capacity of BCG to induce glycolysis in T cells andinnate immune cells such as monocytes/macrophages[49]. Glycolysis fuels proliferation and differentiationof CD4+ T cells towards Th1 and Th17 responses, but it isalso necessary to generate specific populations of inducible regulatory T cells (iTreg) cells from conventional T(Tconv) cells in humans [50]. While differentiated Treg
cells show a mixed metabolic profile being able to engage
both glycolysis and lipid oxidation, engagement ofglycolysis appears to be necessary to generate iTreg cells
from Tconv cells [50,51]. Specifically, this process
appeared tightly linked with the timing and strength of
T cell receptor (TCR) engagement; indeed only TCR
stimulation determined a consistent engagement of glycolysis able to sustain the appropriate induction andmaintenance overtime of the Foxp3 gene, whose expression is crucial for Treg cell maintenance and suppressivefunctions [50,51,52].

Figure 3

Autoimmunity(a)DamageTh1 cells

Treg cells

Tconv cells

Central nervous system
Glycolysis
DamageProliferating
Tconv
Auto Ag-TCR
stimulation

Th17 cells

Low induction of Treg cells andconsequent higher generationof pathologic effector Th subsets

Pancreatic-cells

Immune tolerance(b)Treg cells

Tconv cells
Th17 cells

High glycolysis

Central nervous system
Th1 cells

Auto Ag-TCR
stimulation

BCG
vaccination

Proliferating
Tconv cells

Induction of Treg cells
Control of pathogenic effector
Th subsets

Pancreatic-cells

Current Opinion in Immunology

Hypothetical pro-tolerogenic capacity of BCG vaccination via enhancement of glycolysis. (a) In autoimmune disorders such as MS and T1D there
is a reduced engagement of glycolysis in Tconv cells which leads to reduced iTreg cell induction and consequent enhancement of pro-pathogenic
Th1/Th17 responses. (b) BCG vaccination is able to boost engagement of glycolysis in Tconv cells upon TCR activation and consequently
increase iTreg cell generation which in turn control pro-pathogenic Th1/Th17 responses.
deleterious microglial response to a neuronal insul
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Current Opinion in Immunology 2018, 55:89–96

94 Autoimmunity

It is also interesting to note that autoimmune disorders
such as MS and T1D associate with reduced engagementof glycolysis by T cells [50,53]. From these considerations, it is possible to speculate that glycolysis is part ofa ‘double edged’ process in which while effector
responses towards BCG require glucose oxidation to
warrant Th1/Th17 differentiation, also iTreg cells generate in the same process, whose function is necessary tocontrol the inflammatory process and avoid possible‘collateral damage’ during inflammation driven by
BCG infection. Thus, with this view, immunologicaltolerance could be considered as an inducible processduring ongoing immune responses, and thus infections
and inflammatory processes that consistently engage
glycolysis warrant also a better induction of Foxp3+ T
cells which in turn control inflammation and tissue
damage (Figure 3). These cells, can contribute toimprovement of inflammation in autoimmune diseases
such as MS and T1D, but also could enhance tissueremodeling and regeneration in both myelin and betacells, through mediators such as TGF-b. It is clear thatfurther research is needed to better dissect at cellularand molecular level this hypothesis, but the ongoingdirection of current basic and pre-clinical research ishighly suggestive of this novel concept.
We ‘converged’ on the clinical evaluation of BCG in MS
and T1D from different premises. This is remarkable, as
it supports the broad rationale that a benign exposure to
microbes (especially the ‘old friends’ that have coevolved with human beings for millennia) [54] may help
antagonize conditions sharing chronic inflammation andtissue damage. The beneficial effect of BCG vaccination,
at least in some autoimmune disorders, adds an intriguing
viewpoint for studying the host-microbe interplay in thepathophysiology and treatment of immune-mediateddiseases.

Conflict of interest statement
Nothing declared.

Acknowledgements
We thank Fortunata Carbone for figures.
Fundings: GM is supported by the European Research Council“menTORingTregs” grant no. 310496, the Fondazione Italiana Sclerosi
Multipla grant no. 2016/R/18 and the Telethon grant no. GGP17086. GR is
supported by Fondazione Italiana Sclerosi Multipla, grant no. 2016/S/
1. DLF is supported by The Iacocca Foundation.

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96 Autoimmunity

49. Arts RJW, Carvalho A, La Rocca C, Palma C, Rodrigues F,
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trained immunity. Cell Rep 2016, 17:2562-2571.
This is a key report showing that BCG is able to boost glycolysis in T cells.

52. De Rosa V, La Cava A, Matarese G: Metabolic pressure and thebreach of immunological self-tolerance. Nat Immunol 2017,
18:1190-1196.
This paper suggests metabolism as key novel determinant in the controlof immune tolerance to self.

50. De Rosa V, Galgani M, Porcellini A, Colamatteo A, Santopaolo M,
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et al.: Glycolysis controls the induction of human regulatory T
cells by modulating the expression of FOXP3 exon 2 splicing
variants. Nat Immunol 2015, 16:1174-1184.
This is the first paper linking glycolysis engagement and induction of iTreg
cells in humans, particularly those containing the Foxp3 exon2 splicing
variants.

53. La Rocca C, Carbone F, De Rosa V, Colamatteo A, Galgani M,
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in glycolysis and mitochondrial respiration. Metabolism 2017,77:39-46.
This is the first report showing that in MS there is a reduced engagementof glycolysis which is reversible by treatment with beta-interferon.

51. Procaccini C, Carbone F, Di Silvestre D, Brambilla F, De Rosa V,Galgani M, Faicchia D, Marone G, Tramontano D, Corona M et al.:The proteomic landscape of human ex vivo regulatory andconventional T cells reveals specific metabolic requirements.
Immunity 2016, 44:406-421.
This report, suggests that ex vivo human Treg cells engage glycolysis andwhen put in vitro change dynamically their metabolism engaging bothglycolysis and lipid synthesis.

54. Reber SO, Siebler PH, Donner NC, Morton JT, Smith DG,
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stress resilience in mice. Proc Natl Acad Sci U S A 2016, 113:
E3130-3139.

Current Opinion in Immunology 2018, 55:89–96

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