A icle
S onge induc ion o ained immuni y by mucosal
BCG o MTBVAC accina ion compa ed o s anda d
in ade mal accina ion
G aphical Abs ac
Highligh s
dNonhuman p ima es ecapi ula e ained immuni y upon li e
a enua ed TB accina ion
dIn a enous BCG induces changes in H3K27 ace yla ion and
enhances cy okine p oduc ion
dMucosal BCG imp o es induc ion o ained immuni y o
monocy es o e in ade mal BCG
dThe M. ube culosis-de i ed candida e accine MTBVAC
appea s equally po en as BCG
Au ho s
Michel P.M. Vie boom, Ka in Dijkman,
Claudia C. Somb oek, ..., Nacho Aguilo,
Ca los Ma in, F ank A.W. Ve eck
Co espondence
ie boom@bp c.nl
In B ie
Vie boom e al. demons a e he
induc ion o ained immuni y in blood
and bone ma ow monocy es a e
accina ion wi h li e a enua ed TB
accines in nonhuman p ima es. Mucosal
espi a o y deli e y o BCG o MTBVAC
induces ained immuni y mo e e icien ly
compa ed o s anda d in ade mal
accina ion.
Vie boom e al., 2021, Cell Repo s Medicine 2, 100185
Janua y 19, 2021 ª2020 The Au ho (s).
h ps://doi.o g/10.1016/j.xc m.2020.100185 ll
A icle
S onge induc ion o ained immuni y
by mucosal BCG o MTBVAC accina ion
compa ed o s anda d in ade mal accina ion
Michel P.M. Vie boom,
1,8,
*Ka in Dijkman,
1
Claudia C. Somb oek,
1
Sam O. Ho man,
1
Cha elle Boo ,
1
Richa d A.W. Ve enne,
1
K is a G. Haans a,
1
Maa en an de Sande,
2
Liesbe h an Ems ,
3
Jo ge Domı
´nguez-And e
´s,
3
Simone J.C.F.M. Moo lag,
3
Clemens H.M. Kocken,
1
Jelle Thole,
4
Es eban Rod ı
´guez,
5
Eugenia Puen es,
5
Joos H.A. Ma ens,
2
Reinou an C e el,
3
Mihai G. Ne ea,
3
Nacho Aguilo,
6,7
Ca los Ma in,
6,7
and F ank A.W. Ve eck
1
1
Biomedical P ima e Resea ch Cen e, Rijswijk, he Ne he lands
2
Depa men o Molecula Biology, Facul y o Science, Nijmegen Cen e o Molecula Li e Sciences, Radboud Uni e si y, Nijmegen, he
Ne he lands
3
Radboud Uni e si y Medical Cen e, Nijmegen, he Ne he lands
4
TuBe culosis Vaccine Ini ia i e, Lelys ad, he Ne he lands
5
Bio ab i, Pon e ed a, Spain
6
Depa men o Mic obiology, Facul y o Medicine, IIS A ago
´n, Uni e si y o Za agoza, Za agoza, Spain
7
CIBERES, Ins i u o de Salud Ca los III, Mad id, Spain
8
Lead con ac
*Co espondence: ie boom@bp c.nl
h ps://doi.o g/10.1016/j.xc m.2020.100185
SUMMARY
BCG accina ion can s eng hen p o ec ion agains pa hogens h ough he induc ion o epigene ic and me a-
bolic ep og amming o inna e immune cells, a p ocess called ained immuni y. We and o he s ecen ly
demons a ed ha mucosal o in a enous BCG be e p o ec s hesus macaques om Mycobac e ium
ube culosis in ec ion and TB disease han s anda d in ade mal accina ion, co ela ing wi h local adap i e
immune signa u es. In line wi h p io mouse da a, he e, we show in hesus macaques ha in a enous BCG
enhances inna e cy okine p oduc ion associa ed wi h changes in H3K27 ace yla ion ypical o ained immu-
ni y. Al e na i e deli e y o BCG does no al e he cy okine p oduc ion o un ac iona ed b onchial la age
cells. Howe e , mucosal bu no in ade mal accina ion, ei he wi h BCG o he M. ube culosis-de i ed
candida e MTBVAC, enhances inna e cy okine p oduc ion by blood- and bone ma ow-de i ed monocy es
associa ed wi h me abolic ewi ing, ypical o ained immuni y. These esul s p o ide suppo o s a egies
o imp o ing TB accina ion and, mo e b oadly, modula ing inna e immuni y ia mucosal su aces.
INTRODUCTION
Canonical accinology aims a he induc ion o long-li ed adap-
i e memo y in an igen-speci ic lymphocy e popula ions and
T and/o B cells. Inna e immuni y in accinology is p ima ily
acknowledged in he con ex o accine o mula ion and he
quin essen ial inna e immune ac i a ion o p o essional
an igen-p esen ing cells (APCs) ha will p ime and skew he
adap i e esponse o lymphocy es.
1
Fo yea s, inna e immune
esponses and myeloid monocy e/mac ophage unc ion we e
conside ed cons an and in a iable o e ime, bu i is now widely
ecognized ha also inna e immune cells can adap and display
memo y-like pheno ypes. This inna e immune ‘‘memo y’’ is ypi-
cally e e ed o as ained immuni y.
2
Induc ion o ained immuni y is de ined by a unc ional adap-
a ion o inna e immuni y a e a p ima y insul esul ing in a mo e
e ec i e esponse upon a ‘‘seconda y’’ encoun e wi h an un e-
la ed pa hogen.
2
I is cha ac e ized by inc eased cy okine and/o
chemokine p oduc ion,
3
me abolic ewi ing,
4
and epigene ic e-
p og amming o inna e immune cells.
5
T ained immuni y is no
associa ed wi h he cha ac e is ic immune ecep o gene ea -
angemen s ha a e associa ed wi h he an igen speci ici y o
adap i e memo y, bu a he is d i en by he epigene ic con ol
o speci ic immune gene clus e s esul ing in he up egula ion
o pa icula p o-in lamma o y cy okines.
6
An epigene ic ma k
in pa icula associa ed wi h ained immuni y in monocy es,
and used in he p esen s udy, is he ace yla ion o his one 3 a
he posi ion o lysine 27 (H3K27ac).
7
Recen wo k in mice has
shown ha Bacillus Calme e-Gue
´ in (BCG)-media ed aining
o he myeloid monocy e/mac ophage lineage is sus ained by
he epigene ic ep og amming o hema opoie ic s em cells
(HSCs), he myeloid p ecu so s in bone ma ow (BM).
8
Mycobac e ium bo is-de i ed BCG, cu en ly he only a ail-
able accine o igh ube culosis (TB),
9
is he p o o ypical bio-
logical agen o which ained immuni y has been demons a ed.
T ained immuni y has been linked o he bene icial he e ologous
o - a ge e ec o in an BCG accina ion, which om epidemi-
ological analyses appea s associa ed wi h educed childhood
Cell Repo s Medicine 2, 100185, Janua y 19, 2021 ª2020 The Au ho (s). 1
This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
ll
OPEN ACCESS
mo ali y om causes o he han TB.
10–13
Eme ging e idence
suppo s he hypo hesis ha no only adap i e memo y bu
also he memo y-like plas ici y in inna e immuni y posi i ely con-
ibu es o immune p o ec ion agains and ea ly clea ance o
M. ube culosis (M b) in ec ion.
14,15
Al hough no es ablished
as ea u es o inna e aining, a compa a i e analysis in macaque
species sugges s ha ma ke s o inna e immuni y and myeloid
monocy e unc ion a e associa ed wi h di e en ial TB disease
suscep ibili y.
16
Despi e he widesp ead use o li e a enua ed BCG as p ophy-
laxis agains TB, i is only pa ially e icacious and by and la ge
ails o p o ec adolescen s and adul s om he in ec ious
pulmona y mani es a ion o M b in ec ion.
