Elucidating different pattern of immunoregulation in BALB/c and C57BL/6 mice and their F1 progeny

1 Vol.:(0123456789) Scien i ic Repo s | (2021) 11:1536 | h ps://doi.o g/10.1038/s41598-020-79477-7 www.na u e.com/scien i ic epo s Elucida ing di e en pa e n o immuno egula ion in BALB/c and C57BL/6 mice and hei F1 p ogeny Wiebke Ha mann1*, Bi e Blankenhaus1,3, Ma ie‑Luise B unn1, Jana Meine s1 & Minka B eloe 1,2 Helmin hs a e la ge mul icellula pa asi es ha in ec one qua e o he human popula ion. To p olong hei su i al, helmin hs supp ess he immune esponses o hei hos s. S ongyloides a i delays i s expulsion om he gu by induc ion o egula o y ci cui s in a mouse s ain‑speci ic manne : deple ion o Foxp3+ egula o y T cells (T eg) imp o es he an i‑S. a i immuni y in BALB/c bu no in C57BL/6 mice. In he cu en s udy we compa e he hie a chy o immuno egula o y pa hways in BALB/c, C57BL/6 mice and hei F1 p ogeny (BALB/c × C57BL/6). Using mul icolo low cy ome y, we show ha S. a i induces a dis inc pa e n o inhibi o y checkpoin ecep o s by Foxp3+ T eg and Foxp3− T cells. In ensi y o exp ession was highes in C57BL/6 and lowes in BALB/c mice, while he F1 c oss had an in e media e pheno ype o esembled BALB/c mice. T eg subse s expanded du ing in ec ion in all h ee mouse s ains. Simila o BALB/c mice, deple ion o T eg educed in es inal pa asi e bu den and inc eased mucosal mas cell ac i a ion in S. a i‑in ec ed F1 mice. Ou da a indica e ha T eg domina e he egula ion o immune esponses in BALB/c and F1 mice, while mul iple egula o y laye s exis in C57BL/6 mice ha may compensa e o he absence o T eg. Helmin hs a e la ge mul icellula pa asi es ha cause ch onic in ec ions wi h an impai ed p o ec i e immune esponse. S. a i is a well-es ablished mu ine model o in es inal nema ode in ec ions wi h issue mig a ing s ages. In ec i e hi d s age la ae (iL3) ei he ac i ely pene a e he skin o hei oden hos o a e injec ed subcu aneously. Wi hin 2days he la ae mig a e ia incomple ely de ined pa hways o he head and a e swal- lowed. By day 3 pos in ec ion (p.i.) iL3 each he in es ine and de elop ia a ou h la al s age in o emale adul s. Pa asi es li e embedded in he mucosa o he in es ine and ep oduce ia pa henogenesis by day 5 p.i. Eggs and al eady ha ched i s s age la ae a e eleased in he en i onmen wi h he eces. BALB/c and C57BL/6 mice bo h de elop pa en in ec ions ha a e clea ed in he con ex o a ype 2 immune esponse a e 2–4weeks. While he kine ics o inal clea ance is simila , C57BL/6 mice usually display highe in es inal pa asi e bu dens a he peak o in ec ion compa ed o BALB/c mice1–3. Helmin hs exploi hei hos s’ immune egula o y pa hways and induce he expansion o se e al egula o y cells and ecep o s in o de o p olong hei su i al4. Regula o y T cells ha e been desc ibed o be c i ical in p e en ing se e e immune pa hology5,6 and in dampening he an i-helmin h immune esponses in mice and humans3,7–12. Regula o y T cells can be classi ied in CD4+ Foxp3+ T eg and IL-10 p oducing CD4+ Foxp3− ype 1 egula o y (T 1) T cells. We ha e p e iously shown ha S. a i in ec ion induces expansion o Foxp3+ T eg in BALB/c and C57BL/6 mice3. Using he deple ion o egula o y T cells (DEREG) mouse model on hese wo gene ic backg ounds, T eg deple ion imp o es expulsion o S. a i selec i ely in BALB/c mice, bu no in C57BL/6 mice. Mechanis ically, imp o ed esis ance in T eg-deple ed BALB/c mice is media ed by inc eased IL-9-d i en mas cell ac i a ion. Mas cell de iciency o IL-9 blockade, bu no applica ion o an an i-G -1 an ibody in T eg- deple ed BALB/c DEREG mice ab oga es his pheno ype3. Ou da a indica e ha T eg con ol he ac i a ion o mas cells in BALB/c mice ia egula ion o IL-9, while G -1+ g anulocy es such as neu ophils and eosinophils13 a e dispensable. Despi e inc eased ype 2 immune esponses in T eg-deple ed C57BL/6 mice, he ele a ion o S. a i-speci ic IL-9 p oduc ion is no su icien o igge inc eased mas cell ac i a ion and accele a e pa asi e clea ance, sugges ing he p esence o addi ional egula o y elemen s. Indeed, we obse ed ha up egula ion o he OPEN 1Helmin h Immunology G oup, Be nha d Noch Ins i u e o T opical Medicine, Hambu g, Ge many. 2Depa men o Biology, Uni e si y o Hambu g, Hambu g, Ge many. 3Ins i u o de Medicina Molecula João Lobo An unes, Lisbon, Po ugal. *email: [email p o ec ed] 2 Vol:.