Analysis of adaptive immune cell populations and phenotypes in the patients infected by SARS-CoV-2

A n alys is of a dap ti ve im mu ne c ell po pul a tio ns and p he noty pe s i n th e p atie nts in fec te d by S AR S-C o V-2 X i ao f en g Y an g 1# , T o ng xi n D a i 2# , Xi a obo Z ho u 1 , Hon gbo Q i an 2 , Rui G u o 2 , Lei L ei 1 , Xin gzh e Zh an g 1 , Dan Zh ang 1 , Li n Shi 1 , Y a nbi n Che ng 1 , J ins on g H u 1* , Y aling Guo 2* , Ba oju n Z ha ng 1,4,5* 1. D epa rt me nt of P a t ho ge nic Mic ro bio l og y a nd I m mun ol o gy, Sch o ol o f Ba sic Medi cal Scie nc e s, X i' an Ji aot on g Un ive rsity H e alth Sci enc e C ent er, Xi 'a n , Sh aa nxi , 7 100 6 1, Ch i n a 2. D epa rt me nt of Cli ni cal L ab or ato ry, T he 8th h ospit al of Xi' an, Xi' an, Sh aa nxi , 710 0 1 6 Ch i n a 3. D epa rt me nt of Cell B io l ogy and G en et ics , Xi' an Ji a ot ong U nive rs ity H e alt h S cienc e Ce n te r , X i'a n, S ha a nx i , 7100 61 , C h in a 4. Ke y Lab or ato ry of Env i ro nm ent a nd Gen e s Rel a te d t o Di se ases , X i ’a n Ji aot o ng U nive rsity Heal t h S c ie nc e Ce nte r, Xi' an , S ha anxi , 7100 61 , C h i na 5. Insti t ute of inf ecti on and i m muni ty, T r ans la tion al M edic i ne I ns tit ut e, Xi’a n Ji aot on g U nive rsity Heal t h S c ie nc e Ce nte r, X i ’a n , S ha anxi, 7 100 61 , C hi na # T hes e a u th ors co ntri but ed eq ually to this w ork. * C o rre spo n d enc e to : B aoj un Z ha ng, bj .zh an g @m ail .xjt u.e du.c n; Yali n g G u o, 15 1 0 29 15638 @ 1 2 6. com ; o r J in s ong Hu , ji ns on g.h u@xjt u.e du. c n A b str act C or o n avi rus dise as e -2 019 ( C OV I D - 1 9) , c aus ed by SA RS- Co V-2 , ha s ra pi dly spre ad t o m o s t o f c ou ntr ie s i n th e w or l d , t hr ea t en i n g t he he a lth a nd li v e s o f man y pe op le . U nfo rtu nat ely, i nfo rm ati o n reg ar din g the i mm u n olo gica l c ha rac te risti cs i n CO VI D- 19 patie n ts re mai ns l i mite d. Here w e col l ecte d t he bl oo d s am ples fro m 18 he al th y d on o r s (H D ) and 38 COV ID -19 pa t i ent s t o a na l y z e cha ng e s i n t h e ad ap t i ve im mu n e c e l l pop ul ati ons an d p he no typ es. I n c om paris on to HD , t he l ym p h ocyte p erc e nt age w as sl ightly d ec reas ed, t he p er c ent ag es of CD 4 an d C D8 T cell s in ly mp hoc y tes are si mil ar , w here as B c ell p erc ent ag e inc re ase d in C O VI D -19 p atie n ts. T cells , es p eci all y C D 8 T c e ll s , s how ed a n e nh an c ed e x pr es si on o f la t e ac t iv at i o n m a rk e r CD 2 5 an d e x h au s t i o n mar ker P D-1. I mpo rta ntly, S AR S-C o V-2 in d uc ed a n i ncr eas ed p erc ent ag e of T f ollic u la r h e l pher (T fh ) - and g er m i na l ce n te r B-l i ke (GC B - l i k e ) ce l ls in t he b l ood . H owe v er , th e par am ete rs i n CO V D-19 p atie nts r e m ain ed un cha ng ed ac ro ss vari ous a ge gro ups . Th ere for e, w e d em on s tra te d t hat t he T a nd B c el ls ca n be ac ti vat ed n or mall y and e xhibi t funct i on al fe atu