17
Thus, TB con inues
o be a majo h ea o global human heal h, and a be e
accina ion s a egy is u gen ly needed o elie e he bu den o
TB.
18,19
While in ade mal injec ion is he s anda d ou e o
BCG accina ion, his o ical da a in hesus macaques ha e shown
ha mucosal o in a enous adminis a ion p o ided supe io sig-
nals o p o ec ion agains expe imen al in ec ion.
20
We ha e
ecen ly demons a ed ha pulmona y mucosal BCG accina ion
educed disease in a coho o hesus macaques in which in a-
de mal BCG ailed o show any p o ec i e e ec .
21
In a subse-
quen s udy, exploi ing epea ed limi ing dose (RLD) a he han
single dose M b challenge, we ha e shown ha mucosal bu
no in ade mal BCG p o ides signals o p e en ion o in ec ion.
22
Mos ecen ly, Da ah and colleagues
23
ha e co obo a ed he su-
pe io p o ec i e capaci y o BCG when gi en in a enously. Bo h
s udies did no iden i y an immune bioma ke in he blood ha
co ela ed wi h he p o ec i e e icacy o ei he mucosal o in a-
enous BCG accina ion. Locally, howe e , an igen-speci ic
in e leukin-17A-posi i e (IL-17A
+
) poly unc ional T helpe (Th)
cells and IL-10 sec e ion in he ai ways did co ela e wi h p o ec-
ion,
22
and he a icle by Da ah e al.
23
sugges s ha issue- esi-
den lymphocy es may be pi o al in con e ing p o ec ion. While
om hese non-human p ima e (NHP) s udies uncon en ional
adminis a ion o BCG appea s o be a mos in e es ing lead o
imp o ing ou cu en TB p ophylaxis, i emains o be es ab-
lished, howe e , i and how al e na i e ou ing o BCG a ec s
inna e and ained immuni y.
Va ious candida e TB accines, anging om subuni o mula-
ions o li e a enua ed and ecombinan mycobac e ia, a e a
a ious s ages o clinical es ing in he cu en accine-de elop-
men pipeline (www. b i.eu/wha -we-do/pipeline-o - accines/).
O he li e a enua ed candida es, MTBVAC is unique in ha i
is de i ed om a clinical isola e o M b (M 103) a he han
om M. bo is (which is he pa en al s ain o BCG and ecombi-
nan BCG candida es).
24,25
MTBVAC is a enua ed by dele ion o
he phoP and he adD26 gene. The eby, he exp ession o i u-
lence ac o s is c i ically dis up ed, while an in insically b oade
an igen epe oi e in compa ison o BCG is p ese ed.
26
Aguilo
e al.
27
demons a ed ha he exp ession o ESAT6 and CFP10
by MTBVAC is key o i s p o ec i e capaci y in a mouse models
o TB. MTBVAC has shown p eclinical e icacy be e han BCG
in animal models.
24,27
MTBVAC accina ion p o ec s hesus
macaques agains ae osol challenge wi h M b and induces im-
mune signa u es analogous o hose obse ed in clinical
s udies.
28
I has es ed sa e and immunogenic in adul and in an
human popula ions so a .
9,29
Simila o BCG, MTBVAC is able o
gene a e ained immuni y h ough he induc ion o glycolysis
and glu aminolysis, and he accumula ion o his one me hyla ion
ma ks a he p omo e s o p oin lamma o y genes. Recen ly,
Ta anco
´n e al.
30
showed ha MTBVAC-induced he e ologous
p o ec ion agains a le hal challenge wi h S ep ococcus pneu-
moniae in an expe imen al mu ine model o pneumonia.
In he p esen s udy, we sough o in es iga e he impac o
li e a enua ed mycobac e ial accina ion ia he espi a o y
mucosa on ained inna e immuni y in NHPs. To his end,
we ollowed a 2 32 ac o ial design s a egy o an immuno-
genici y analysis in adul hesus macaques, accina ing ani-
mals by in ade mal injec ion o by endob onchial ins illa ion
ei he wi h BCG o wi h MTBVAC. We con i med he dis inc-
i e adap i e esponse p o ile in he ai ways upon pulmona y
mucosal deli e y o BCG and MTBVAC, and analyzed he
p oduc ion o inna e and adap i e cy okines a e in i o
s imula ion o cells ob ained om lung, blood, and BM. We
pilo ed he induc ion o ained immuni y a e in a enous
BCG injec ion, demons a ing he inc eased acquisi ion o
H3K27ac ma ks and esul ing in p ominen inc eased cy o-
kine p oduc ion a e he e ologous s imula ion in monocy es
isola ed om blood and BM. We subsequen ly demons a ed
ha espi a o y mucosal adminis a ion o li e a enua ed
mycobac e ial accines mo e e icien ly induced ained im-
muni y in blood and BM monocy es, compa ed o in ade mal
immuniza ion, whe eby MTBVAC was equally po en as BCG.
Ou indings unde pin he inna e immune s imula o y po en ial
o he candida e TB accine, MTBVAC. Fo li e a enua ed
mycobac e ial accines in gene al, hey sugges he enhance-
men o inna e immune aining ia espi a o y mucosal ac-
cine adminis a ion.
RESULTS
In he p esen s udy, we se ou o in es iga e whe he
mucosal adminis a ion o li e a enua ed TB accines in he-
sus macaques could modula e inna e immune esponses and
induce ained immuni y. To his end, we selec ed heal hy,
pu pose-b ed adul hesus macaques (Macaca mula a),
bo h males and emales, and s a i ied hem in o compa able
g oups o 6 animals each (Table S1). By andom assignmen
in a 2 32 ac o ial design s a egy, hese g oups ecei ed
ei he a s anda d human dose o BCG o he equi alen
dose o he li e a enua ed candida e accine, MTBVAC, by
in ade mal injec ion (BCG.id; MTBVAC.id) o by endob on-
chial ins illa ion in o he lowe igh lung lobe (BCG.muc;
MTBVAC.muc). Fo he assessmen o adap i e and/o inna e
immuni y, ele an samples we e collec ed be o e and a e
accina ion om he ai ways by b onchoal eola la age
(BAL), om he blood by enipunc u e, and om he BM by
needle aspi a ion. Be o e analyzing he in ade mal and
mucosal accina ion g oups, and since i had been epo ed
as an e ec i e esea ch s a egy o measu ing ained immu-
ni y in mice,
8
a sepa a e g oup o 3 hesus macaques we e
accina ed by in a enous BCG injec ion (BCG.i ) o pilo -
ained immuni y p o iling o monocy es (2 weeks a e acci-
na ion). A schema ic diag am o accina ion and sampling
o e ime is displayed in Figu e 1.
2Cell Repo s Medicine 2, 100185, Janua y 19, 2021
A icle
ll
OPEN ACCESS
BCG and MTBVAC a e immunogenic and bo h display an
adap i e immune p o ile speci ic o he ou e o
accina ion
To con i m he e iciency o accina ion, we pe o med a speci ic
in e e on-g(IFN-g) enzyme-linked immunoso ben spo
(ELISpo ) assay a e in i o ecall s imula ion o pe iphe al blood
mononuclea cells (PBMCs) wi h he p o ein-pu i ied de i a i e
(PPD) o M b (o cul u e medium as a nega i e con ol). Fo
bo h BCG and MTBVAC and by ei he ou e o deli e y, we ound
an inc ease in an igen-speci ic IFN-g elease a e p ima y acci-
na ion. In line wi h p e ious obse a ions,
21,22
he accine-
induced IFN-g esponse was highe a e in ade mal o e
mucosal BCG deli e y, and a compa able esponse pa e n
was obse ed wi h MTBVAC (Figu es 2A and 2B). When s imu-
la ing wi h a ecombinan usion p o ein o ESAT6 and CFP10,
wo an igens encoded in he egion o dele ion (RD)1 ha is
absen om M. bo is BCG bu p esen in MTBVAC, posi i e
IFN-g elease signals we e ob ained only om MTBVAC- acci-
na ed animals, as expec ed (Figu e 2C).
