A icle
Pulmona y MTBVAC accina ion induces immune
signa u es p e iously co ela ed wi h p e en ion o
ube culosis in ec ion
G aphical Abs ac
Highligh s
dPulmona y MTBVAC deli e y con e s immune signa u e
co ela ing wi h TB p o ec ion
dThis signa u e sp eads h ough he lung wi hou a ecall
esponse in he skin
dVaccine-induced T cells ha e inc eased mucosal homing and
issue esidency ma ke s
dVaccine-induced an ibodies enhance phagocy osis o
M. ube culosis
Au ho s
Ka in Dijkman, Nacho Aguilo,
Cha elle Boo , ..., Eugenia Puen es,
Ca los Ma in, F ank A.W. Ve eck
Co espondence
[email p o ec ed]
In B ie
Dijkman e al. show ha pulmona y
immuniza ion wi h he M. ube culosis-
de i ed accine candida e MTBVAC
con e s a local mucosal an igen-speci ic
signa u e—poly unc ional Th1/Th17 cells
exhibi ing inc eased homing and issue
esidency ma ke exp ession, IL-10, and
phagocy osis-p omo ing
immunoglobulins— ha has been
associa ed p e iously wi h p o ec ion
om TB in ec ion and disease in hesus
macaques.
Dijkman e al., 2021, Cell Repo s Medicine 2, 100187
Janua y 19, 2021 ª2020 The Au ho (s).
h ps://doi.o g/10.1016/j.xc m.2020.100187 ll
A icle
Pulmona y MTBVAC accina ion induces
immune signa u es p e iously co ela ed
wi h p e en ion o ube culosis in ec ion
Ka in Dijkman,
1
Nacho Aguilo,
2,3
Cha elle Boo ,
1
Sam O. Ho man,
1
Claudia C. Somb oek,
1
Richa d A.W. Ve enne,
1
Clemens H.M. Kocken,
1
Dessisla a Ma ino a,
2,3
Jelle Thole,
4
Es eban Rod ı
´guez,
5
Michel P.M. Vie boom,
1
K is a G. Haans a,
1
Eugenia Puen es,
5
Ca los Ma in,
2,3
and F ank A.W. Ve eck
1,6,
*
1
Biomedical P ima e Resea ch Cen e (BPRC), Rijswijk, he Ne he lands
2
Depa men o Mic obiology, Facul y o Medicine, IIS A agon, Uni e si y o Za agoza, Za agoza, Spain
3
CIBERES, Ins i u o de Salud Ca los III, Mad id, Spain
4
TuBe culosis Vaccine Ini ia i e (TBVI), Lelys ad, he Ne he lands
5
Bio ab i, Pon e ed a, Spain
6
Lead con ac
*Co espondence: e eck@bp c.nl
h ps://doi.o g/10.1016/j.xc m.2020.100187
SUMMARY
To igh ube culosis, be e accina ion s a egies a e needed. Li e a enua ed Mycobac e ium ube culosis-
de i ed accine, MTBVAC, is a p omising candida e in he pipeline, p o en o be sa e and immunogenic in
humans so a . Independen s udies ha e shown ha pulmona y mucosal deli e y o Bacillus Calme e-Gue
´-
in (BCG), he only ube culosis (TB) accine a ailable oday, con e s supe io p o ec ion o e s anda d in a-
de mal immuniza ion. He e we demons a e ha mucosal MTBVAC is well ole a ed, elici ing poly unc ional T
helpe ype 17 cells, in e leukin-10, and immunoglobulins in he ai way and yielding a b oade an igenic p o-
ile han BCG in hesus macaques. Beyond ou p e ious wo k, we show ha local immunoglobulins, induced
by MTBVAC and BCG, bind o M. ube culosis and enhance pa hogen up ake. Fu he mo e, a e pulmona y
accina ion, bu no M. ube culosis in ec ion, local T cells exp essed high le els o mucosal homing and is-
sue esidency ma ke s. Ou da a show ha pulmona y MTBVAC adminis a ion has he po en ial o enhance
i s e icacy and jus i ies u he explo a ion o mucosal accina ion s a egies in p eclinical e icacy s udies.
INTRODUCTION
Despi e ad ances in ea men and ca e, ube culosis con inues
o cause app oxima ely 1.6 million dea hs and an addi ional 10
million cases o ac i e disease annually.
1
Con ol o his ongoing
epidemic is complica ed by a lack o accu a e diagnos ics,
leng hy ea men egimens, and an inc ease in d ug- esis an
ube culosis (TB) incidence. An e ec i e accina ion s a egy
p e en ing TB in ec ion o disease is he e o e o c i ical impo -
ance o con olling he con inuing TB epidemic. Un o una ely,
he only p ophylac ic accine cu en ly a ailable, Bacillus Calm-
e e-Gue
´ in (BCG), despi e p e en ing dissemina ion o he dis-
ease, is no o iously a iable in p o ec ing adul s and adolescen s
om pulmona y TB. Pulmona y disease is he majo cause
o mo bidi y and mo ali y and he d i e o TB sp ead.
2
Geog aphical loca ion, p io non- ube culous mycobac e ium
(NTM) exposu e, and o e -a enua ion o BCG ha e been
implied in he a iable BCG e icacy.
2–4
Rega dless o he unde -
lying mechanisms o his a ia ion in e icacy, i is e iden ha a
mo e eliable accine s a egy is u gen ly needed.
Cu en ly, mul iple no el TB accines a e being de eloped
o eplace BCG a bi h o o se e as a (he e ologous) boos e
on op o p io BCG accina ion.
5,6
One o hese new candi-
da e accines is MTBVAC, a li e a enua ed whole-cell ac-
cine designed as a po en ial eplacemen o neona al BCG
accina ion. MTBVAC was gene a ed by gene ic modi ica ion
o a clinical Mycobac e ium ube culosis (M b)isola eo he
lineage 4 Eu o-Ame ican geno ype and ha bo s dele ions in
wo i ulence genes, phoP and adD26.
7,8
These dele ions
in e e e wi h ansc ip ion, syn hesis, and/o sec e ion o mul-
iple i ulence ac o s, including ea ly sec e o y an igenic
a ge 6 (ESAT6) and ph hioce ol dimycoce osa es (PDIMs).
9
Because MTBVAC is M b de i ed, i con ains genomic egions
o di e ence (RDs) ha a e absen om M. bo is,andin
pa icula also RD1, ha is lacking om M. bo is-de i ed
BCG.