res. Th ese da ta p rov id e a clue t hat t he a da pti ve i mm uni t y in m ost p eopl e coul d b e p r i me d to in duc e a si gni fic an t im m u ne r es p ons e ag ains t S A RS- Co V -2 i nf ecti o n upo n r ece i ving s ta nd ar d m edic al c are . Ke y words : SA R S- Co V-2, C O VI D-19 , A dap tive i mm unit y , A c tiv atio n, Ly m ph ocy te A sev er e pne um o ni a- assoc i at ed re sp irato ry s yn dr om e be gan i n W u ha n o f Chi na i n D ece mb er 2 019 , c all in g t he at te ntio n o f WH O , w hich su b s equ entl y dec l are d t he dise as e a s a p ub l ic h ea l t h em er g en cy o f int er na t i o na l c onc er n . T he nov e l c or on a v i r u s s t ra i n w as offic ially na me d as s eve re acu t e r espi ra tory s y nd ro me c or ona v iru s 2 ( S AR S- Co V-2) [ 1, 2 ]. C or o n avi rus es i nfe ctio ns s uch as se v er e ac ute r espi rat ory s yn dr om e (SA RS ) and Mi ddl e E as t r esp i r at o ry s yn dr om e (M E RS) , c a n c au se sev er e r e spi ra t or y d is ea s e [ 3 , 4 ]. S AR S-C o V-2 is an e nve l op ed p ositiv e -str an d RN A vi rus , w hich belo ng s to t he fa mil y of c o r on a vir u se s a lo ng w i th SA RS- C oV an d ME RS -CoV a cco rd i ng to t he g en ome sim i l ar i t y [2, 5 -7 ]. A n umbe r o f s t ud i e s de mo nst r at ed that th e a da pt i ve i m m un i t y r e sp on d s t o c o r on a v ir u s and i s r eq uire d f or effic i ent cl ea ra nce of the v iru s . I n p at ie nts i nf ec te d with SAR S - C o V , th e acut e p has e o f inf e cti on i n h um ans wa s a ss oci ate d w i t h a s ev er e r ed uct i on of T cell num be rs i n the bl oo d, i nv ol ving a d ra m a tic l os s of CD4 a nd CD 8 T c ells i n co mpa ris o n to healt h y co ntr ol i n divi du al s [8, 9 ]. T his s ugg ests th at SA RS- Co V i nfe c tio n i mp a i red c ellu la r imm u nity in th e ea rly s ta ges of t he dis ea s e. W i th th e pr o l on ge d rec ov ery ti m e of S AR S- i nfec te d pa tien ts, ac tiv at ed T c ell ma rke rs su ch a s CD6 9 an d CD2 5 expr essi on decr eas ed [10 , 11 ], i n dicati ng t hat T c ell activ a tion i n r es p ons e to t he vi rus i s i m pair ed [ 12] . Wit h t he i m pr ove me nt of the dis ea se , th e r atio of C D4 t o C D8 T c ells inc rea se d, i ndic ati n g that CD 4 T cel ls rec ove re d fa ste r th an CD 8 T cell s [ 13] . I n ad di tio n, w it hi n t he 9 2 % of cure d SA R S p atie nts w h os e B c el ls d eclin ed fir st an d t he n i ncr eas ed o r c ontin ue d to inc r e a se d uri ng th e c o u r s e of t he dis e a se, only 8 % of t he m ha d a c ons ta nt or decr easi ng c e ll co un t [14] . S im i l ar to SAR S i nf e ct ion i n hu ma ns , le uko pe n i a and l ym ph o peni a a re a l so obse rv e d i n M E RS p ati en ts , al bei t to a l ess e r d eg re e t ha n t hat o bs e rve d in S A RS pa tien ts . A d etail e d cli ni cal s t udy sho w ed tha t 14 % o f M ER S p atie nts w ere l euk op enic w hile 3 4% o f the pati ent s h ad l y mp ho pe nia [ 15] . M E RS- Co V-i nfec ted p at ie nts t hat e x