To u he con i m he adap i e esponse p o ile ha we egis-
e ed p e iously a e pulmona y mucosal BCG as a co ela e o
he p e en ion o M b in ec ion, we assessed he cy okine p o-
duc ion p o ile o CD4
+
T lymphocy es om he ai ways by
low cy ome y
22
a e ex i o s imula ion wi h PPD. As expec ed,
we de ec ed he mos p ominen le els o poly unc ional Th17
cells (p oducing IL-17A, IFN-g, umo nec osis ac o a
[TNF-a], and IL-2) in BALs ha we e collec ed 8 weeks a e
p ima y accina ion wi h BCG ia he pulmona y mucosal a he
han he in ade mal ou e (Figu e 2D). M b-de i ed MTBVAC
was ound o induce a simila esponse pa e n and o be a leas
as po en as BCG. Rega dless o he accine, signi ican
numbe s o poly unc ional Th17 cells we e also eco e ed om
he lowe le lung lobe (i.e., opposi e o he si e a ge ed by en-
dob onchial immuniza ion), sugges ing ha he poly unc ional
Th17 esponse is no con ained o he si e o accina ion bu dis-
semina es locally.
Es ablishing ained immuni y in monocy es a e
in a enous immuniza ion wi h BCG
To e alua e he induc ion o ained immuni y a e s anda d in-
ade mal e sus mucosal accina ion wi h li e a enua ed TB
accines, we es ablished he easibili y o he de ec ion o
ained immuni y in he nonhuman p ima e model. Fo his, we
immunized h ee animals wi h BCG in a enously, a p o ocol
ha was success ully explo ed in mice by Kau mann e al.
8
To
minimize he in e e ence o a de eloping adap i e immune
esponse while s ill being able o de ec obus inna e immune e-
sponses, we ook samples om di e en compa men s a week
2 a e accina ion. Nex o he analysis o BAL cells om he
lung, we pe o med a mo e selec i e analysis o myeloid
CD14
+
monocy es pu i ied om esh PBMCs (PBMC.mo) and
BM (BM.mo), he p o o ypic cells in which he ained immuni y
pheno ype has been desc ibed. CD14
+
monocy e popula ions
we e ob ained by posi i e selec ion (using magne ic bead-cell
so ing) and ypically eached a pu i y o ±90% (Figu e S1).
Bo h un ac iona ed BAL cells and en iched monocy es om
blood and BM we e subsequen ly analyzed o signals o ained
immuni y a e in i o es imula ion wi h M b-de i ed whole-cell
lysa e (WCL) o he he e ologous Esche ichia coli de i ed Toll-
like ecep o (TLR)-4 agonis lipopolysaccha ide (LPS). A e 24
h, supe na an s we e ha es ed, ozen, and analyzed la e o
cy okine p oduc ion, indica i e o ained inna e immuni y,
including TNF-a, IL-6, and IL-1b.
A e in a enous BCG accina ion, we ound ma ginal sup-
p ession o cy okine sec e ion, i any e ec a all, a e compa ing
M b o LPS s imula ion o BAL (Figu es 3A and 3B). S imula ion o
PBMC.mo and BM.mo wi h M b e ealed ma kedly inc eased
le els o TNF-a2 weeks a e BCG.i (Figu e 3A), bu no appa en
modula ion o IL-6 and IL-1bsec e ion le els. Howe e , he e ol-
ogous s imula ion wi h LPS demons a ed p ominen inc eased
p oduc ion o all h ee cy okines by bo h monocy es om he
blood and BM (wi h he excep ion o a single animal wi h high
baseline le els in BM.mo; Figu e 3B, PBMC.mo and BM.mo).
Me abolic ewi ing, speci ically a swi ch om oxida i e phos-
pho yla ion o glycolysis unde ae obic condi ions,
4
has been
desc ibed as an ini ia ing e en unde lying ained immuni y.
The e o e, we measu ed lac a e, a me aboli e o glycolysis,
le els in he supe na an s o s imula ed blood- and BM-de i ed
monocy es as a su oga e ma ke o me abolic ewi ing, and
hus ained immuni y. Lac a e p oduc ion was obse ed o
2 o 3 LPS-s imula ed BM.mo, bu unde ec able o PBMC.mo
(Figu e 3C).
Ul ima ely, we de e mined whe he BCG.i accina ion e-
sul ed in epigene ic ep og amming, ano he hallma k o ained
inna e immuni y, in blood-de i ed monocy es (PBMC.mo).
The e o e, we examined he dynamics o his one modi ica ion
H3K27ac (an epigene ic ma k associa ed wi h ac i e ch oma in
in he con ex o ained immuni y
7
) a e BCG.i by ch oma in
immune p ecipi a ion, ollowed by high- h oughpu sequencing
(ch oma in immunop ecipi a ion sequencing [ChIP-seq]) o
assess he changes associa ed wi h BCG.i accina ion.
H3K27ac changes we e de ec ed a many loci (Figu es 3D–3F).
In o al, we de ec ed 792 egions inc eased and 646 egions
dec eased in H3K27ac (Figu e 3D). While he egions showing
inc eased ace yla ion we e associa ed wi h genes ela ed o
he cell cycle, signaling, and ac i a ion o he immune esponse,
Figu e 1. Schema ic ep esen a ion o he
imeline o accina ion in he di e en acci-
na ion s a egies
The BCG.i g oup (n = 3) was analyzed a weeks 1
and 2. The g oups ha we e immunized wi h BCG.id
(n = 6), BCG.muc (n = 6), MTBVAC.id (n = 6), and
MTBVAC.muc (n = 6) we e analyzed a weeks 1
and 8. Only om mucosally accina ed animals BAL
was collec ed om he igh lung as well as he le
lung. See also Table S1.
Cell Repo s Medicine 2, 100185, Janua y 19, 2021 3
A icle ll
OPEN ACCESS
egions dec eased in ace yla ion we e associa ed wi h genes
in ol ed in cellula di e en ia ion (Figu e 3E). Examples o
bo h an ac i e egion, IFN egula o y ac o 3 (IRF3), and sup-
p essed egion, syndecan 2 (SDC2) a e gi en (Figu e 3F). These
da a a e in line wi h accina ion-induced changes in he ace y-
lome and he cellula p og am ela ed o he induc ion o ained
immuni y.
P o-in lamma o y cy okine p oduc ion a e s imula ion
o BAL cells wi h M b o LPS
We hen sough o compa e he induc ion o ained immuni y a -
e s anda d in ade mal e sus mucosal accina ion wi h he li e
a enua ed TB accines BCG and MTBVAC. We analyzed cy o-
kine/chemokine p oduc ion in BAL cells a e ex i o s imula ion
wi h homologous M b WCL o he e ologous LPS. Using a
ailo ed mul iplex ki o cy okine and chemokine measu emen ,
we assessed he p oduc ion o molecules ha ha e p e iously
been associa ed wi h lymphocy e ac i a ion and unc ion
(IFN-g, IL-2, IL-17A), in lamma ion, leukocy e di e en ia ion,
mig a ion, and/o wi h ained immuni y (TNF-a, IL-6, IL-1b,
g anulocy e-mac ophage-colony-s imula ing ac o [GM-CSF],
mac ophage in lamma o y p o ein 1a[MIP-1a; CCL3], and IFN-
g-induced p o ein 10 [IP-10; CXCL10]).