(1234567890) Scien i ic Repo s | (2021) 11:1536 | h ps://doi.o g/10.1038/s41598-020-79477-7 www.na u e.com/scien i ic epo s/ inhibi o y ecep o BTLA by T cells nega i ely egula es he an i-helmin h immune esponse in S. a i-in ec ed C57BL/6 mice14. C57BL/6 mice ei he de icien o B and T lymphocy e a enua o (BTLA) o i s ligand he pes i us en y media o (HVEM) ha e educed numbe s o pa asi ic adul s in he small in es ine, educed la al ou pu h oughou he in ec ion as well as inc eased IL-9-d i en mas cell ac i a ion. The aim o he cu en s udy was o compa e he hie a chy o immune egula o y pa hways du ing in ec ion wi h S. a i in BALB/c, C57BL/6 mice and he F1 c oss o bo h mouse s ains (BALB/c × C57BL/6). Immune checkpoin molecules a e egula o s o he immune sys em and a e c ucial o he unc ion o T eg popula ions. We analyzed he exp ession o he immune checkpoin s cy o oxic T lymphocy e an igen-4 (CTLA-4), BTLA, p og ammed dea h-1 (PD-1), V-domain Ig-con aining supp esso o T cell ac i a ion (VISTA), CD39, lympho- cy e ac i a ion gene-3 (LAG-3), immunoglobulin-like ansc ip 3 (ILT3) and T cell immunoglobulin-3 (TIM-3) unde s eady s a e condi ions and a he peak o S. a i in ec ion. CTLA-4 (CD152) binds wi h high a ini y o CD80/CD86 and he eby p e en s CD28-dependen co-s imula o y signals, while BTLA (CD272) deli e s nega- i e signals in o he cell a e binding o i s ligand HVEM (CD270). The exp ession o bo h immune checkpoin s is up egula ed by CD4+ T cells om S. a i-in ec ed mice as we ha e p e iously shown9,14. PD-1 (CD279) and VISTA, also known as PD-H1, a e non- edundan inhibi o y e ec o molecules egula ing T cells esponses15. PD-1 binds o PD-L1 (CD274) o PD-L2 (CD273), while VISTA may ei he unc ion as a ligand o as a ecep o 16. LAG-3 (CD223) binds wi h high a ini y o MHC-II. This in e ac ion p e en s he binding o CD4 o MHC-II and di ec ly blocks T cell ac i a ion17. In addi ion, LAG-3 in combina ion wi h CD49b iden i ies T 1 cells18. CD39 is an ec onucleo idase ha con ibu es o he supp essi e capaci y o T eg19. ILT3 (CD85k) is a ansmemb ane glycop o ein ha con ains in acellula immuno ecep o y osine-based inhibi o y mo i s. ILT3+ T eg ha e been desc ibed o p omo e he ma u a ion o Th2 cells-inducing dend i ic cells and hus a e poo egula o s o ype II immuni y20,21. Recen ly CD166 was iden i ied as a ligand o ILT322. Fou Ligands (galec in-9, ca cinoemb yonic an igen cell adhesion molecule 1, Phospha idylse ine and high-mobili y g oup p o ein B1) ha e been epo ed o bind o he ecep o TIM-3 (CD366). TIM-3 lacks inhibi o y mo i s in i s cy oplasmic egion and has depending on he con ex co-s imula o y o co-inhibi o y unc ions23. Ou analysis e ealed dis inc exp ession pa e ns in BALB/c, F1 and C57BL/6 mice. The eby, he majo i y o he immune checkpoin s including BTLA we e highes in CD4+ Foxp3+ T eg and CD4+ Foxp3− T cells om in ec ed C57BL/6 mice and lowes in in ec ed BALB/c mice. F1 mice ei he esembled he pheno ype o BALB/c mice o showed in e media e exp ession le els compa ed o BALB/c and C57BL/6 mice. Simila o ou p e ious da a3 deple ion o T eg esul ed in an accele a ed mas cell ac i a ion and expulsion o S. a i in BALB/c, bu no in C57BL/6 mice while he F1 mice phenocopied BALB/c mice. In summa y, ou da a indica e ha Foxp3+ T eg a e cen al in he egula ion o immune esponses in BALB/c and he BALB/c x C57BL/6 F1 mice. By con as , in C57BL/6 mice mul iple laye s o egula ion exis ha may compensa e o he absence o T eg. Resul s Lowe pa asi e bu den in F1 and BALB/c mice han in C57BL/6 mice. The aim