9
MTBVAC, he e o e, has a b oade an igenic epe oi e
ha is linked o i s enhanced p o ec i e capaci y.
10
Al hough
RD1 encodes no o ious i ulence ac o s, such as ESAT6,
sec e ion is igh ly egula ed and in e up ed by he a ge ed
phoP dele ion in MTBVAC.
9
Acco dingly, in ea ly-s age clinical
e alua ion in adul s and in an s, in ade mal MTBVAC
immuniza ion has been ound o ha e an accep able sa e y
p o ile compa able wi h BCG, co obo a ing i s a enua ed
pheno ype.
11,12
Cell Repo s Medicine 2, 100187, Janua y 19, 2021 ª2020 The Au ho (s). 1
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In p eclinical s udies MTBVAC p esen ed wi h an abili y o p o-
ec om expe imen al TB in ec ion and disease be e han
BCG.
7,13
I has been shown o con e imp o ed p o ec i e e i-
cacy agains M b in newbo n mice a a single dose a bi h.
13
In
guinea pigs, e accina ion wi h MTBVAC a e BCG p iming e-
sul ed in a u he educ ion o M b bu den in he lung compa ed
wi h BCG alone,
14
and non-human p ima e da a on he e icacy o
( e) accina ion wi h in ade mal MTBVAC ha e been es ablished
ecen ly.
15
Al hough ini ially designed as a accine o newbo ns,
MTBVAC is also conside ed o e accina ion o BCG-p imed
adolescen s and adul s.
12
Phase2 dose- inding, sa e y, and
immunogenici y s udies in neona es (NCT03536117) and Quan i-
FERON-nega i e and -posi i e adul s (NCT02933281) a e in
p og ess, and a subsequen phase 3 e icacy s udy in neona es
is scheduled o s a in 2021.
The indica ed ou e o adminis a ion o BCG and MTBVAC is
he skin, which induces limi ed immune esponses a he pulmo-
na y mucosa, he p ima y si e o in ec ion wi h M b. A g owing
body o da a gene a ed in p eclinical models o TB shows ha
al e ing he ou e o BCG adminis a ion o he pulmona y mu-
cosa signi ican ly imp o es i s p o ec i e e icacy.
16–19
In p e i-
ous wo k, we showed ha pulmona y bu no in ade mal BCG
accina ion could p o ec highly suscep ible hesus macaques
(Macaca mula a) om epea ed low-dose M b in ec ion and
TB-associa ed pa hology.
20
Macaques a e conside ed o be a
p edic i e model o TB accine de elopmen because o hei
close phylogene ic ela ionship o man and highly simila TB dis-
ease de elopmen .
21,22
In his model, he p o ec ion con e ed
by mucosal BCG s a is ically co ela ed wi h induc ion o poly-
unc ional in e leukin-17A (IL-17A)+ CD4+ T cells a he pulmo-
na y mucosa and IL-10 p oduc ion by b onchoal eola la age
(BAL) cells, whe eas ele a ed le els o an igen-speci ic immuno-
globulins we e ound in associa ion wi h mucosal BCG immuni-
za ion as well.
Al hough he mucosa o he ai ways can be conside ed an
en i onmen o obus inna e hos de ense o wa an homeo-
s a ic balance, po en ially esul ing in apid clea ance and poo
immunogenici y o li e a enua ed accines, ou da a om
mucosal deli e y in non-human p ima es (NHPs) ha e shown
local pe sis ence o BCG and p o ec ion-associa ed immuni y
in he ai ways in he absence o o e espi a o y ad e si y.
20
In e es ingly, on his no e, p io exposu e o BCG o al eola lin-
ing luid om nai e animals in i o has been desc ibed o
enhance i s p o ec i e e icacy when adminis e ed pe iphe ally
o mice.
23
Also, in o he NHP s udies explo ing pulmona y deli -
e y o BCG, no ad e si y has been epo ed.
24,25
Howe e , o
pulmona y mucosal deli e y o MTBVAC, he ole abili y and
immunogenici y emain o be es ablished.
In ligh o his, he e we se ou o assess he ole abili y and
immunogenici y o pulmona y mucosal deli e y o MTBVAC in
hesus macaques. Using a wo-by- wo ac o ial design s a egy,
we compa ed MTBVAC wi h BCG by s anda d in ade mal injec-
ion and endob onchial ins illa ion o a s anda d human dose.
Co obo a ing ou ea lie obse a ions ega ding al e na i e
BCG deli e y, we show ha pulmona y MTBVAC adminis a ion
was well ole a ed and induced local IL-17A-p oducing T cells,
IL-10 p oduc ion, and M b-speci ic immunoglobulin A (IgA).
Compa ed wi h BCG, accina ion wi h MTBVAC esul ed in
mo e apid induc ion o immune esponses and b oade an i-
genic speci ici y. Beyond wha we ha e epo ed p e iously o
mucosal BCG, we iden i ied inc eased exp ession o mucosal
homing ma ke s o pu i ied p o ein de i a i e (PPD)-speci ic
T cells in lung wash samples o mucosally accina ed bu no
M b-in ec ed animals. Fu he mo e, we show ha accine-
induced mucosal an ibodies a e unc ional in binding o li e
M b and acili a ing pa hogen up ake by phagocy es. In ou
a emp o iden i y an immune co ela e o pulmona y whole-
cell TB accina ion ha is assessable by pe iphe al sampling,
we exploi ed in i o ecall s imula ion by ube culin skin es ing
(TST) bu could no iden i y meaning ul esponses in skin o
skin-d aining axilla y lymph nodes. This s udy co obo a es
and ex ends beyond p e ious indings and p o ides a a ionale
o u u e explo a ion o mucosal adminis a ion o MTBVAC
wi h he pe spec i e o imp o ing ou p ophylaxis agains TB
in ec ion and disease.
RESULTS
Local immune signa u es a e mucosal MTBVAC
accina ion
To in e oga e whe he he M b-de i ed MTBVAC accine candi-
da e, like M. bo is BCG, is ole a ed well and induces unique
immune ea u es upon pulmona y mucosal adminis a ion, we
designed a dedica ed sa e y/immunogenici y s udy (wi hou
in ec ious challenge) in hesus macaques, ep esen ed sche-
ma ically in Figu e 1. We accina ed wi h MTBVAC using a single
(human) dose simila o BCG (5 310
5
colony- o ming uni s
[CFUs]/dose) h ough he s anda d in ade mal ou e (M.id) o
endob onchial ins illa ion (M.muc) o di ec compa ison wi h
in ade mal o mucosal BCG accina ion (B.id and B.muc,
espec i ely; Figu e 1).