3,11,31
In line wi h he
low cy ome ic de ec ion o poly unc ional Th17 cells, we ound
signi ican ly ele a ed le els o IL-17A, IFN-g, IL-2, and TNF-a
sec e ion om M b WCL-s imula ed BAL cells—in pa icula
a e mucosal accina ion wi h BCG o MTBVAC (Figu e 4A). A
signi ican inc ease in he sec e ion o IL-2 and TNF-a(bu no
IL-17A and IFN-g) was also measu ed a e in ade mal accina-
ion. As he esponse le els o BCG and MTBVAC in his ega d
we e indis inguishable, we ha e depic ed esul s and pe o med
s a is ical analyses on he 12 animals o bo h BCG and MTBVAC
ABC
D
Figu e 2. Mucosal accina ion es ablishes a unique local adap i e signa u e de ined by poly unc ional Th17 cells
(A–C) Indi idual IFN-gELISpo esponses a e in i o ecall s imula ion o PBMCs agains PPD (A) be o e accina ion o (B) 8 weeks a e accina ion o wi h (C)
ESAT6-CFP10 8 weeks a e accina ion. Ho izon al lines in ba s indica e g oup medians; n = 6 animals/g oup. ANOVA adjus ed o mul iple compa isons; Dunn’s
mul iple compa ison es .
(D) The pe cen ages o poly unc ional IL-17A
+
PPD-speci ic CD4
+
T cells (also p oducing IFN-g, TNF-a, and IL-2) we e de e mined by low cy ome ic analysis o
BAL cells be o e (PRE) and 8 weeks a e in ade mal (.id) o mucosal (.muc) accina ion. Cells we e ypically collec ed om he lowe igh lung lobe (R); a ge ed
o accina ion) and o mucosally accina ed animals also om he le lung (L) o es ablish dissemina ion o he immune esponse. The da a a e p esen ed as
medium con ol co ec ed (cc); n = 6 animals/g oup; Wilcoxon ma ched-pai s signed ank es . ULD, uppe limi o de ec ion; LLD, lowe limi o de ec ion.
See also Figu e S7.
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AB
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E
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a ms oge he , pe ou e o adminis a ion. The elease o p o-in-
lamma o y IL-6, a e in i o s imula ion wi h M b WCL,
compa ing p e- and pos - accina ion ime poin s, was a he
he e ogeneous. In con as , IL-1b, GM-CSF, and MIP-1a
(CCL3) sec e ion was, by end o wi h s a is ical signi icance,
dec eased in mucosally accina ed animals (Figu e 4A). CXCL-
10 (IP-10), howe e , was ma kedly and signi ican ly inc eased
upon M b WCL s imula ion, ega dless o he na u e o he ac-
cine o he immuniza ion ou e.
This cy okine/chemokine sec e ion p o ile o BAL cells a e
s imula ion wi h a p epa a ion as c ude as he WCL o M b, con-
aining bo h p o ein an igens and (non-p o ein) inna e ecep o
ligands, is he ne esul o adap i e and inna e ac i a ion o all
subse s ha a e p esen in he un ac iona ed BAL cell popula-
ion. In addi ion, i mus be no ed ha he ela i e abundance
o immune cell subse s in BAL is a ec ed by pulmona y mucosal
bu no by in ade mal BCG accina ion owa d a ela i e in-
c ease in T lymphocy es o e al eola mac ophage (Figu e S2).
To a leas ci cum en he adap i e componen o his esponse
p o ile and owa d he assessmen o ained immuni y by he e -
ologous inna e s imula ion, we also s imula ed BAL cells wi h
LPS o measu e he ensuing cy okine p oduc ion capaci y.
Upon LPS, and in con as o M b WCL s imula ion, IL-17A,
IFN-g, and TNF-aconcen a ions a e accina ion we e no
di e en om wha was ound a baseline (Figu e 4B). Mos likely
as a esul o he bys ande ac i a ion o T lymphocy es speci -
ically ec ui ed in o he ai ways a e mucosal accina ion, IL-2
elease upon LPS s imula ion was inc eased wi h BAL cells
om mucosally accina ed animals (Figu e 4B), bu o a lesse
ex en compa ed o M b WCL s imula ion (Figu e 4A). Also
compa ed o M b WCL s imula ion, he accina ion-modula ed
GM-CSF sec e ion o BAL cells was los wi h LPS, and ha o
CXCL-10 was much lowe and he e ogeneous (Figu e 4B).
LPS-s imula ed IL-6 le els we e compa ably he e ogeneous as
wi h M b WCL s imula ion. IL-1band MIP-1a, as wi h M b WCL
s imula ion, showed diminished sec e ion upon he LPS s imula-
ion o BAL cells a e accina ion, ega dless o he ou e o ac-
cine deli e y (Figu e 4B).
This cy okine/chemokine sec e ion analysis o un ac iona ed
BAL cells upon he e ologous LPS (o homologous M b WCL)
s imula ion did no e eal a signal o ained immuni y in he ai -
ways a e (mucosal) li e a enua ed mycobac e ial accina ion.
Induc ion o ained immuni y in monocy es a e
mucosal accina ion wi h BCG and MTBVAC
Since BAL cells did no p o ide a signal indica i e o ained im-
muni y, we sough o compa e he induc ion o ained immuni y
h ough ex i o he e ologous LPS s imula ion o blood- and
BM-de i ed monocy es, as was shown o BCG.i be ween in-
ade mal and mucosal immunized animals, wi h li e a enua ed
BCG o MTBVAC. To demons a e ha he cy okine p oduc ion
is due o ex i o he e ologous LPS s imula ion and exclude he
possibili y o con inued inna e ac i a ion by he (pe sis ing) ac-
cine, uns imula ed medium con ols we e included ha showed
minimal o no p oduc ion o cy okines (Figu e S3).
While T cell-de i ed cy okines IL-17A, IFN-g, and IL-2 we e no
de ec ed upon monocy e s imula ion (no shown), he enhance-
men o LPS-s imula ed sec e ion o TNF-a, IL-6, and IL-1bwas
e iden in some bu no all animals ha we e accina ed by in a-
de mal injec ion (Figu e 5A). Speci ically, 4 o 11 animals (2 om
each o he BCG- and he MTBVAC- accina ed g oups) showed
ele a ed TNF-ap oduc ion wi h an e ec size (independen
o baseline p oduc ion) o >2- old inc ease a e BCG.id/
MTBVAC.id (Figu e 5B; due o a echnical e o , we missed he
baseline alues o a single animal o he MTBVAC.id g oup).
Simila ly, 4 o 11 showed he opposi e e ec o dec eased
TNF-ale els a e BCG.id/MTBVAC.id, while 3 o 11 showed
no conside able modula ion o LPS-s imula ed TNF-aa e in a-
de mal accina ion (Figu e 5B). O no e, M b-de i ed MTBVAC
appea ed equally as po en as BCG in he induc ion o ained
immuni y in PBMC.mo (Figu e S4A). No ably, he sec e ion le els
o p o-in lamma o y IL-6 and IL-1bshowed a pa e n simila o
ha o TNF-a. The a io o he p oduc ion o he 3 signa u e cy-
okines (TNF-a, IL-6, and IL-1b) did no de ia e o e a pe iod o
8 weeks in a coho o un accina ed animals (Figu e S4C).
In con as o in ade mal BCG/MTBVAC accina ion, he
mucosal deli e y o hese li e a enua ed accines esul ed in a
obus and signi ican enhancemen o TNF-a, IL-6, and IL-1b
p oduc ion a e LPS s imula ion in 10 o 12 animals (Figu es
5A and 5B; 5 animals om he BCG and he MTBVAC accina ed
g oup each). The median e ec size was 15.1, 6.2, and 5.9 o he
old inc ease in TNF-a, IL-6, and IL-1bp oduc ion, espec i ely
(Figu e 5B). Fo GM-CSF and MIP-1a, 2 cy okines ha ha e
been associa ed wi h ained immuni y, a simila pa e n o
enhanced sec e ion a e p io mucosal a he han in ade mal
accina ion was ob ained (Figu es 5A and 5B). This enhance-
men , howe e , was less p ominen han o TNF-a, IL-6, and
IL-1b, wi h a median old-inc ease o GM-CSF and MIP-1aa e
BCG.muc/MTBVAC.muc o 2.1 and 2.9, espec i ely (Figu e 5B).