o he cu en s udy was o compa e he egula ion o he immune esponse agains S ongyloides a i in ec ion in C57BL/6,and BALB/c miceand he F1 c oss o bo h mouse s ains (BALB/c × C57BL/6). Bo h pa en al mouse s ains de elop a pa en S. a i in ec ion ha is clea ed wi h simila kine ics. Howe e , C57BL/6 mice display highe in es inal wo m bu den a he peak o in ec ion compa ed o BALB/c mice1–3. To quan i y he suscep ibili y o hei F1 c oss we in ec ed all h ee geno ypes s.c. in he hind oo pad wi h S. a i and coun ed he numbe o pa asi ic adul s a day 6 p.i. The numbe o adul s in he in es ine was signi ican ly highe in C57BL/6 mice compa ed o BALB/c mice as obse ed be o e1,3. Pa asi e bu den in F1 mice was simila o BALB/c mice (Fig.1). T eg and T 1 cell numbe s inc ease du ing in ec ion wi h S. a i. We ha e p e iously shown ha in ec ion wi h S. a i ele a ed he numbe s o T eg3. We now compa ed he equency o egula o y T cell subse s (Foxp3+ T eg and CD49b+ LAG-3+ Foxp3− T 1 cells) in he mesen e ic lymph nodes (mes LN) om BALB/c, F1 and C57BL/6 mice (see ga ing s a egy Supplemen a y Fig.1). In line wi h ou p e ious da a in ec ion o BALB/c mice wi h S. a i did no al e he equency o con en ional T eg, de ined as Foxp3+ T cells wi hin he CD4+ T cells, while he equency o Foxp3+ cells inc eased in in ec ed F1 and C57BL/6 mice compa ed o non- in ec ed mice (Fig.2a). The equency o T 1 cells expanded in all h ee in ec ed mu ine geno ypes compa ed o he non-in ec ed mice (Fig.2b). In ec ion wi h S. a i esul ed in an inc ease o o al mes LN cells in all h ee mu ine geno ypes o a simila ex en (Fig.2c). The eby numbe s o CD4+ T cells in he mesLN inc eased du ing in ec ion in BALB/c (a e age numbe : 1.0 × 107 cells) and C57BL/6 mice (5.5 × 106 cells) and by end (p = 0.1) in F1 mice (a e age numbe : 7.9 × 106 cells). S. a i-in ec ed C57BL/6 mice displayed he lowes numbe s o CD4+ T cells (Fig.2d) compa ed o in ec ed BALB/c and F1 mice. In line wi h his in ec ed BALB/c and F1 mice displayed sligh ly highe numbe s o T eg (BALB/c: 1.2 × 106 T eg, F1: 1.0 × 106 T eg) in he mes LN compa ed o in ec ed C57BL/6 mice (7.4 × 105 T eg) (Fig.2e). The numbe o T 1 cells (Fig.2 ) inc eased in in ec ed BALB/c, F1 and C57BL/6 mice compa ed o non-in ec ed mice, al hough due o high a iance i did no each s a is ical signi icance in BALB/c mice (p = 0.051). Dis inc exp ession pa e ns by T eg and Foxp3− T cells om BALB/c, F1 and C57BL/6 mice. Using mul icolo low cy ome y, we nex analyzed he exp ession o he inhibi o y immune checkpoin molecules CTLA-4, BTLA, PD-1, VISTA, LAG-3, and TIM-3 by Foxp3+ T eg in he mes LN. Fi s , we pe o med an unbiased -dis ibu ed s ochas ic neighbo embedding ( -SNE) analysis o isualize cell clus e s wi hin he Foxp3+ T eg (Fig.3). T-SNE is a dimensionali y educ ion plugin om FlowJo, ha was applied o p e-ga ed CD4+ Foxp3+ T cells. Dead cells and double s we e excluded (see ga ing s a egy Supplemen a y Fig.2). The gene a ed -SNE plo s om T eg did no e eal dis inc popula ions in BALB/c, C57BL/6 and F1 mice (Fig.2). 3 Vol.:(0123456789) Scien i ic Repo s | (2021) 11:1536 | h ps://doi.o g/10.1038/s41598-020-79477-7 www.na u e.com/scien i ic epo s/ We obse ed a coexp ession o he ma ke CTLA-4 wi h CD49b (Fig.3, g ey ga e) o LAG-3 (Fig.3, blue ga e) in all mouse s ains, while all o he ma ke s showed a pa chy dis ibu ion. Exp ession o VISTA o PD-1 only pa - ially o e lapped wi h o he ma ke s. The majo i y o PD-1+ o VISTA+ T eg did no coexp ess CTLA-4, BTLA, LAG-3 o CD49b. S ikingly, T eg om C57BL/6 mice showed an inc eased exp ession o CTLA-4, CD49b and VISTA compa ed o T eg om F1 mice and e en mo e p onounced compa ed o T eg om BALB/c mice, which was con i med by s a is ical analysis (Fig.4). The ecep o TIM-3 was ba ely de ec able (Supplemen a y Fig.2b) and exp ession le els we e simila by T eg om BALB/c, F1 and C57BL/6 mice (Fig.3). S a is ical analysis o Foxp3+ T eg showed he highes exp ession