On a daily basis, animals we e moni o ed o changes in con-
di ion and well-being (including bu no limi ed o ale ness,
appe i e, and espi a ion), bu no de ia ion om no mal beha io
was obse ed ha would indica e accine ad e si y. Mo eo e ,
he e we e no signals o se ological inc ease in C- eac i e p o-
ein (CRP) le els du ing he s udy ha would indica e an ad e se
sys emic in lamma o y esponse ela ed o ea men (Figu e S1).
Al hough we aimed o add ess accine pe sis ence by cul u ing
om lung wash samples, ou e o ailed because o echnical
e o ; he e o e we could no con i m pe sis ence o MTBVAC
in he ai way like we ha e shown p e iously o BCG.
20
In
Figu e 1. S udy design schema ic
Shown is a schema ic o e iew o accina ion
s a egies and pos - accina ion sampling (o pe-
iphe al blood and BAL). O no e, BALs we e ha -
es ed bila e ally only o he mucosally accina ed
g oups 3 and 8 weeks a e accina ion.
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summa y, and wi hin limi s o obse a ion, li e a enua ed M b-
de i ed MTBVAC, also by pulmona y mucosal deli e y, was
well ole a ed by hesus macaques.
Flow cy ome y p o iling o T cells in BAL e ealed obus induc-
ion o PPD-speci ic CD4+ T cells in bo h mucosally accina ed
g oups, wi h MTBVAC elici ing highe esponses han BCG o
almos all cy okines, especially ea ly a e accina ion (Figu es
2A–2D). Al hough in ade mal BCG and MTBVAC showed an in-
c ease in in e e on g(IFNg)-, umo nec osis ac o alpha (TNF-
a)-, and IL-2-p oducing T cells in he ai ways in he weeks ollowing
accina ion (Figu es 2A–2C), IL-17A p oduc ion was uniquely
obse ed in he mucosally accina ed g oups (Figu e 2D). These
IL-17A+ T cells also p oduced IFNg,TNF-a, and IL-2, con i ming
induc ion o a local, quad uple-posi i e, CD4+ T cell popula ion
(Figu e 2E), ound p e iously o be associa ed in his species
wi h p o ec ion om TB in ec ion and disease.
20
Li le PPD-spe-
ci ic cy okine p oduc ion was obse ed in BAL CD8+ T cells (Fig-
u es S2A–S2E). Local lymphocy e p oli e a ion was obse ed p e-
dominan ly a e mucosal accina ion (Figu e S2F).
Al hough, by endob onchial ins illa ion, he accine was a -
ge ed o he lowe igh lobe, we in es iga ed whe he accine-
induced immune esponses would dissemina e o be es ained
o he a ge ed lobe only. 3 and 8 weeks a e accina ion, we
bila e ally collec ed BAL o immune p o iling and ound ha
PPD-speci ic, cy okine-p oducing T cells we e p esen in lowe
igh and lowe le lung lobes, albei a a somewha lowe e-
quency in he non- a ge ed lobe (Figu e 2F). In ei he lobe,
MTBVAC induced ea lie and highe esponses compa ed wi h
BCG, including poly unc ional CD4+ Th17 cells.
We also p o iled immune esponses in lung-d aining lymph no-
des, he canonical si e o T cell p iming o espi a o y an igenic
challenge. PPD-speci ic IFNg, TNF-a, and IL-2 p oduc ion by
CD4+ T cells was obse ed mos p ominen ly a e mucosal
accina ion bu was also de ec able a e in ade mal accina-
ion (Figu e S2G). In e es ingly, an igen-speci ic IL17A+ CD4+
T cells we e no appa en in hese lymph nodes (Figu e S2G)
because he p ecu so equency o hese cells was oo low o
be de ec ed o because Th17 p iming occu s elsewhe e; o
ins ance, in e ia y lymphoid s uc u es in he lung.
26
Because we p e iously also iden i ied IL10 p oduc ion by un-
ac iona ed BAL cells as a co ela e o p o ec ion, we in es i-
ga ed, by low cy ome y analysis, whe he IL-10 p oduc ion
could be T cell de i ed. Al hough we con i med high le els o
PPD-speci ic IL10 p oduc ion in s imula ed BAL cell supe na-
an s a e mucosal accina ion wi h MTBVAC as well as BCG
(Figu e 2G), by low cy ome y, only e y low equencies o IL-
10+ CD4+ T cells we e de ec ed in he BAL o mucosally acci-
na ed animals (Figu e 2H). Al hough he equencies a e low and
conclusions he e o e li le obus , on a e age, only 2% o IL-
17+CD4+ BAL T cells ob ained om mucosally accina ed ani-
mals we e ound o be IL-10+ (da a no shown). Thus, i seems
ha he high le els o IL-10 may no be T cell de i ed bu p o-
duced by local inna e immune cells in esponse o inna e ecep-
o liga ion by mycobac e ial compounds in he PPD p epa a ion.
Pulmona y accina ion wi h MTBVAC induces ypically as e
and highe poly unc ional CD4+ T cell esponses and IL-10
sec e ion signals associa ed p e iously wi h p o ec ion by pul-
mona y BCG accina ion.
Pe iphe al immuni y a e pulmona y accina ion
In pa allel o he pulmona y immune esponses, we p o iled pe-
iphe al T cell immuni y in sea ch o po en ial co ela es o p o-
ec ion wi h a pe spec i e o ansla ion o clinical se ings.
Howe e , as be o e, when assessing adap i e PPD-speci ic
CD4+ and CD8+ T cell cy okine esponses by low cy ome y,
no disc imina ing quali a i e signals could be iden i ied ha
dis inguished mucosally om in ade mally accina ed animals.
CD4+ T cell cy okine p oduc ion was obse ed om week 3
pos - accina ion onwa d and was mos p ominen in in ade -
mally accina ed animals and he M.muc g oup (Figu e 3A; Fig-
u es S3A–S3D). A sligh inc ease in PPD-speci ic CD8+ T cell
cy okine p oduc ion was only appa en in he in ade mally
accina ed g oups 6 weeks pos - accina ion (Figu e 3B; Figu es
S4A–S4D). No di e ences in CD4+ and CD8+ T cell poly unc-
ionali y could be de ec ed.