CXCL-10 was no de ec ed om hese monocy e s imula ion
analyses (Figu e 5A). Compa ing BCG o MTBVAC wi hin each
ou e o deli e y (id e sus muc) s a egy demons a ed ha
bo h accines we e equally po en in media ing ained immuni y
(Figu e S5A). We explo ed he mycobac e icidal ac i i y wi h
Figu e 3. T ained immuni y a e in a enous BCG accina ion
(A and B) F eshly isola ed BAL cells ( om he igh lung) and monocy es om blood (PBMC.mo) and bone ma ow (BM.mo) we e s imula ed o 24 h wi h (A) M b
whole-cell lysa e (25 mg/mL) o (B) LPS (0.1 mg/mL) be o e and 2 weeks a e in a enous accina ion wi h BCG. The da a a e p esen ed as medium con ol
co ec ed.
(C) Lac a e p oduc ion as an indica o o me abolic ewi ing was measu ed in 24-h supe na an om LPS-s imula ed monocy es pu i ied om blood (PBMC.mo)
o BM (BM.mo). See also Figu e S1.
(D) Hea map o H3K27ac eads ( ed) o e BCG-speci ic peaks. The in ensi y o e he cen e o he peak ±12 kb is depic ed.
(E) The op GO pa hways associa ed wi h he nea es genes o dynamic H3K27ac, wi h adjus ed p alues.
(F) H3K27ac dynamics a in e e on egula o y ac o 3 (IRF3) and syndecan 2 (SDC2) locus o PBMC.mo p e- and pos - accina ion. The comple e lis o genes
and alues can be ound a GEO: GSE159046.
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PBMC.mo isola ed om esidual ozen PBMCs, bu wi hin he
limi s o ou e o s, we did no de ec mycobac e icidal ac i i y
by ained monocy es (Figu e S6).
Nex o PBMC.mo, we add essed he LPS-s imula ed cy o-
kine/chemokine sec e ion po en ial o BM-de i ed CD14
+
monocy es (BM.mo) be o e and a e BCG o MTBVAC
Figu e 4. Cy okine p oduc ion by BAL cells
F eshly isola ed BAL cells we e s imula ed (A) wi h M b whole cell lysa e o (B) LPS, be o e (PRE) and 8 weeks a e in ade mal (.id) o mucosal (.muc) accina ion
(wi h ei he BCG, in ci cles, o MTBVAC, in iangles). Cells we e ypically collec ed om he lowe igh lung lobe (R), he lobe a ge ed by accina ion, and o
mucosally accina ed animals also om he le lung (L), o es ablish dissemina ion o he immune esponse. The da a a e p esen ed as medium con ol co ec ed
(mcc); n = 12 animals/g oup; Wilcoxon ma ched-pai s signed ank es . p alues a e indica ed a he op o each g aph. See also Figu e S2.
A
B
Figu e 5. T ained immuni y a e mucosal accina ion in pe iphe al blood monocy es
Cy okine p oduc ion was measu ed a e 24 h o s imula ion wi h LPS (0.1 mg/mL).
(A) Pai ed display o cy okine le els be o e and 8 weeks a e accina ion. The da a a e p esen ed as medium con ol co ec ed (mcc; BCG in ci cles; MTBVAC in
iangles). S a is ical signi icance was calcula ed by Wilcoxon’s non-pa ame ic pai ed analysis es (n = 12 animals/g oup; PRE e sus 8 weeks pos - acci-
na ion). p alues a e indica ed a he op o each g aph.
(B) Median old inc ease in cy okine p oduc ion is indica ed in i alics; signi ican p < 0.05 is indica ed in bold; n = 12 animals/g oup; Wilcoxon signed ank es .
See also Figu es S1,S3,S4,S5, and S6.
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STAR+METHODS
KEY RESOURCES TABLE
REAGENT o RESOURCE SOURCE IDENTIFIER
An ibodies
an i-CD3 – AF700 (clone SP34-2) BD Biosciences Ca #: 557917; RRID: AB_396938
an i-CD4 – Pe CP.Cy5.5 (clone L200) BD Biosciences Ca #: 552838; RRID: AB_394488
an i-CD8a– APC-H7 (clone SK1) BD Biosciences Ca #: 641400; RRID: AB_164536
an i-CD14 – BV421 (clone M5E2) BD Biosciences Ca #: 301830; RRID: AB_10959324
an i-CD20 – BV421 (clone 2H7) Biolegend Ca #: 302330; RRID: AB_10965543
an i-CD28 – ECD (clone CD28.2) IOTes Ca #: 6607111; RRID: AB_1575955
an i-CD45RA – PE-CF594 (clone 5H9) BD Biosciences Ca #: 565419; RRID: AB_2739229
an i-CD95 – BV605 (clone DX2) Biolegend Ca #: 305628; RRID: AB_2563825
an i-IL-2 – AF488 (clone MQ1-17H12) Biolegend Ca #: 500314; RRID: AB_493368
an i-IL-17A – PE-Cy7 (clone ebio64DEC17) Biolegend Ca #: 25-7179-42; RRID: AB_11063994
an i-TNF-a– BV650 (clone Mab11) BD Biosciences Ca #: 502938; RRID: AB_2562741
an i-IFN-g– BV711 (clone 4S.B3) BD Biosciences Ca #: 502540; RRID: AB_2563506
an i-CD3ε– FITC (clone SP34) BD Biosciences Ca #: 556611; RRID: AB_396484
an i-CD14 – V450 (T€
uk4) Mil eny Bio ec Ca #: 130-113-152; RRID: AB_10831023
an i-CD20 – BV605 (clone 2H7) Biolegend Ca #: 302334; RRID: AB_2563398
an i-CD45 – BV786 (clone D058-1283) BD Biosciences Ca #: 563861; RRID: AB_2738454
an i-CD3 – BV421 (clone SP34-2) BD Biosciences Ca #: 562877; RRID: AB_2737860
an i-CD14 – BV786 (clone M5E2) BD Biosciences Ca #: 301840; RRID: AB_2563425
an i-CD16 – PE-CY7 (clone 3G8) BD Biosciences Ca #: 560716; RRID: AB_1727433
an i-CD20 – BV605 (clone 2H7) Biolegend Ca #: 302334; RRID: AB_2563398
an i-CD45 – AF700 (clone D058-1283) BD Biosciences Ca #: 561288; RRID: AB_10613813
an i-CD66 – PE (clone TET2) Mil eny Bio ec Ca #: 130-093-133; RRID: AB_871699
an i-CD206 – Pe CP (clone 15-2) BioLegend Ca #: 321122; RRID: AB_10899411
B bu e BioLegend Ca #: 563794; RRID: AB_2869750
VIVID – BV421 The mo ishe Ca #: L34955
Li e/Dea h – eFluo 506 eBioscience Ca #: 65-0866-14
an i-H3K27ac Diagenode Ca #: pab-196-050; RRID: AB_2637079
Bac e ial and i us s ains
BCG (s ain So ia; 5 310
5
CFU) In e Vax L d Ca #: no applicable
MTBVAC (M. ub s ain; 5 310
5
CFU) Bio ab i Ca #: no applicable
M b E dmann BEI Resou ces Ca #: NR-50781
Chemicals, pep ides, and ecombinan p o eins
Pu i ied P o ein De i a e (PPD; M. ub) AJ Vaccines Ca #: 2391
Pu i ied P o ein De i a e (PPD; M.bo ) Li e Technologies NV Ca #: 760060
Pu i ied P o ein De i a e (PPD; M.a ) Li e Technologies NV Ca #: 760065
Whole Cell Lysa e (WCL; M b HN878) BEI Resou ces Ca #: NR-14824
Lipopolysaccha ide (0111:B4 s ain; Ul apu e) In i ogen Ca #: l l-3pelps
PMA Sigma-Ald ich Ca #: P8139
Ionomycine Sigma-Ald ich Ca #: I0634
Roswell Pa k Memo ial Ins i u e (RPMI) Li e Technologies NV Ca #: 52400041
Lymphop epTM Axis-Shield Ca #: AXI-1114547
Fe al Cal e Se um (FCS) Li e Technologies NV Ca #: 10270106
(Con inued on nex page)
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RESOURCE AVAILABILITY
Lead con ac
Fu he in o ma ion and eques s o esou ces and eagen s should be di ec ed o and will be ul illed by he Lead Con ac Michel PM
Vie boom ([email p o ec ed]).