o CTLA-4 (Fig.4a), BTLAhigh (Fig.4b), CD49b (Fig.4c), and VISTA (Fig.4d) in C57BL/6 mice compa ed o BALB/c mice. Mes LN om F1 mice exhibi ed an in e media e pheno ype o we e simila o BALB/c mice a day 0 and day 6 p.i. Mu ine geno ype o in ec ion did no al e he exp ession le el o LAG-3 by T eg (Fig.4e). Su p isingly, he exp ession o PD-1 did no inc ease du ing in ec- ion wi h S. a i (Fig.4 ). Cons i u i e PD-1 exp ession was lowes in mes LN om F1 mice and signi ican ly highe in T eg de i ed om S. a i-in ec ed BALB/c and C57BL/6 mice. Nex , we analyzed he exp ession o ILT3 (ga ing s a egy Supplemen a y Fig.3). ILT3 exp ession by Foxp3+ T eg om C57BL/6 mice was al eady highe a s eady s a e (day 0) (Fig.4g). Du ing in ec ion ILT3 inc eased only sligh ly in C57BL/6 mice, bu was signi ican ly highe compa ed o in ec ed BALB/c and F1 mice. By con as , in ec ion wi h S. a i did no al e he ILT3 exp ession by T eg om BALB/c and F1 mice. T-SNE analysis om CD4+ Foxp3− T cells e ealed mo e p onounced di e ences in he exp ession le els o all checkpoin ecep o s be ween he h ee mu ine geno ypes (Fig.5a–c). We obse ed coexp ession o he ma ke s CD49b and LAG-3 (Fig.5, g ey ga e) indica ing he p esence o T 1 cells. CD49b+ LAG3+ T 1 cells we e pa ially posi i e o CTLA-4 and BTLA (Fig.5, g ey ga e). The majo i y o LAG-3+ Foxp3− T cells did no coexp ess CD49b (Fig.5, blue ga e). Cells wi h he highes BTLA exp ession coexp essed PD-1 and ac ions we e posi i e o LAG-3 and CTLA-4 (Fig.5, blue ga e). Foxp3− T cells om all mouse s ains we e highly posi i e o VISTA. The exp ession o he ma ke s TIM-3 and CD39 was low in BALB/c, F1 and C57BL/6 mice (Fig.5 and Supple- men a y Fig.2b). The s a is ical analysis con i med ha he exp ession o CTLA-4 (Fig.6a), BTLAhigh (Fig.6b), CD49b (Fig.6c), LAG-3 (Fig.6d), and PD-1 (Fig.6e) was highes in C57BL/6 mice and lowes in BALB/c mice. Foxp3− T cells om F1 mice had in e media e exp ession le els o hese ma ke s. VISTA exp ession was simila in all mouse s ains and was no up egula ed compa ed o non-in ec ed con ol mice (Fig.6 ). As shown o Foxp3+ T eg (Fig.4g) ILT3 exp ession by Foxp3− T cells om C57BL/6 mice was al eady highe a s eady s a e (Fig.6g) compa ed o BALB/c mice and by end o F1 mice. The exp ession o ILT3 inc eased du ing in ec ion in C57BL/6 mice and was signi ican ly highe compa ed o in ec ed BALB/c and F1 mice while he exp ession emained unchanged by Foxp3− T cells om BALB/c and F1 mice. In summa y, analysis o immune checkpoin s e ealed dis inc exp ession pa e ns by Foxp3+ T eg and Foxp3− T cells du ing in ec ion and al eady by end unde s eady s a e condi ions. S ikingly, T cells om C57BL/6 mice exp essed signi ican ly highe le els o he majo i y o immune checkpoin s compa ed o BALB/c mice. T cells om F1 mice ei he phenocopied he exp ession le els om BALB/c mice o showed an in e media e exp es- sion. Pa asi e bu den om F1 mice esembled BALB/c mice as well. In line wi h he mo e p onounced laye s o egula ion, C57BL/6 mice had inc eased numbe s o adul s in he in es ine compa ed o BALB/c and F1 mice. Figu e1. F1 mice ha e lowe numbe s o adul s in he in es ine han C57BL/6 mice. BALB/c, F1 and C57BL/6 mice we e in ec ed wi h 2000 iL3 s.c. in he oo pad. Pa asi ic adul s in he small in es ine we e coun ed a day 6 p.i. Shown a e combined esul s om 2–3 expe imen s (BALB/c: n = 14 mice, F1: n = 13 mice and C57BL/6: n = 8 mice). Each symbol ep esen s he wo m bu den om a single mouse. Ho izon al lines ep esen he mean. Da a we e analyzed by 1-way ANOVA wi h Tukey’s mul iple compa isons es . As e isks indica e signi ican di e ences o he mean (****p ≤ 0.0001). 4 Vol:.