We used an IFNgenzyme-linked immune abso ben spo (ELI-
SPOT) assay o assess he b ead h o immune esponses
induced by MTBVAC in compa ison wi h BCG. In line wi h he
low cy ome y da a, a e s imula ion wi h PPD, which con ains
an igens sha ed by BCG and MTBVAC, in ade mal MTBVAC
was indis inguishable om in ade mal BCG by IFNgsec e ion
(Figu e 3C). A e s imula ion wi h ESAT6 and CFP10, an igens
p oduced by MTBVAC bu absen om BCG, we obse ed
IFNgp oduc ion only by pe iphe al blood mononuclea cells
(PBMCs) o MTBVAC- accina ed animals ega dless o accina-
ion ou e (Figu e 3D). O no e, mucosal MTBVAC appea ed o be
equally po en in inducing PPD-speci ic IFNgsignals (Figu e 3C).
Al hough we p e iously obse ed compa able pe iphe al im-
mune esponses a e mucosal and in ade mal BCG accina-
ion, he e mucosal BCG accina ion appea ed o be less po en
in inducing pe iphe al cy okine p oduc ion and p oli e a ion (Fig-
u es 3A–3C and 3E, espec i ely).
Figu e 2. Pulmona y mucosal accina ion wi h MTBVAC induces immune signa u es associa ed wi h p o ec ion
Shown is an o e iew o BAL cell immune esponses a e mucosal o in ade mal accina ion wi h BCG o MTBVAC.
(A–D) Flow cy ome y analysis o e ime o (A) IFNg, (B) TNF-a, (C) IL-2, and (D) IL-17A CD4+ T cell esponses a e accina ion.
(E) S acked ba g aphs depic ing CD4+ T cell cy okine poly unc ionali y o e ime a e PPD ecall s imula ion (by g oup median alues).
(F) PPD-speci ic cy okine p oduc ion o CD4+ T cells in he lowe igh and lowe le lung lobes a week 3 and week 8, indica ing p ima y and dissemina ed
accine esponses.
(G) Sec e ion o IL-10 by un ac iona ed BAL cells s imula ed wi h PPD a week 8, plo ed as cul u e medium con ol-co ec ed alues.
(H) Flow cy ome y analysis o IL-10 p oduc ion by CD4+ T cells o e ime a e accina ion.
All g aphs show 6 animals pe g oup. In (A)–(D) and (H), + indica es PPD-s imula ed samples, and indica es uns imula ed, cul u e medium-incuba ed samples
as con ols. Ho izon al lines wi hin ba s indica e g oup medians. Signi icance o g oup di e ences was de e mined by wo-sided Mann-Whi ney es adjus ed o
mul iple compa isons. Holms-adjus ed p %0.05 is depic ed. Colo coding pe indi idual is consis en h oughou he pape .
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As an al e na i e app oach, we in es iga ed he capaci y o
accine-induced T cells by an in i o ecall s imula ion in TST.
To his end, 8 weeks a e accina ion, we in ade mally injec ed
saline (Sal) o old ube culin (Tub) on opposi e a ms o each an-
imal and ook biopsies o he injec ion si es 3 days la e . The skin
biopsies we e subsequen ly p ocessed and cha ac e ized by
low cy ome y o measu e he delayed ype hype sensi i i y
(DTH) esponse. By isual inspec ion o he local skin eac ion,
edness and swelling appea ed a e in ade mal bu no
mucosal accina ion (da a no shown). Acco dingly, a ube cu-
lin-speci ic in lux o an igen-speci ic CD3+ T cells was exclu-
si ely obse ed in in ade mally accina ed animals (Figu e 3F).
These T cells showed highe equencies o cy okine-p oducing
subse s a e in ade mal accina ion (Figu e 3G). When assess-
ing an igen-speci ic T cells om axilla y lymph nodes ha d ain
he TST-DTH skin si e, IFNg-, TNF-a-, and IL-2-p oducing
CD4+ T cells we e de ec able in in ade mally bu no mucosally
accina ed animals (Figu e S5). So, al hough mucosal accina-
ion does esul in a pe iphe al blood esponse (by low cy ome-
y and IFNgELISPOT), i does no enable hese cells o mig a e
o he si e o a skin challenge and, he e o e, ules ou hei anal-
ysis o a po en ial bioma ke assay.
The supe io induc ion o local immune esponses by mucosal
MTBVAC o e mucosal BCG was also e lec ed in he pe iphe y,
and hese esponses appea ed o co e a b oade ange o an i-
gens, including ESAT6/CFP10. Howe e , wi hin he limi s o ou
analyses, no pe iphe al adap i e esponses disc imina ing be-
ween mucosal e sus in ade mal immuniza ion could be
iden i ied.
Mucosal homing ma ke exp ession a e accina ion
In ou sea ch o pe iphe al co ela es o p o ec ion, we also
conside ed he possibili y ha pulmona y a he han in ade mal
accina ion would imp in pe iphe al T cells wi h a highe exp es-
sion o pulmona y mucosal homing ma ke s. To his end, we as-
sessed he exp ession o CD103, CXCR3, and CCR5, all known
o be in ol ed in homing o he pulmona y mucosa,
27
on pe iph-
e al CD4+ T cells by means o low cy ome y.
P io o accina ion, app oxima ely 25% o ci cula ing CD4+
T cells exp essed one o mo e o hese homing ma ke s. A e
accina ion, ei he pe iphe ally o mucosally, his pe cen age
did no change, no was he pa e n o homing ma ke co-
exp ession no ably al e ed in he mucosally accina ed g oups
(Figu e 4A; Figu e S6). Because only a small ac ion o all pe iph-
e al CD4+ T cells is accine speci ic (Figu e 3A), we also as-
sessed homing ma ke exp ession o cy okine-p oducing
CD4+ T cells (IFNgand/o IL-17A). Howe e , because o he
low numbe o cy okine-posi i e e en s in he pe iphe y, i was
no possible o measu e obus and eliable pe cen ages o hom-
ing ma ke exp ession o e ime. Using a cu o o a minimum o
100 cy okine-posi i e e en s, we only ound obus equencies
8 weeks pos - accina ion. The equency o homing ma ke s ex-
p essed by PPD-speci ic T cells was compa able wi h ha in he
o al CD4+ T cell popula ion, al hough cy okine+ CD4+ T cells
om all accina ed g oups consis ed o mo e CCR5 single-pos-
i i e cells (Figu e 4B). When compa ing homing ma ke exp es-
sion be ween g oups, again, no ma ked di e ences be ween
he mucosally and in ade mally accina ed g oups we e
appa en .