Ma e ials a ailabili y
This s udy did no gene a e new unique eagen s.
Da a and code a ailabili y
Raw da a iles o he ChIPseq analysis on BCG ained monocy es bego e and a e accina ion ha e been deposi ed in he NCBI
Gene Exp ession Omnibus unde accession numbe GEO: GSE159046.
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Animals and e hics
All animal expe imen s we e in acco dance wi h Du ch Law on animal expe imen a ion, which is in acco dance wi h he EU Di ec i e
2010/63/EU on he p o ec ion o animals used o scien i ic pu poses. The s udy was app o ed by he ins i u ional animal
wel a e body (in Du ch: Ins an ie oo Die welzijn, I D; accession numbe CCD009D) o BPRC. A coho o 8 non accina ed
con ol animals was used o Figu e S4C. The s udy in which hese animals we e used was app o ed by he ins i u ional animal
Con inued
REAGENT o RESOURCE SOURCE IDENTIFIER
Pe chlo ic acid Sigma-Ald ich Ca #: 244252-1L
Fo maldehyde (16%) The mo Scien i ic Ca #: 28906
P o ease Inhibi o Cock ail Sigma-Ald ich Ca #: P8465
Dynabeads p o ein A In i ogen Ca #: 10002D
Dynabeads p o ein G In i ogen Ca #: 10004D
BSA Sigma-Ald ich Ca #: A7030
Tagmen DNA bu e Nex e a Ca #: 20034197
AMPu eXP beads Beckman Coul e Ca #: A63880
KAPA HiFi Ho s a Ready Mix KAPA Biosys ems Ca #: KK2601
Nex e a DNA Lib a y P ep Ki Illumina Ca #: FC-121-1031
KAPA Lib a y P epa a ion Ki KAPA Biosys ems Ca #: KK8400
TMB (ELISPOT subs a e) Mab ech Ca #: 3651-10
C i ical comme cial assays
CD14 mic obeads Mil eny Bio ec Ca #: 130-091-097
Cus om NHP Legendplex (11-plex) Biolegend Ca #: 92919
Monkey IFN-gELISPOT U-CyTech Ca #: CT610-10
Lac a e-ELISA P omokine Ca #: PK-CA577-K627
Deposi ed da a
ChIP sequencing da a be o e and a e
BCGi accina ion
This pape GEO: GSE159046
Expe imen al models: o ganism/s ain
Rhesus monkeys (Indian); male and emale;
adul (> 4 yea s); pu pose b ed
BPRC b eeding colony no applicable
So wa e and algo i hms
Eli.Analyze (ELISPOT; 6.1) A.EL.VIS GmbH no applicable
FACSDi a So wa e 8.0.1 (BD LSRII) BD Biosciences SCR_001456
Flowjo so wa e 10 T ees a SCR_000410
LEGENDplexTM Da a Analysis So wa e (V8.0) Biolegend/Vigene Tech no applicable
G aphPad P ism 8.4.2 G aphPad So wa e h ps://www.g aphpad.com
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wel a e body (Accession numbe CCD009F). The BPRC is acc edi ed by he Ame ican Associa ion o Acc edi a ion o Labo a o y
Animal Ca e (AAALAC) and has an app o ed Assu ance (#A5509-1) o he ca e and use o animals on ile a he Na ional Ins i u es
o Heal h (NIH).
The hesus monkeys we e housed in socially compa ible pai s a he Biomedical P ima e Resea ch Cen e (BPRC; animal biosa e y
le el ABSL-3). The animals we e o e ed a daily die consis ing o monkey ood pelle s (Hope Fa ms, Woe den, he Ne he lands), ui
and ege ables o he season, and b ead. D inking wa e was a ailable ad libi um ia au oma ic wa e sys ems.
The s udy was ca ied ou in 27 heal hy pu pose-b ed, pedig eed, male and emale hesus monkeys (Macaca mula a) o Indian
o igin (5.99 – 13,50 kg; see Table S1). All animals we e sc eened o be nega i e o p e-exis ing immuni y agains mycobac e ial an-
igens as de e mined by in e e on gamma (IFN-g) ELISpo assay a e in i o ecall s imula ion wi h Pu i ied P o ein De i a e (PPD) o
M. ube culosis,M.a ium and M.bo is.
All animal handling and biosampling was pe o med unde ke amine seda ion (10 mg kg1, by in amuscula injec ion). Fo b on-
choal eola la age (BAL) and in ab onchial accina ion inocula ion wi h BCG o MTBVAC ke amine (5 mg kg1) was supplemen ed
wi h in amuscula mede omidine (0.04 mg kg1) and an analgesic sp ayed in o he la ynx.
Vaccines and accina ion
BCG s anda d dose & MTBVAC s anda d dose. Animals immunized wi h Bacillus Calme e–Gue in (BCG) s ain So ia (In e Vax L d.)
ecei ed a single s anda d adul human dose o 5 310
5
CFU. Animals immunized wi h MTBVAC ecei ed an equi alen human dose
o 5 310
5
CFU. Vaccines we e deli e ed in a olume o 0.1 mL in ade mally and 10 mL in a enously o in ab onchially. Mucosal
accina ion in he lung was pe o med by in ab onchial deli e y o he same dose in 10mL o s e ile saline solu ion o a segmen al
b onchus in he lowe igh lung lobe using a b onchoscope. Vaccina ion was execu ed o all animals in a single session in andom
o de wi hin 2-3 hou s om accine p epa a ion
METHOD DETAILS
Biological sample collec ion and p ocessing
Cells om he pulmona y mucosa we e eco e ed by BAL a week 1 o all g oups om he lowe igh lung lobe; a week 2 a e
in a enous BCG and week 8 a e in ade mal BCG om lowe igh lung lobe and week 8 a e mucosal accina ion om bo h he
lowe igh and le lung lobe. Th ee olumes o 20 mL o p ewa med 0.9% saline solu ion we e consecu i ely ins illed and eco e ed.
BAL luid was ha es ed by cen i uga ion o BAL samples o 10 min a 400 g a e 100 mm il a ion. Supe na an was subsequen ly
decan ed and s o ed a 80C pending u he analysis. The BAL cell pelle was aken up in Roswell Pa k Memo ial Ins i u e (RPMI)
medium supplemen ed wi h 10% FCS, glu aMAX, and penicillin/s ep omycin ( om now on e e ed o as R10) and used eshly in
downs eam assays. Hepa inized blood o immune moni o ing, was collec ed by enipunc u e. Bone ma ow was ob ained h ough
needle ex ac ion o he capu hume us. PBMCs and BM cells we e ob ained by densi y g adien cen i uga ion wi h Lymphop ep
lymphocy e sepa a ion medium (Axis-Shield) and esuspended in R10 o u he analysis.
Mycobac e ium up ake and killing assay
To assess mycobac e ium up ake, PBMC.mo (30.000 cells/well) we e incuba ed o 1h a 37C wi h M b E dmann a a MOI o 5.