hibit ed distinc tivel y high fr eq ue nc ies of ME RS c oro navi r us– r eacti ve C D8 T cell s were a sso c iate d w i t h sev e re/ mo de rat e i lln ess , w here as C D4 T c el l res p on se was mi nim al ly de te cted at t his stag e . I n t he c onv a les cen t p h as e, sli ght ly mo re C D4 T c e lls w ere de tec ted [15] . C ur r entl y, ve ry f e w st udi e s sh o w e d that C O VI D - 19 pati ent s u nde r we nt devel opi ng ly mp hop eni a a nd ris in g pr o-i nfl a m m ato ry c yt okin e s in sev e r e cas es [ 16-18]. T h e infor ma ti o n is s till v e ry lim i ted ab out ho w i mm un e cells c ha nge an d fu nctio n in resp ons e t o SA RS - COV - 2 i nf e ct io n. K now i ng th at T a nd B c e ll s re spo nd to t h e i nf ec t ion an d p la y criti cal rol es in de fe ndin g a ga i nst vi r us i nfecti o n, syst em a tic ally st udyi ng t he ch ang es in T and B c ells in CO V I D- 19 pati ent s will h el p u nc ove r th e im mu ne res po nse a gai ns t S AR S-C O V-2 inf ecti o n and p rovi de i nsi ghts fo r CO VI D-19 d i ag nosis an d t re at men t. In t his stu dy, we an aly z ed th e bloo d s a mple s fro m 18 HD an d 38 pa tie nts an d focus e d o n t h e c h ar a c te r i za t ion o f a da p t i v e i mm une c e l l p op u la t ion s an d ph e n o ty p e s upon S AR S-C o V-2 i nfe ctio n. We s ho w ed t ha t upo n inf ec tio n, ly m ph ocyt e p erc en t ag e decli n e d, the p erc en tag es o f CD4 an d CD8 T ce lls w ithi n th e l ym ph ocy t e p opul atio n rem ai n ed u n c hang ed , a nd B ce l l p e rce nt ag e w a s r el at i v e ly i n cr e ase d. C D4 a n d C D8 T ce l l s exhi bi ted a mil d a nd st ro ng a cti va tion phe not y pe , re spe ctiv el y. Not ably , th e p erce nt age s of Tf h- a nd G C B - l ike c ell s incr eas ed . Simil ar p he not y pe s am on g th e pati ent s in vari ou s a g e gr ou p s i nd i cat e t ha t a ge d i nd i vi dua l s a r e al s o c ap ab l e t o r es po nd t o S AR S -C oV -2 infec ti on. Ou r dat a s up p or t th e n o tio n t hat ad aptiv e i mm unity c o uld be no rm all y activ a te d and de fen d agai nst S A RS - C o V -2 i nfec t i on. R e sult s T h e p erc en tag e a n alysi s of T an d B c ells i n C O V ID - 19 pa tie nts To d ete rmi ne t he c han ge i n the c om p o sitio n of a da ptiv e i mm un e cells , we a nalyz ed th e perc en tag e s of T an d B cells i n t he bl o od fr om HD and pati ents usin g fl o w cyt om e try. I n com p ar i son to H D , ly mp hoc yte p e rc e nta ge in t he w hole bl o od w a s no t s igni fic antly c h a nged , t ho u gh e x h ibi t ed a d e cr ea s ing tr end in p a t ie nt s ( F ig. 1A ) . W it h i n th e l y m ph o c y t e pop ul ati on, th e p erc ent age s of C D4 + a nd CD 8 + T c el ls w e re co m pa ra ble (F i g. 1B an d C) , w here as B cell pe rc e nta ge sig ni fica ntl y incre as e d (F i g.1D) in CO VI D-19 p ati e nt s. A n ac tiv at ed ph en oty pe o f T c ells i n CO VID -1 9 pati en ts T o e va l ua te T ce ll st a tu s in r e sp onse to SAR S‐C oV- 2 i n fe c t i on, w e ana l y zed t he expr es si on o f CD6 9, C D 2 5 , P D - 1, C D45 R A, CD 45 R O a nd CX CR 3 in b ot h C D4 + and CD 8 + T c e l l s . I n C D 4 + T cells of CO VI D-19 pati ent s , th e ex pres sio n of CD 69 and CD 2 5 (Fig . 