Al hough he a o emen ioned homing ma ke s did no e eal a
pe iphe al co ela e ei he , we wen on o analyze hei exp ession
on cy okine-p oducing T cells in he ai ways a e mucosal acci-
na ion as well as a e expe imen al pulmona y M b in ec ion. Fo
he la e , samples we e ob ained om ano he , independen
in ec ion s udy o cha ac e ize p o ec i e e sus pa hogenic BAL
esponses. Again, we analyzed he exp ession o CD103,
CXCR3, and CCR5 o PPD-speci ic IFNgand/o IL-17A-p oducing
Tcells.A e M b in ec ion, a high local CD4+ T cell cy okine
esponse is induced, simila o mucosal MTBVAC and highe
han mucosal BCG adminis a ion (Figu e 4C). Howe e , he
exp ession o homing ma ke s was signi ican ly lowe in PPD-spe-
ci ic T cells om M b-in ec ed animals compa ed wi h animals
accina ed mucosally wi h BCG o MTBVAC (Figu es 4Dand
4E). Al hough, a e M b in ec ion, app oxima ely 20% o cy o-
kine-p oducing cells exp essed a combina ion o CD103,
CXCR3, and CCR5, 80% o accina ion-induced T cells we e pos-
i i e o one o mo e o hese ma ke s (Figu e 4D). In addi ion o
p o iling chemokine ecep o exp ession, we also measu ed
CD69 and PD-1 co-exp ession on cy okine-posi i e CD4+
T cells as an indica o o a unc ional issue- esiden pheno ype.
28
P e iously, we ha e ound ha CD69 exp ession o BAL CD4+
T cells was highe a e mucosal o e in ade mal BCG accina ion
and, sepa a ely, ha IFNg+TNF-a+IL-2+IL-17A+ T cells ex-
p essed highe le els o PD-1.
20
When assessing co-exp ession
o hese wo ma ke s on IFNg-and/o IL-17A-p oducingCD4+
T cells induced by mucosal accina ion, a subs an ial po ion
(15%–50%) o hese cells was ound o co-exp ess bo h ma ke s.
Con a ily, a e M b in ec ion, CD69 and PD1 co-exp ession on
PPD-speci ic T cells was signi ican ly lowe ( ypically less han
10%) (Figu e 4F).
Pulmona y accina ion, associa ed p e iously wi h enhanced
p o ec ion, esul s in he p esence o an igen-speci ic T cells
Figu e 3. Pe iphe al immune esponses a e accina ion
Shown is a cha ac e iza ion o he heigh and b ead h o pe iphe al immune esponses a e accina ion.
(A and B) S acked ba g aphs depic ing (A) CD4+ and (B) CD8+ T cell cy okine poly unc ionali y o e ime (by g oup median alues) a e PPD s imula ion.
(C and D) PBMC IFNgp oduc ion in esponse o s imula ion wi h (C) PPD o (D) ESAT6-CFP10 usion p o ein, measu ed by ELISPOT o e ime.
(E) PPD-speci ic p oli e a ion o PBMCs, plo ed as a s imula ion index ( he a io o an igen- o e medium con ol-s imula ed alues) o e ime.
(F) T cell numbe s in skin biopsies aken (3 days) a e in ade mal injec ion o saline (Sal) o old ube culin (Tub) 8 weeks a e accina ion.
(G) PPD-speci ic cy okine p oduc ion by T cells om Tub skin biopsies ( igh panel).
The do ed line in (C) indica es he maximum limi o de ec ion. In (G), + indica es PPD-s imula ed samples, and indica es uns imula ed, cul u e medium-
incuba ed samples as con ols. All g aphs show 6 animals pe g oup, excep o (G), whe e he e a e 5 animals o he B.muc, M.id, and M.muc g oups. Ho izon al
lines indica e g oup medians. Signi icance o g oup di e ences was de e mined by wo-sided Mann-Whi ney es adjus ed o mul iple compa isons. Holms-
adjus ed p %0.05 is depic ed. Colo coding pe indi idual is consis en h oughou .
6Cell Repo s Medicine 2, 100187, Janua y 19, 2021
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wi h a dis inc issue esidency and mucosal homing pheno ype.
The obse a ion ha M b in ec ion does no elici his pheno ype
sugges s ha hese cells could be in ol ed in p o ec ion.
Mucosal an ibody le els and unc ionali y
In ou p e iously epo ed accina ion and RLD M b in ec ion
s udy, mucosal BCG accina ion esul ed in pulmona y PPD-
speci ic Ig esponses.
20
This obse a ion, in combina ion wi h
he ecen in e es in he ole o Igs in p o ec ion om TB,
29,30
p omp ed us o in es iga e he humo al immune esponse in
his s udy in mo e de ail.
As obse ed p e iously o BCG, mucosal accina ion wi h
MTBVAC also esul ed in a ma ked inc ease in M b-speci ic
IgA, IgG, and IgM le els in BAL luid, as de ec ed by ELISA,
and in a modes inc ease in some o he in ade mally
MTBVAC- accina ed animals (Figu e 5A). Like he cellula im-
mune esponses, humo al immuni y was ound o dissemina e
om he accine- a ge ed lobe (Figu e 5B).
A
BC
E
D
F
Figu e 4. Exp ession o mucosal homing ma ke s a e in ade mal and pulmona y accina ion
(A) Exp ession o CCR5, CD103, and CXCR3 on ex i o CD4+ T cells om pe iphe al blood o e ime, depic ed as g oup median alues.
(B) CCR5, CD103, and CXCR3 exp ession by PPD-speci ic (IFNg+ and/o IL-17A+) CD4+ T cells om PBMCs a week 8 a e accina ion.
(C–E) Compa ison o PPD-speci ic T cells om BALs om pulmona y BCG- accina ed, MTBVAC- accina ed, and M b-in ec ed animals.
(C) F equencies o IFNg+ and/o IL-17A+ CD4+ T cells a e s imula ion wi h PPD.