Non-phagocy osed mycobac e ium was washed away and monocy es we e incuba ed wi h high dose (30 mg/mL) gen amycin o
10-minu es. PBMC.mo we e subsequen ly lysed wi h 0.05% SDS in wa e , i a ed and pla ed on o 7H10 aga o bac e ial enume -
a ion. To asses killing, PBMC.mo we e incuba ed wi h M b E dmann a a MOI o 5 o 1h a 37C and subsequen ly cul u ed wi h low
gen amycin (5 mg/mL) o 24 h . Bac e ia we e enume a ed as desc ibed be o e. As a con ol, bac e ia we e incuba ed wi hou
PBMC.mo. Da a a e p esen ed as % o con ol (Figu e S5).
Monocy e isola ion
CD14+ cells (monocy es) we e labeled wi h magne ic CD14 Mic oBeads. The cell suspension was loaded on o a MACSColumn
which is placed in he magne ic ield o a MACS Sepa a o . The magne ically labeled CD14+ cells a e e ained in he column. The
unlabeled cells un h ough, his cell ac ion is deple ed o CD14+ cells. A e emo al o he column om he magne ic ield, he
magne ically e ained CD14+ cells can be elu ed as he posi i ely selec ed cell ac ion. Pu i y was analyzed by lowcy ome y
(Figu e S1).
IFN-gELISPOT
A NHP speci ic IFN-gELISpo assay was used on PBMC, acco ding o he manu ac u e p o ocol (U-CyTech, U ech ), o de e mine
he equency o an igen speci ic IFN-gp oducing cells. In b ie , 200.000 eshly isola ed PBMC we e incuba ed in iplica e o 24 h
wi h speci ied an igens o con ol s imuli. Subsequen ly, supe na an was collec ed and s o ed (80C), and cells we e ans e ed o
speci ic an i-IFN-gcoa ed il e pla es (PVDF, Millipo e) o an addi ional o e nigh (18 h) incuba ion. Cells we e disca ded and
memb ane bound IFN-gwas de ec ed using bio inyla ed an i-IFN-gan ibody, s ep a idin-ho se adish pe oxidase conjuga e
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and e ame hylbenzidine (TMB) subs a e ( he la e om MAbTech, S ockholm). Spo s we e quan i ied using an au oma ed eade
(AELVIS, Hanno e ).
Flow cy ome y
An igen-speci ic T cell esponses we e de e mined a WK-1 (PRE) and week 8 indica ed ime poin s by low cy ome y locally using
BAL cells. Cells we e s imula ed o e nigh in he p esence o Golgiplug anspo inhibi o . The ollowing mAb s aining T cell panel
was used: In acellula s aining: CD3
AF700
(SP34-2; BD), TNF-a
PE-CY7
(Mab11; BD), IL-2
BV510
(MQ1-17H12; BioLegend), IFN-g
APC
(4S.B3; BD); IL-17A
PE-Cy7
(ebio64DEC17; The mo ishe ). Su ace s aining: CD4
Pe CP-Cy5.5
(L200; BD), CD8a
APC-H7
(SK1; BD),
CD14
BV421
(M5E2; BD), CD20
BV421
(2H7; BioLegend), CD45
BV786
(D058-1283; BD).
Ga ing s a egy o T cells (Figu e S7; Rela ed o Figu e 2D)
T cells we e ga ed as Single s/Lymphocy es/Viable/ CD14-CD20-/CD45+ /CD3+ be o e CD4 and CD8 ga ing was applied. A e
double exclusion, lymphocy es we e ga ed based on size and g anula i y. Any anomaly indica i e o uns able signal acquisi ion
was excluded using he ime pa ame e . E en s om he combined ime-ga es we e plo ed agains he dump channel con aining
he iabili y, CD14 and C20 ma ke s and subsequen ly ga ed as iable, CD14- and CD20-. Cells we e u he selec ed o CD45
and CD3 posi i i y be o e CD4 and CD8 ga ing was applied. Boolean ga ing o any cy okine exp ession o IL-2/IFN-g/TNFa/IL-17A.
BAL cell composi ion analysis
Cells we e s imula ed o e nigh wi h WCL. The ollowing mAb s aining panel was used o analyze BAL cell composi ion: CD45
AF700
(D058-1283; BD); CD66
PE
(TET2; Mil enyi); CD206
Pe CP
(15-2;Biolegend); CD16
PE-CY7
(3G8; BD); CD3
BV421
(SP34-2;BD); CD20
BV605
(2H7; Biolegend); CD14
BV786
(M5E2; BD).
Ga ing s a egy o BAL cell composi ion (Figu e S2B)
Single s/ Leucocy es (CD45)/Viable/Time ga e: CD206+/CD14+ (Al eola Mac ophages); CD3 (T cells); CD20 (B cells); CD16+
(NK-cells) and CD66+ (Neu ophils). BAL cell composi ion is exp essed as % om he CD45+/ iable ac ion.
Isola ion o CD14+ monocy es
PBMC o BM cells we e incuba ed wi h CD14 magne ically labeled Mic obeads (Mil enyi Bio ec; Ne he lands). The cell suspension is
loaded on o a MACSColumn which is placed in he magne ic ield o a MACS Sepa a o . The magne ically labeled CD14+ cells a e
e ained in he column. The column is washed once. The column was emo ed om he magne ic ield and he magne ically e ained
CD14+ cells we e hen elu ed as he posi i ely selec ed cell ac ion. Pu i y o CD14+ ac ion was subsequen ly es ed by low
cy ome y.
Ga ing s a egy o CD14+ monocy es (Figu e S1B)
Single s/CD45+/ Viable/CD14+. CD14+ cells a e exp essed as % om he CD45+/ iable ac ion.
Mul iplex cy okine assay
Cells om di e en sou ces (PBMC.mo, BM.mo and BAL we e s imula ed wi h WCL and i a ed amoun o LPS (E.coli; Ul apu e).
F eshly isola ed cells 25000 cells we e incuba ed ex- i o o 24 h wi h each s imulus a e which he supe na an was collec ed.
Medium alone was aken along o con ol. Cy okine p oduc ion was assessed wi h mul i-analy e low assay ki using a cus om
nonhuman p ima e Legendplex
TM
(Biolegend; USA). Assays we e pe o med acco ding o he manu ac u e ’s ins uc ions. B ie ly,
he supe na an s o s imula ed cells we e incuba ed wi h beads coa ed wi h cy okine-speci ic an ibodies. Bound cy okines we e
isualized using bio in-coupled de ec ion an ibodies and R-phycoe y h in-conjuga ed s ep a idin. Beads we e acqui ed on 3 lase ,
14 colo LSR-II low cy ome e (BD Biosciences). A NHP cus om made 11-plex consis ed o TNF-a, IL-6, IL-1bcha ac e izing ained
immuni y, GM-CSF, CCL3 (MIP-1a), CXCL10 (IP-10) and cy okines associa ed wi h adap i e immuni y IFN-g, IL-2, IL-17A.
Lac a e measu emen s
Supe na an collec ed om he 24 h inna e s imula ion was analyzed o he p oduc ion o lac a e as a su oga e ma ke o he me a-
bolic ewi ing (Wa bu g-e ec ) unde lying ained immuni y. P o eins in he supe na an , in e e ing wi h he lac a e analysis, we e
dena u ed p ecipi a ed wi h pe cholo ic acid (PCA) and emo ed by high speed cen i uga ion. The clea dep o einized supe na an
was collec ed and neu alized wi h NaOH.
Samples we e subsequen ly dilu ed 15- old and analyzed in a L-Lac a e Assay Ki I (P omokine; P omoCell GmbH; Ge many) ac-
co ding o manu ac u e ’s ins uc ion.