2 A a nd B ) a nd t he pe rce nta ge o f re gul at o r y T ce lls ( T re gs), mar ked by CD 3 + CD 4 + CD 25 + CD127 - ( F i g. 2 F ) w ere si milar t o tha t of HD. C D 2 5 expr essi o n upr eg ulat e d signi fi ca ntly i n CD 8 + T cell po p ulati on of th e p atie nts (Fi g. 3 B ) . T he pr op orti on of n aïv e a nd eff ect or/ me mo ry c ells i n b oth CD 4 + T ce ll s (F i g. 2D an d E ) an d C D 8 + T cells (Fig . 2 E and F ) of the t wo gr oup s w ere no t si g nifica ntl y diffe re nt. P D - 1 exp ressi o n upr eg ulat e d d ra ma tic all y i n bot h CD 4 + T c e l l s ( F i g . 2 C ) a n d C D 8 + T c e l l s ( F i g . 3 C ) o f t h e patie n ts. T he dat a d em ons tra ted a wea k activ a tio n in C D4 + T cell s, bu t a s tro n g ac tiv ati o n in C D 8 + T c ell s d uri ng S A RS‐ Co V- 2 in f ecti o n. A n inc re ase in ge rmi nal c en te r-lik e c e lls i n C O VID -19 p a tie nts T f ollic ul ar hel pe r (Tf h) cell s ca n help B cel l activ ate a nd diff er enti ate int o eff ecto r c ells , pro duc e high -af fini ty a ntib od y a nd fo rm g er min al c ent ers (G C )[ 19]. T o s t u dy w het her CO VI D- 19 p atie nts p rod uc e effic i ent adap tive i mm u ne res p on se, we an alyz e d the expr es si on of P D -1 a nd C XC R 5 i n CD4 + T cel ls , a nd th e exp res sion of Fas a nd G L 7 in B c e ll s . A s sh own i n F i gu r e 4A an d B , t h er e w a s a s ig n i f i c a nt in cr ea s e o f bot h Tf h - and GC B-l ike cel ls i n t h e blo od of t he pat i en ts co m p ar e d t o HD gr ou p. C o rrela tio n a naly sis b etw ee n a cti va tio n s i gn at ure a nd pati en t a ge To stu dy w het he r age a ffec ts ada ptiv e i m mun e c ell pop ul a tion a nd eff ect or f eat ure s, w e per for me d co rr elati on a nalys i s bet w ee n T cell a cti vati on mar ker s an d ag e. I n Fi g ure 5 , no d r am at i c ch an ge o c c ur re d w i t h i ncr eas i n g age . Th e re s u lt i nd i cat e s t ha t i n t he age d indi vid ual s i nfec ted by SA RS -Co V -2, t h ere is no d ef ect in CD 8 + T cel l activa t i on, as well as T fh- a nd G CB-lik e c ell dif fe ren ti atio n . D is cus si on S AR S-C o V-2 i nf ectio n i s q uick ly s pr ea di ng ar oun d th e w o rl d. T h e p atie nts pre se nt t ypic al sy mp to ms o f p ne um oni a , s uch as dry c oug h, dys pn e a, fev er, an d bil ate ral l u ng i nfiltr ate s on i magi ng [20 ]. Alt hou gh th e p o stm or tem stu dy r eve ale d int en si ve i nfla m m a tion i n th e patie n t’s lu ng, littl e is kn o wn a bo ut th e i mm u nol ogic al fea tu res in r esp ons e to t his ne w vi r us . In th e p res ent s tu dy, w e hav e focus ed on c h ar acte rizi n g a da ptiv e i mm un e cell pop ul ati ons an d p h e not ype s i n CO VI D -19 p ati ents . Lym p ho pe nia w a s s ho wn i n CO V I D p a tients f ro m pr evio us s