(D and E) Exp ession o CCR5, CD103, and CXCR3 by cy okine+ CD4+ T cells, (D) depic ed as g oup median alues (s acked ba g aph) o (E) as indi idual
equencies o sepa a e ma ke s.
(F) Pe cen age o CD69 and PD1 double-posi i e cells o cy okine+ CD4+ T cells.
Fo all g aphs, n = 6 animals pe g oup, wi h he excep ion o (B) whe e n = 5 o he BCG.muc g oup. Ho izon al lines indica e g oup medians. Signi icance o
g oup di e ences was de e mined by wo-sided Mann-Whi ney es adjus ed o mul iple compa isons. Holms-adjus ed p %0.05 is depic ed. Colo coding pe
indi idual is consis en h oughou .
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A
B
C
D
(legend on nex page)
8Cell Repo s Medicine 2, 100187, Janua y 19, 2021
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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 #: 565283; RRID: AB_2739154
an i-CD20 – BV421 (clone 2H7) Biolegend Ca #: 302334; RRID: AB_10965543
an i-CD28 – ECD (clone CD28.2) IOTes Ca #: 6607111; RRID: AB_1575955
an i-CD45 – BV786 (clone D058-1283) BD Biosciences Ca #: 563861; RRID: AB_2738454
an i-CD45RA – PE-CF594 (clone 5H9) BD Biosciences Ca #: 565419; RRID: AB_2739229
an i-CD69 – APC (clone FN-50) Biolegend Ca #: 310910; RRID: AB_314845
an i-CD69 – BV785 (clone FN-50) Biolegend Ca #: 310932; RRID: AB_2563696
an i-CD95 – BV605 (clone DX2) Biolegend Ca #: 305628; RRID: AB_2563825
an i-CD103 – FITC (clone Be -ACT8) Biolegend Ca #: 350204; RRID: AB_10639865
an i-CCR5 – APC-H7 (clone 3A9) BD Biosciences Ca #: 560748; RRID: AB_1937308
an i-CCR7 – BV650 (clone G043H7) Biolegend Ca #: 353234; RRID: AB_2563867
an i-CXCR3 – PE-Cy7 (clone G025H7) Biolegend Ca #: 353720; RRID: AB_11219383
an i-PD1 – BV510 (clone EH12.2H7) Biolegend Ca #: 329932; RRID: AB_2562256
an i-IFN-g– BV711 (clone 4S.B3) BD Biosciences Ca #: 502540; RRID: AB_2563506
an i-IL-2 – AF488 (clone MQ1-17H12) Biolegend Ca #: 500314; RRID: AB_493368
an i-IL10 – PE (clone JES3-9D7) Biolegend Ca #: 501404; RRID: AB_315170
an i-IL-17A – PE-Cy7 (ebio64DEC17) Biolegend Ca #: 25-7179-42; RRID: AB_11063994
an i-IL-17A – BV605 (clone BL168) Biolegend Ca #: 512326; RRID: AB_2563887
an i-TNF-a– BV650 (clone Mab11) BD Biosciences Ca #: 502938; RRID: AB_2562741
VIVID – BV421 The mo ishe Ca #: L34955
GolgiPlug BD Biosciences Ca #: 555029; RRID: AB_2869014
Cy o ix/Cy ope m BD Biosciences Ca #: 554714; RRID: AB_2869014
Goa An i-Human IgM(mchain) An ibody, Alkaline
Phospha ase (AP) Conuga e, A ini y pu i ied
In i ogen Ca #: A18838; RRID: AB_2535615
Goa An i-Human IgA(achain) An ibody, Alkaline Phospha ase
(AP) Conuga e, A ini y pu i ied
In i ogen Ca #: A18784; RRID: AB_2535561
An i-MONKEY IgG (gamma chain)(GOAT) An ibody
Pe oxidase Conjuga ed
Rockland Inc Ca #: 617-103-012; RRID: AB_218715
IgA bio inyla ed de ec ion Ab (MT57) Mab ech 3860-4; RRID: AB_10736549
IgG bio inyla ed de ec ion Ab (MT78) Mab ech 3850-6; RRID: AB_10666158
IgM bio inyla ed de ec ion Ab (MT22) Mab ech 3880-6; RRID: NA
Bac e ial and i us s ains
M. ube culosis s ain E dman K01 BEI Resou ces Ca #: NR-50781
BCG, s ain So ia (5 310
5
CFU/dose) In e Vax L d Ca #: N/A
MTBVAC (5 310
5
CFU/dose) Bio ab i Ca #: N/A
Chemicals, pep ides, and ecombinan p o eins
Lymphop ep
TM
Axis-Shield Ca #: AXI-1114547
Pe chlo ic acid Sigma-Ald ich Ca #: 244252-1L
Fo maldehyde (16%) The mo Scien i ic Ca #: 28906
p-Ni ophenyl Phospha e (p-NPP) Alkaline Phospha ase
Subs a e
Me ck Millipo e Ca #: ES009-500mL
(Con inued on nex page)
Cell Repo s Medicine 2, 100187, Janua y 19, 2021 e1
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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 , F ank
Ve eck ([email p o ec ed]).
Ma e ials a ailabili y
This s udy did no gene a e new unique eagen s. Howe e , any emaining bioma e ials om his s udy can be made a ailable and
shipped a ecei e ’s cos upon speci ic eques o he Lead Con ac , o which we equi e comple ion o a Simple Le e Ag eemen
o T ans e o Ma e ials.
Da a and code a ailabili y
All da a he e is, a e p esen ed in his pape (including supplemen als) and can be made a ailable in di e en o ma s upon easonable
eques . This s udy did no gene a e any unique code.
EXPERIMENTAL MODEL AND SUBJECT DETAILS
E hics & Animal Handling
All housing and animal ca e p ocedu es we e pe o med a he Biomedical P ima e Resea ch Cen e (BPRC) in Rijswijk, he
Ne he lands, and in compliance wi h Eu opean di ec i e 2010/63/EU as well as he ‘‘S anda d o Humane Ca e and Use o Labo-
a o y Animals by Fo eign Ins i u ions’’ p o ided by he Depa men o Heal h and Human Se ices o he US Na ional Ins i u es o
Heal h (NIH, iden i ica ion numbe A5539-01). BPRC is acc edi ed by he Ame ican Associa ion o Acc edi a ion o Labo a o y An-
imal Ca e (AAALAC). An e hical amewo k app o al om he independen , cen al animal expe imen s au ho i y in he Ne he lands (in
Du ch: Cen ale Commissie Die p oe en, CCD) was in place, and be o e s a he s udy plan was app o ed by BPRC’s ins i u ional
animal wel a e body (in Du ch: Ins an ie oo Die welzijn, I D). The BCG and MTBVAC immunogenici y s udy was egis e ed unde
CCD.009.D (while he M b-in ec ion samples used o immunological c oss-compa ison, we e aken om a s udy egis e ed unde
CCD.009.C; see also u he below).