Ch oma in immunop ecipi a ion
Cell pelle s we e dissol ed in PBS and c osslinked in solu ion by using o maldehyde (1% inal olume, shaking 10 min a oom
empe a u e). Glycine (0.125 M) was added o quench he eac ion. Cells we e hen washed wo imes wi h cold PBS. Cell pelle s
we e lysed in a olume o 110ml using lysis bu e (20 mM HEPES pH 7.6, 1% SDS, 1 3P o ease Inhibi o Cock ails). Samples
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we e sonica ed using he Bio up e Pico (Diagenode) wi h eigh cycles (30 s on/30 s o ). A e wa d, he samples we e spun a
16,000 3g o 5 min a oom empe a u e and he supe na an was s o ed a 80C.
Ch oma in om 100,000 cells was used o ChIPmen a ion. ChIPmen a ion was pe o med as desc ibed by Schmidl e al.,
46
wi h
se e al modi ica ions. In sho , he ch oma in was incuba ed o e nigh a 4C o a ing in dilu ion bu e (1% T i on X-100, 1.2 mM
EDTA, 16.7 mM T is pH 8, 167 mM NaCl), 1 3P o ease Inhibi o Cock ail
47
and 1mg o an ibody [H3K27ac (C15410196, Diagenode)]
in a o al olume o 300 ml. The nex day pe ChIP 10ml p o ein A Dynabeads and 10ml p o ein G Dynabeads (bo h In i ogen) we e
added. Beads we e washed be o e use wi h dilu ion bu e (+0.15% SDS, +0.1% BSA) and incuba ed wi h he ch oma in and
an ibody mix o 60 min a 4C o a ing. A e wa d, he beads we e washed a 4C once wi h ChIP wash bu e 1 (2 mM EDTA,
20 mM T is pH 8, 1%T i on X-100, 0.1% SDS, 150 mM NaCl), wice wi h ChIP washbu e 2 (2 mM EDTA, 20 mM T is pH 8, 1% T i on
X-100, 0.1%SDS, 500 mM NaCl), and wice wi h TE (1 mM EDTA, 10 mM T ispH 8). Beads we e esuspended in 24 ml Tagmen DNA
bu e (Nex e a) and 1ml Tn5 enzyme (p oduced in-house) and incuba ed o 10 min a 37C wi h 550 pm shaking. A e wa d, he
beads we e washed wice wi h WBI (20 mM HEPES, 150 mM NaCl, 0.1% SDS, 0.1% DOC,1% T i on X-100, 1 mM EDTA,
0.5 mM EGTA) and wice wi h WBIV (20 mM HEPES, 1 mM EDTA, 0.5 mM EGTA) wi h 5 min o a ing a oom empe a u e in
be ween. Samples we e dec osslinked o 1 h a 55C 1,000 pm shaking ollowed by an o e nigh incuba ion a 65C using elu ion
bu e (0.5% SDS, 300 mM NaCl,5 mM EDTA, 10 mM T is pH 8) and p o einase K. Samples we e incuba ed one addi ional hou he
nex day wi h elu ion bu e and p o einase K o 1 h a 55C. The samples we e pu i ied using 2 3SPRI AMPu eXP beads. qPCR was
used o de e mine he su icien amoun o PCR cycles needed o ampli y he lib a y. The lib a ies we e ampli ied using he KAPA
HiFi Ho s a Ready Mix (KAPA Biosys ems) and Nex e a Index Ki 1 (i7) and 2 (i5) p ime s (Illumina). Ampli ied lib a ies we e
pu i ied using a 0.65 3SPRI AMPu eXP beads incuba ion ollowed by a 1.8 3SPRI AMPu eXP beads incuba ion. Lib a y
concen a ion was measu ed using he KAPA Lib a y Quan i ica ion Ki (KAPA Biosys ems); lib a y size was de e mined using he
BioAnalyze High Sensi i i y DNA Ki (Agilen ). Sequencing was pe o med using an Illumina HiSeq 2000, and 50-bp pai ed-end eads
we e gene a ed.
ChIP-seq analysis
Sequenced eads we e quali y and adap o immed wi h im galo e and cu adap (K uege , Felix, T im galo e. ‘‘A w appe ool
a ound Cu adap and Fas QC o consis en ly apply quali y and adap e imming o Fas Q iles 516 (2015): 517. ‘‘Pica d Toolki .’’
2020. B oad Ins i u e, Gi Hub; Ma in, Ma cel. Cu adap emo es adap o sequences om high- h oughpu sequencing eads.
EMBne .jou nal). These eads we e aligned agains he UCSC Rhesus e e ence genome (RheMac10) wi h Bu ows-Wheele
Aligne (BWA) p og am
48
wi h de aul pa ame e s. Duplica e eads we e emo ed wi h pica d Ma kDuplica es. Peaks we e called
wi h MACS2 and de aul se ings
49
and used o de ec he binding si es o ChIP-seq acks. Tags wi hin a gi en egion we e
coun ed and adjus ed o ep esen he numbe o ags wi hin a 1 kb egion. Subsequen ly he pe cen age o hese ags as a mea-
su e o he o al numbe o sequenced ags o he sample was calcula ed and displayed as hea maps using lu .
50
To de e mine
genomic loca ions o binding si es, he peak ile was analyzed using a sc ip ha anno a es binding si es acco ding o all Re Seq
genes.
Nec opsy and pos -mo em e alua ion
Rhesus monkeys we e scheduled by p o ocol o eu hanasia and nec opsy a 2 weeks (3 animals o BCG
IV
) o 8 weeks (BCG
ID
,
BCG
MUC
, MTBVAC
ID
, MTBVAC
MUC
) a e accina ion. A he end o he expe imen blood (< 45 ml) and bone ma ow was collec ed
be o e eu hanasia wi h sodium pen oba bi al (> 50 mg/kg). Samples we e s o ed a 80C o analysis a la e ime poin .
QUANTIFICATION AND STATISTICAL ANALYSIS
Da a handling o measu emen s o cy okine p oduc ion by Mul iplex
The Legendplex
TM
is a bead-based assay o low cy ome y allowing o he measu emen o mul iple cy okines in he same sam-
ple. Bead coun pe analy e was ±300/sample. Da a ob ained wi h a bead coun a bi a ily se a < 25 was excluded. S anda d
cu e (pg/mL) aken along on each pla e: 10000, 2500, 625, 156, 39.1, 9.8, 2.4 pg/mL. PRE and WK2/WK8 samples om indi idual
animals we e measu ed on he same pla e o a oid in e -pla e a ia ion. Samples om animals om di e en ea men g oups
we e andomly dis ibu ed o e pla es. I lowe limi o quan i ica ion (LLOQ) was < 2.4 pg/mL, he lowes de ec able concen a ion
was se a 2.4 pg/mL. I LLOQ was > 2.4 pg/mL, he lowes de ec able concen a ion was se a LLOQ. This da a p ocedu e
ensu ed a conse a i e es ima ion o he old change be ween PRE and WK2/WK8 pos accina ion.
S a is ics compa ing ou es o accina ion, cy okine, and lac a e p oduc ion
S a is ical pa ame e s including numbe o animals, pe cen age o cells le els o cy okine p oduc ion, old inc ease o cy okine
p oduc ion and absolu e inc ease in lac a e p oduc ion a e epo ed in he Figu es and Figu e legends. Da a was analyzed
by ANOVA adjus ed o mul iple compa isons, Wilcoxon’s non-pa ame ic pai ed analysis es , Wilcoxon signed ank es o
whe e applicable. S a is ical analyses we e conduc ed using G aphPad P ism 7 so wa e ( e sion 7.0d; G aphPad So wa e,
La Jolla, CA).
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S a is ics epigene ics
Peaks we e called wi h MACS2 and de aul se ings
49
and used o de ec he binding si es o ChIP-seq acks. All peaks
we e me ged using bed ools and o each sample ags pe peak we e coun ed. Di e en ial peaks we e called by compa ing
he wo se s o samples using hese c i e ia: he sum o coun ed ags wi hin one peak using all samples should be > 200 and
he old di e ence should be > median plus 2xSD. The es ing o gene se /pa hway en ichmen s was done using Fishe ’s exac
es ing.
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