Pulmonary MTBVAC vaccination induces immune signatures previously correlated with prevention of tuberculosis infection

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 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 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. 2Cell Repo s Medicine 2, 100187, Janua y 19, 2021 A icle ll OPEN ACCESS A B C D E F H G (legend on nex page) Cell Repo s Medicine 2, 100187, Janua y 19, 2021 3 A icle ll OPEN ACCESS 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 . 4Cell Repo s Medicine 2, 100187, Janua y 19, 2021 A icle ll OPEN ACCESS A B C D E FG (legend on nex page) Cell Repo s Medicine 2, 100187, Janua y 19, 2021 5 A icle ll OPEN ACCESS 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 A icle ll OPEN ACCESS 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 . Cell Repo s Medicine 2, 100187, Janua y 19, 2021 7 A icle ll OPEN ACCESS A B C D (legend on nex page) 8Cell Repo s Medicine 2, 100187, Janua y 19, 2021 A icle ll OPEN ACCESS 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 A icle ll OPEN ACCESS 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 e2 Cell Repo s Medicine 2, 100187, Janua y 19, 2021 A icle ll OPEN ACCESS 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 Cell Repo s Medicine 2, 100187, Janua y 19, 2021 e3 A icle ll OPEN ACCESS 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 A icle ll OPEN ACCESS