Con inued
REAGENT o RESOURCE SOURCE IDENTIFIER
S ep a idine-FITC Biolegend Ca #: 405202
TMB, ELISA subs a e MT Diagnos ics Ca #: SB04/B
TMB, ELISPOT subs a e Mab ech Ca #: 3651-10
C i ical comme cial assays
NHP speci ic IFN-gamma ELISPOT an ibody pai s U-CyTech Ca #: 610-10
BOVIGAM
TM
Tube culin PPD s imula ing an igen, Bo ine;
Pu i ied P o ein De i a i e (M. bo is)
Li e Technol. NV Ca #: 760060
BOVIGAM
TM
Tube culin PPD s imula ing an igen, A ian;
Pu i ied P o ein De i a i e (M. a ium)
Li e Technol. NV Ca #: 760065
Milliplex NHP Cy okine Magne ic Bead Panel Me ck Millipo e Ca #: PRCYTOMAG 40K
Expe imen al models: o ganisms/s ains
pu pose-b ed Macaca mula a ( hesus macaques); adul
(> 4 yea s o age) males and emales; Indian-geno ype
BPRC N/A
So wa e and algo i hms
Eli.Analyze (ELISPOT; 6.1) A.EL.VIS GmbH N/A
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 N/A
G aphPad P ism 8.4.2 G aphPad So wa e h ps://www.g aphpad.com:443/
O he
Old Tube culin Synbio ics, Inc N/A
Mycobac e ium Tube culosis - ube culine PPD o in i o
use; Pu i ied P o ein De i a i e (M. ube culosis)
AJ Vaccines Ca #: 2391
M. ube culosis s ain HN878 Whole Cell Lysa e BEI Resou ces Ca #: NR-14824
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Twel e emale and wel e male Indian- ype hesus macaques (Macaca mula a) we e selec ed om BPRC’s b eeding colonies and
s a i ied by gende , age, body weigh and social indica o s o pai wise housing in o 4 g oups o 6 animals. T ea men was andomly
assigned o each g oup.
Selec ed animals we e nega i e o p io exposu e o mycobac e ia, as assessed by ube culin skin es ing wi h Old Tube culin
(Synbio ics Co po a ion, San Diego, CA) and an IFNgELISPOT using Pu i ied P o ein De i a i e (PPD) om Mycobac e ium bo is,
Mycobac e ium a ium (bo h Li e Technologies NV) o Mycobac e ium ube culosis (AJ Vaccines, Copenhagen, Denma k) o
in i o ecall s imula ion o PBMC.
Animals we e housed pai wise a biosa e y le el 3 h oughou he expe imen and p o ided wi h en ichmen in he o m o ood and
non- ood i ems on a daily basis. Animal wel a e was moni o ed daily. Animal weigh was eco ded p io o each blood collec ion e en .
All animal handling and bio-sampling was pe o med unde ke amine seda ion (10 mg/kg, by in a-muscula injec ion). Fo endo-
b onchial ins illa ion ke amine seda ion (5mg/kg) was supplemen ed wi h in amuscula mede omidine (0.04 mg/kg) and an analgesic
applied o he la ynx.
Eigh weeks a e accine adminis a ion animals eached s udy endpoin by p o ocol and we e eu hanized by in a enous injec ion
o pen oba bi al (200 mg/kg) unde ke amine seda ion. All animal ca e and e e ina y pe sonnel we e blinded o expe imen al
ea men .
Vaccines, Vaccine P epa a ion & Adminis a ion
Animals we e accina ed ei he wi h Bacillus Calme e Gue
´ in s ain So ia (In e Vax L d., On a io) o wi h MTBVAC (Bio ab i, Spain),
and ei he ia he skin o he pulmona y mucosa. The in ade mally accina ed g oups ecei ed a s anda d, adul human dose o 1.5-
6.0 x10
5
CFU BCG o 3.0-17.0 x10
5
CFU MTBVAC in 0.1 mL econs i u ed accine in he skin (abb e ia ed as BCG.id o MVAC.id).
The mucosally accina ed g oups we e adminis e ed he same dose, bu in 10 mL o s e ile saline solu ion by endob onchial
ins illa ion in o he lowe igh lung lobe (abb e ia ed as BCG.muc/B.muc o MTBVAC.muc/M.muc). The accines, ega dless o
adminis a ion ou e, we e p epa ed om a single, pooled mix o eshly econs i u ed ials, immedia ely p io o adminis a ion.
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.
M b In ec ion
Fo he compa ison o homing ma ke exp ession by cy okine posi i e pulmona y mucosal T cells, BALs om M b in ec ed animals
we e ob ained 11 weeks a e endob onchial ins illa ion o 3 o 15 CFU o M b E dman (NR-50781, BEIResou ces). In ec ion was
con i med o all animals by he induc ion o M b-speci ic IFNgp oduc ion by PBMCs and by pos -mo em pa hology assessmen
(da a no shown).
Biosample Collec ion & P ocessing
Cells om he pulmona y mucosa we e eco e ed a speci ic ime poin s by b oncho-al eola la age (BAL), a ge ing ei he he lowe
igh o lowe 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 minu es a 400 g a e 100 mm il a ion. Supe na an was subse-
quen ly decan ed and s o ed a 80C pending u he analysis. The BAL cell pelle was aken up in RPMI supplemen ed wi h
10% e al bo ine se um (FBS), glu amax and penicillin/s ep omycin ( om he eon e e ed o as R10) and used in downs eam as-
says. BAL luid was il e -s e ilized by cen i uga ion h ough 0.2 mm PVDF memb ane pla es (Fishe Scien i ic) be o e analysis.