(1234567890) Scien i ic Repo s | (2021) 11:1536 | h ps://doi.o g/10.1038/s41598-020-79477-7 www.na u e.com/scien i ic epo s/ T eg a e cen al in he egula ion o he an i‑helmin h immune esponse in F1 mice. Since we ha e p e iously shown ha T eg signi ican ly supp ess he an i-S. a i immune esponse in BALB/c mice3, we now aimed o compa e he impac o Foxp3+ T eg in BALB/c and C57BL/6 mice and hei F1 gene a ion. To his end, we made use o he DEREG mice, which allow he deple ion o T eg by applica ion o diph he ia oxin (DT)24. To gene a e F1 DEREG mice, C57BL/6 DEREG mice we e c ossed wi h BALB/c mice and ice e sa. We Figu e2. T eg and T 1 cells inc ease du ing in ec ion wi h S. a i. BALB/c, F1 and C57BL/6 mice we e le naï e (day 0) o in ec ed wi h 2000 iL3. Six days la e mes LN we e isola ed and s ained wi h a panel o egula o y ecep o s. F equencies o (a) Foxp3+ cells in CD4+ T cells and (b) T 1 cells in Foxp3− CD4+ T cells a e shown. Numbe s o mes LN (c), CD4+ T cells (d), Foxp3+ T eg (e), and CD49b+ LAG-3+ T 1 cells ( ). Each symbol ep esen s alues om a single mouse. Shown a e combined esul s om 2–3 expe imen s (BALB/c day 0 p.i.: n = 3, day 6 p.i.: n = 14; F1 day 0 p.i.: n = 5, day 6 p.i.: n = 13; C57BL/6day 0 p.i.: n = 4 mice, day 6 p.i.: n = 8). Da a we e analyzed by an unpai ed - es . As e isks indica e signi ican di e ences o he mean (*p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.001). 5 Vol.:(0123456789) Scien i ic Repo s | (2021) 11:1536 | h ps://doi.o g/10.1038/s41598-020-79477-7 www.na u e.com/scien i ic epo s/ Figu e3. Simila exp ession pa e n o egula o y ma ke s by T eg om BALB/c, F1 and C57BL/6 mice. BALB/c, F1 and C57BL/6 mice we e in ec ed wi h 2000 iL3. Six days la e mes LN we e isola ed and s ained wi h a panel o egula o y ecep o s. -SNE calcula ion was pe o med wi h 40,000 Foxp3+ CD4+ T eg using FlowJo Plugins. Rep esen a i e -SNE hea maps de i ed om T eg om BALB/c (a), F1 (b) and C57BL/6 (c) showing he exp ession o BTLA, CTLA-4, LAG-3, CD49b, TIM-3, VISTA, PD-1 and Foxp3. 6 Vol:.(1234567890) Scien i ic Repo s | (2021) 11:1536 | h ps://doi.o g/10.1038/s41598-020-79477-7 www.na u e.com/scien i ic epo s/ ended up wi h wo di e en geno ypes, F1 mice wi h he e ozygous exp ession o he DT ecep o and F1 li e - ma es ha did no exp ess he ansgene. All g oups we e ea ed wi h DT and subsequen ly in ec ed wi h S. a i. T ansien T eg deple ion esul ed in imp o ed e adica ion o S. a i in BALB/c DEREG mice while C57BL/6 DEREG micedid no bene i om T eg deple ion (Fig.7a) as we ha e shown be o e3. The educed numbe o wo ms in T eg-deple ed BALB/c micewas accompanied by an inc eased mas cell deg anula ion (Fig.7b). The amoun o he mas cell-speci ic mouse mas cell p o ease-1 (mMCPT-1) in he se um o in ec ed C57BL/6 mice was compa able in T eg-deple ed and non-deple ed DEREG mice. The T eg-deple ed DEREG F1 gene a ion o BALB/c and C57BL/6 micehad educed pa asi e bu den (Fig.7A) and inc eased mas cell deg anula ion (Fig.7b) and hus phenocopied he T eg-deple ed BALB/c mice. Taken oge he ou da a show a simila pheno ype o F1 and BALB/c mice. In bo h mouse s ains he an i- S. a i esponse is con olled by T eg as non- edundan supp esso cells, while in C57BL/6 mice deple ion o T eg did no inc ease mas cell-media ed expulsion o S. a i. In line wi h he missing impac o T eg deple ion, CD4+ T cells om C57BL/6 mice displayed a dis inc and mo e p onounced exp ession o immune checkpoin molecules compa ed o BALB/c and F1 mice. Discussion Inb ed mouse s ains such as C57BL/6 and BALB/c mice a e aluable models o s udy immune esponses du ing in ec ious diseases25. These mouse s ains a e well de ined and a ious cell- and ecep o -de icien mice exis on bo h gene ic backg ounds. In he cu en s udy we compa ed he egula ion o he immune esponse agains he in es inal pa asi e S. a i in C57BL/6, BALB/c mice and he F1 gene a ion “hyb id” (BALB/c × C57BL/6) o bo h mouse s ains. In line wi h p e ious indings1–3 we eco ded inc eased pa asi e bu den in C57BL/6 mice compa ed o BALB/c mice a he peak o in ec ion. In e es ingly, he F1 c oss esembled BALB/c mice wi h ega d o pa asi e bu den. Pe o ming a comp ehensi e low cy ome ic analysis, we eco ded he exp ession o a a ie y o immune checkpoin molecules in hese h ee dis inc gene ic backg ounds. Mice we e analyzed unde s eady s a e con- di ions as well as du ing acu e in ec ion wi h he gas oin es inal nema ode S. a i. We iden i ied p onounced di e ences in he exp ession pa e n o immune checkpoin s by Foxp3+ and Foxp3− CD4+ T cells de i ed om BALB/c and C57BL/6 mice al eady a s eady s a e. Despi e a simila kine ic o S. a i expulsion1–3, ou da a clea ly indica e di e ences in he egula ion o immune esponses in hese wo commonly used labo a o y mouse s ains. We show ha he egula o y pa e n o he F1 c oss om BALB/c and C57BL/6 mice esembled BALB/c mice a he han C57BL/6 mice since (i) he exp ession o egula o y ecep o s by Foxp3+ T eg and Foxp3− T cells in F1 mice mimics mo e he pheno ype o BALB/c mice han C57BL/6 mice and (ii) deple ion o T eg causes an imp o ed mas cell ac i a ion and subsequen e adica ion o S. a i in F1 and BALB/c mice, bu no in C57BL/6 mice. We hypo hesize ha he p onounced exp ession o checkpoin ecep o s by Foxp3− T cells o o he egula- o y cells subse s such as T 1 cells compensa es o he deple ion o Foxp3+ T eg in C57BL/6 mice. The lack o pheno ypic ou come o he T eg deple ion wi h ega d o pa asi e bu den in C57BL/6 mice would emphasize he enhanced egula o y capaci y o he Foxp3− T cell popula ions o in e e e wi h an i-S. a i immune esponses. The impac o T egs on an i-helmin h immune esponses was ecen ly summa ized26, howe e , he e is no consis en analysis compa ing he impac o he gene ic mu ine backg ound on he ole o T eg du ing helmin h in ec ion. T egs p omo e pa asi e su i al du ing in ec ion wi h Schis osoma japonicum27,28, S. a i3,9, T ichu is mu is8 and Li omosoides sigmodon is11,29,30 whe eas hey a e edundan supp esso cells du ing in ec ion wi h Heligmosomoides polygy us5,31 and T ichinella spi alis32. Di e ences in he egula ion migh , howe e , no be pa asi e-speci ic bu ely on he gene ic backg ound o he mice. Two ecen s udies compa ed he ec ui men o T eg du ing in ec ion wi h he gas oin es inal nema ode H. polygy us31 and he ila ial nema ode L. sigmodon is11 in BALB/c and C57BL/6 mice. Bo h s udies did no compa e he impac o ea ly T eg deple ion on he cou se o helmin h in ec ion. Suppo ing ou da a H. polygy us-in ec ed suscep ible C57BL/6 mice had inc eased Foxp3+ T eg equencies compa ed o mo e esis an BALB/c mice, while he o al numbe o Helios+ T eg was highe in BALB/c mice. By con as , a simila ec ui men o T eg was shown o bo h geno ypes du ing in ec ion wi h L. sigmodon is. In his helmin h model, BALB/c mice a e suscep ible o L. sigmodon is and de elop pa en , ch onic in ec ions, while C57BL/6 mice a e semi- esis an and clea he in ec ion be o e he onse o mic o ila emia. In e es ingly, CD4+ Foxp3− T cells om L. sigmodon is-in ec ed C57BL/6 mice had a mo e ac i a ed pheno ype han T e ec o cells om BALB/c mice11. We obse ed an ele a ed equency o CD49b+ LAG-3+ T 1 cells and Foxp3+ T eg in CD4+ T cells and o e all mo e ac i a ed pheno ype o Foxp3− CD4+ T cells in C57BL/6 mice Figu e4. Inc eased exp ession o dis inc checkpoin molecules by T eg om C57BL/6 mice. BALB/c, F1 and C57BL/6 mice we e le naï e (day 0) o in ec ed wi h 2000 iL3. Six days la e mes LN we e isola ed and s ained wi h a panel o egula o y ecep o s (a– ) o ILT3 (g). S a is ical analysis showing he exp ession o CTLA-4 (a), BTLAhigh (b), CD49b (c), VISTA (d), LAG-3 (e), PD-1 ( ), and ILT3 (g) by T eg. The ga ing s a egy is shown in he Supplemen a y Figs.S1 and S2. Each Symbol ep esen s one mouse. Shown a e combined esul s om 2 o 3 expe imen s (a- : BALB/c day 0 p.i.: n = 3, day 6 p.i.: n = 14; F1 day 0 p.i.: n = 5, day 6 p.i.: n = 13; C57BL/6day 0 p.i.: n = 4 mice, day 6 p.i.: n = 8; g: BALB/c day 0 p.i.: n = 3 mice; F1 day 0 p.i.: n = 5 mice; C57BL/6day 0 p.i.: n = 5 mice; n = 8 mice o all in ec ed mouse s ains). Da a (a– ) we e analyzed by 1-way ANOVA wi h Tukey’s mul iple compa isons es . Di e ences