Pe iphe al blood mononuclea cells (PBMC) we e isola ed om hepa inised blood collec ed by enepunc u e. Isola ion o PBMCs
was pe o med by densi y g adien cen i uga ion wi h Lymphop ep lymphocy e sepa a ion medium (Axis-Shield, UK), and PBMCs
we e subsequen ly esuspended in R10 o downs eam immunological assays.
METHOD DETAILS
Flow cy ome y
T cell cy okine p oduc ion and homing ma ke exp ession was assessed by low cy ome y. F eshly isola ed PBMC we e incuba ed
o e nigh wi h M b PPD (5 ug/mL) in he p esence o GolgiPlug anspo inhibi o (BD Biosciences). PMA/ionomycin s imula ed sam-
ples we e aken along as echnical/posi i e con ols. The nex day, cells we e washed and incuba ed wi h he panels lis ed in he Key
Resou ces Table. To acili a e in acellula cy okine s aining, cells we e pe meabilized wi h Cy o ix/Cy ope m (BD Biosciences) be o e
addi ion o cy okine an ibodies. A e o e nigh ixa ion wi h 2% pa a o maldehyde,
66
samples we e acqui ed on a 3-lase , 14-colo
LSR-II low cy ome e (BD Biosciences). Analyses we e pe o med in FlowJo e sion 10 (T ees a ). T cells we e selec ed as Sin-
gle s/Lymphocy es/Viable/CD14-CD20-/CD45+/CD3+ e en s, a e which u he CD4 and CD8 ga ing was applied. Any anomalies
indica i e o uns able signal acquisi ion we e excluded using he ‘‘Time’’ pa ame e . Cy okine posi i i y was de e mined by placemen
o cy okine ga es on he medium con ol samples and subsequen ly applying he ga es o he co esponding PPD s imula ed samples.
Luminex
Cy okine p oduc ion by BAL cells s imula ed o 72 hou s wi h PPD (5 ug/mL, inal concen a ion), was assessed by cus omised Milli-
plex Luminex ki s (Me ck Millipo e, USA). Assays we e pe o med acco ding o manu ac u e ’s p o ocol. In sho : supe na an s o
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s imula ed BAL 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 o an ibodies and PE-labeled s ep a idin. Beads we e acqui ed on a Bioplex 200 sys em and cy okine le els
we e calcula ed wi h Bioplex Manage so wa e e sion 6.1 (bo h Bio ad, CA, USA).
IFNgELISPOT
Non-human p ima e speci ic IFNgELISPOT (U-CyTech, he Ne he lands) was pe o med on PBMC acco ding o manu ac u e ’s p o-
ocol on. B ie ly, 200,000 PBMC we e incuba ed in iplica e o 24 hou s wi h M b-de i ed PPD (AJ Vaccines, Denma k) o ecom-
binan ESAT6-CFP10 usion p o ein (p o ided by Kees F anken om he O enho lab, Leiden Uni e si y Medical Cen e). The nex
day cells we e washed and ans e ed o an i-IFNgcoa ed memb ane pla es (Millipo e). A e 24 hou s, cells we e disca ded and
memb ane-bound IFNgwas isualized using a bio inyla ed an i-IFNgde ec o an ibody, s ep a idin-ho se adish pe oxidase conju-
ga e and e ame hylbenzidine subs a e. Spo s we e quan i ied using an au oma ed eade (AELVIS, Hanno e ).
Immunoglobulin ELISA
An ibody le els in BAL we e de e mined by Enzyme Linked ImmunoSo ben Assay (ELISA). In b ie , 96-well pla es we e coa ed wi h
ei he 5 mg/mL M b s ain HN828 Whole Cell lysa e (BEI Resou ces, VA, USA) in PBS. A e o e nigh blocking wi h 1% BSA, samples
we e added o he wells. Bound an ibodies we e subsequen ly de ec ed ei he wi h ho se adish pe oxidase-conjuga ed an i-IgG
(Rockland, PA, USA), alkaline phospha ase-conjuga ed an i-IgA (Fishe Scien i ic) o alkaline phospha ase-conjuga ed IgM (Sigma),
and he subsequen addi ion o pa a-ni ophenylphospha e subs a e o ELISA colo de elopmen . All samples we e no malized o
a bi a y uni s (AU) agains a se ial dilu ion o a posi i e e e ence sample included in all assays.
M b binding and phagocy osis assay
To assess M b binding o immunoglobulins, 0.05 mL aliquo s o BAL luid ob ained p io o and 8 weeks a e accina ion, we e incu-
ba ed a 37C o 1 hou wi h 10
7
CFU o M b H37R -dsRed. (Ko bee e al., 2018) Subsequen ly, samples we e equally di ided o e
h ee ials and bio inyla ed de ec ion an ibodies speci ic o IgA, IgG o IgM (all om Mab ech) we e added ( inal dilu ion 1:500). A e
30 minu es o incuba ion a oom empe a u e, s ep a idin-FITC ( inal dilu ion 1:400) was added, and samples we e incuba ed o a
u he 60 minu es owa d de ec ion o bound an ibody. Samples we e ixed o e nigh wi h 2% PFA be o e analysis.
Fo he phagocy osis assay, 5x10
5
THP1 cells we e seeded in a 24-wells pla e and ac i a ed by o e nigh incuba ion wi h 10ng/mL
PMA a 37C. The nex day, 10
7
CFU o M b H37R -GFP was incuba ed wi h BAL luid as desc ibed abo e, and subsequen ly added
o he ac i a ed THP-1 cells. A e 4 hou s cells we e dissocia ed wi h ypsin-EDTA and esuspended in 2% PFA o o e nigh ix-
a ion. The binding assay samples we e subsequen ly acqui ed on a 4 lase , FACSA iaIII sys em; samples om he phagocy osis
assay we e acqui ed on a 3 lase , Beckman Coul e Gallios sys em. Fo bo h assays, M b H37R -dsRed o -GFP incuba ed in
PBS only was aken along as a nega i e con ol.
QUANTIFICATION AND STATISTICAL ANALYSIS
S a is ical analysis was pe o med wi h G aphpad P ism e sion 8 and R e sion 3.5.1.
67
Signi icance o di e ences be ween g oups
was calcula ed by wo-sided Mann-Whi ney es ing o which Holm’s adjus ed p alues a e epo ed. Co ela ion s a is ics we e
gene a ed by Spea man’s ank analysis.
e4 Cell Repo s Medicine 2, 100187, Janua y 19, 2021
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