in he ILT3 exp ession (g) we e analyzed by K uskal–Wallis es . Black as e isks a e shown o naï e and in ec ed mice and indica e signi ican di e ences o he mean (*p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.001, ****p ≤ 0.0001, ns = non signi ican ). I in a g aph only a la ge ba wi h ns is depic ed, no s a is ical di e ences be ween he h ee geno ypes exis a his ime poin . G ey as e isks indica e signi ican di e ences be ween day 0 and day 6 p.i. om one mu ine geno ype. ▸ 7 Vol.:(0123456789) Scien i ic Repo s | (2021) 11:1536 | h ps://doi.o g/10.1038/s41598-020-79477-7 www.na u e.com/scien i ic epo s/ 8 Vol:.(1234567890) Scien i ic Repo s | (2021) 11:1536 | h ps://doi.o g/10.1038/s41598-020-79477-7 www.na u e.com/scien i ic epo s/ Figu e5. Exp ession o egula o y ecep o s is mo e p onounced by Foxp3− CD4+ T cells om C57BL/6 mice han BALB/c and F1 mice. BALB/c, F1 and C57BL/6 mice we e in ec ed wi h 2000 iL3. Six days la e mes LN we e isola ed and s ained wi h a panel o egula o y ecep o s. -SNE calcula ion was pe o med wi h 80,000 Foxp3−CD4+ T cells using FlowJo Plugins. Rep esen a i e -SNE hea maps de i ed om BALB/c (a), F1 (b) and C57BL/6 (c) showing he exp ession o BTLA, CTLA-4, LAG-3, CD49b, TIM-3, VISTA, PD-1 and CD39. 9 Vol.:(0123456789) Scien i ic Repo s | (2021) 11:1536 | h ps://doi.o g/10.1038/s41598-020-79477-7 www.na u e.com/scien i ic epo s/ compa ed o BALB/c and F1 mice. The numbe o egula o y T cells, howe e , was no highe in C57BL/6 mice due o lowe o al numbe s o CD4+ T cells in C57BL/6 mice. We now p o ide e idence ha he F1 p ogeny a e c ossing BALB/c and C57BL/6 mice has a mo e BALB/c- like pheno ype: BALB/c and F1 mice displayed simila ly lowe numbe s o adul s in he small in es ine, and T eg deple ion educed in es inal pa asi e bu dens and inc eased mas cell deg anula ion selec i ely in BALB/c and F1 mice, bu no in C57BL/6 mice. Expulsion o S. a i elies in bo h, BALB/c and C57BL/6 mice, on mas cells2 and on IL-9 ha p omo es mas cell ac i a ion ea ly in in ec ion1. Howe e , he mechanisms con olling his c ucial mas cell deg anula ion seem o be di e en in BALB/c, and C57BL/6 mice. In BALB/c mice Foxp3+ T eg supp ess su icien IL-9 p oduc ion o subsequen mas cell deg anula ion du ing S. a i in ec ion in a non- edundan manne , whe eas addi ional egula o y pa hways seem o supp ess mas cell ac i a ion in T eg- deple ed C57BL/6 mice3. The analysis o immune checkpoin s ha we pe o med in his s udy e ealed s iking di e ences in he exp ession pa e n by T cells in he mesen e ic lymph nodes, d aining he si e o in ec ion in C57BL/6 mice compa ed o bo h BALB/c and F1 mice. We obse ed an inc eased exp ession o ILT3 selec i ely in C57BL/6 mice. ILT3 belongs o he immunoglobulin supe amily and con ains immuno ecep o y osine-based inhibi o y mo i s sugges ing an inhibi o y unc ion o ILT333. Indeed, T egs exp essing ILT3 show lowe T cell ecep o signaling han ILT3− T eg and a o he expansion o Th2 cell-inducing dend i ic cells20. ILT3 is exp essed by Foxp3+ T eg20 and in hypo esponsi e Th2 cells34 du ing in ec ion wi h L. sigmodon is. In his s udy hypo espon- si e Th2 cells we e dis inguished om exhaus ed/ane gic cells due o hei speci ic gene exp ession pa e n, which includes LAG-334. We obse e he highes exp ession o ILT3, LAG-3 and se e al o he inhibi o y ecep o s such as BTLA by Foxp3− T e ec o cells om C57BL/6 mice while T cells in he mes LN om BALB/c and F1 mice showed a lowe exp ession. BTLA is a non- edundan egula o o S. a i-induced immune e asion in C57BL/6 mice. Mice lacking BTLA o i s ligand HVEM ha e a educed in es inal pa asi e bu den and accele a ed mas cell deg anula ion compa ed o wild ype mice14. We now epo ha Foxp3+ T eg and especially Foxp3− CD4+ T cells om in ec ed C57BL/6 mice display a signi ican ly highe exp ession o BTLA han BALB/c and F1 mice which is in line wi h he non- edundan unc ion o BTLA in supp essing an i-helmin h immuni y in C57BL/6 mice. In ec ion wi h S. a i did no induce a signi ican up egula ion o TIM-3, CD39 o VISTA in BALB/c, C57BL/6 o F1 mice. Since he exp ession o he ma ke s TIM-3 and CD39 has been desc ibed o occu unde in lamma o y condi ions35–37 he missing exp ession in a helmin h-induced Th2 en i onmen is no unexpec ed. VISTA is a ecen ly disco e ed immune checkpoin molecule ha is cons i u i ely exp essed by T cells38. We show an exp ession o VISTA by Foxp3+ T eg and Foxp3− Te unde s eady s a e condi ions. VISTA was no up egula ed by Foxp3− CD4+ T cells du ing in ec ion in any o he h ee mouse s ains indica ing a mino ole o VISTA in he egula ion o an i-helmin h immune esponses. In summa y, he missing exp ession o CD39, TIM-3 and VISTA shows ha S. a i did no induce b oad unspeci ic up egula ion o all immune checkpoin s bu a he led o a dis inc egula o y pa e n. In gene al, immune checkpoin s a e exp essed by ac i a ed T cells, T eg o exhaus ed T cells, i.e. cells ha a e pe sis en ly exposed o an igen du ing ch onic in ec ion and lose hei unc ional capabili ies39. In he cu en s udy, mice we e acu ely in ec ed o 6days wi h S. a i a he ime poin o analysis. Thus, inc eased exp ession o checkpoin molecules by Foxp3− CD4+ T cells in C57BL/6 mice compa ed o BALB/c o F1 mice is unlikely due o T cell exhaus ion obse ed in ch onic in ec ions. Ou low cy ome y da a mo e likely iden i y ac i a ed T cells exp essing inhibi o y ecep o s sugges ing edundan laye s o egula ion in C57BL/6 mice. Helmin hs, employ mul iple egula o y pa hways o hei hos o modula e he immune esponse and o ensu e hei su i al. In he cu en s udy we use a helmin h ha causes pa en in ec ions in BALB/c and C57BL/6 mice o poin ou ha di e en gene ically de e mined pa e ns o egula ion exis in bo h mouse s ains. These pa e ns we e pa ially isible al eady a s eady s a e and expanded du ing helmin h in ec ion. The eby, he s ain-speci ic di e ences ha became appa en should be conside ed analyzing hos –pa hogen in e ac ions. Using in ec ions wi h S. a i as a model o in es inal helmin h in ec ions, BALB/c ai s a e pa ially dominan and inhe i ed o F1 mice in a con olled and op imized en i onmen such as a “speci ic pa hogen ee” animal acili y while in C57BL/6 mice mul iple laye s o egula ion exis ha may compensa e o he absence o single egula o y ci cui s. The exac gene ic mechanisms, and a pu a i e con ibu ion o X-linked dominan inhe i - ance, causing he di e ences in he egula ion o immune esponses emain unknown and should be add essed in u u e s udies. Inb ed mouse s ains a e homozygous and hus e lec only a mino i y o he gene ic a ia ion p esen in wild mice and in he human popula ion. In he wild, suscep ibili y o in ec ion is addi ionally in luenced by demog aphic ac o s such as gende , age, and in ec ion his o y40. I was p e iously shown ha he immune s a us o C57BL/6 mice g ea ly di e s om wild mice41–43 and is a ec ed by body condi ions and age and o a lesse ex en by he gene ic a ia ions44. Consequen ly, da a ob ained using hese wo inb ed mouse s ains canno be ans e ed di ec ly o gene ically mo e he e ogeneous humans. A ho ough analysis o di e en egula o y pa - e n in labo a o y mouse s ains may allow a be e unde s anding o he a iabili y obse ed in human s udies in e ms o helmin h-induced immunomodula ion. Ma e ial and me hods E hics and mice. Animal expe imen s we e conduc ed in ag eemen wi h he Ge man animal p o ec ion law. Expe imen al p o ocols we e app o ed by Fede al Heal h Au ho i ies o he S a e o Hambu g wi h he pe mission numbe s 54/10 and N103/2018. BALB/c, C57BL/6, F1 (BALB/c × C57BL/6) mice, BALB/c DEREG, C57BL/6 DEREG and F1 DEREG mice we e b ed a he animal acili y o he BNITM and kep in indi idually en ila ed cages unde speci ic pa hogen ee condi ions. Fo he gene a ion o he F1 mice, emale BALB/c mice we e c ossed wi h male C57BL/6 mice. To gene a e F1 DEREG mice, male C57BL/6 DEREG mice we e c ossed