tu dies [2 0], Epi d emio l ogic al inv esti gati o n of c oro na vi r us inf ecti o n sh o wed th at ly mp h op eni a is p res ent i n m ore tha n 8 0 % of p ati ents , a nd s e r i o us decl in e is co rr el ate d t o w ors e p ro gn osi s [ 21]. H o w ev e r , w e di d n ot obse rv e a s i gnific ant de cre as e of l y m phoc y te po pula t io ns i n CO V I D- 19 pati e nts. T his findi n g c ould be at trib ute d t o th e f act t hat mos t of th e p atie nt s i n thi s st udy s ho w e d mil d sy mp to m be s ide s fev er. Int er es tin gly, follo wi ng t he di v isio n of pati ents i nto thos e w i th sy mp to matic a nd asy mp to matic , we o bser ved a d ecr eas e i n ly m pho cyte p o pulati on s i n t h e s ymp to ma t i c pat i e nt s (D ata n o t sh ow n) . PD -1 is a ma r ke r o f e xh au st ed T c e l l s du r i n g chro ni c and ac ute i nfec tio ns [2 2, 23]. A nu mbe r o f stu dies s h o w e d th a t PD -1 + CD 8 + T c ells inc r e a se d in th e p e r i p her al blo od of pati ents wit h a v ari ety o f ac ut e vir al i nfec tio ns s uch as H BV, H I V, a nd E bol a v i rus [ 24, 2 5]. In our s t udy, t he ex pr es sio n of P D-1 wa s upr eg ula t ed in b ot h CD 4 + and CD 8 + T c ells of CO V I D -19 pati ents , wh ich m ay ex plai n t h e o bs erv e d red ucti o n i n th e ly mp hocy te p o pula tion . In res po ns e t o vi ral inf ec ti on, n or mally bo t h C D4 + a n d C D 8 + T ce l l s be com e ac t i v ate d . In CO VI D- 19 p ati e nts, w e ob ser v ed a v e r y mil d acti v ati on in CD 4 + T cell s b ut s tr on ger acti va tion in CD 8 + T c ells ba s ed o n C D25 ex pre ss io n. Thi s r e fl ects th at CD 8 + c e l l s a r e m a j or r es p onde r s of C OV ID- 19 in f e ct i on a nd ar e c o n s is t e n t l y a c t i v a te d . I t i s pos s i b l e t h at CD 4 + T c ells m ay be s t ro ngly ac t ivat ed earli er d urin g i nf ec tio n the n re ver t t o th e quie sc e nt state aft er pr ovidi ng hel per fu ncti o ns, w h ich c ou l d ex pla i n th at l ac k of d ete c tio n of a s tro n g a c t iv at e d p h en ot ype . T hi s co u ld be ex pl a i n ed by t he c om p ar ab l e exp re s s i on of CD 6 9 , an early ac tiv ati o n m ark er i n HD a nd p ati e nts. C D4 + T c ells m ay in dee d b e we akl y activa t e d in re s po ns e to th e v ir us, whi c h wa r r ant s fur th er s tudi es. Du r ing v ir a l in f ec t i on s, th e a nt i g en- s p eci f i c i mm un e r e s p on s e i s ex e cu t ed by Tfh an d GCB cel ls. Tfh c ells hel p B cel l diff ere nti a te int o a ntig en -sp eci fic eff e cto r c ell s to p ro duc e high -af fi nity a ntib odi es a n d f ac ilita te g ermi na l c e nte r for m ati o n [ 19] , w hich a re e ss enti al for i ndu c ing effic i en t vir us cle ar anc e. I n COV I D-19 p ati ents , th er e wa s a n i nc re ase i n b o th Tfh - an d GBC -like c ells i n the bl oo d, r eflec ti ng th at a n anti gen -s pec ific r esp o ns e c a n be acti va ted u pon S A RS - C o V -2 i nfe cti o n. E l de r l y i nd iv i du a ls ty p i ca ll y e x h ibi t a red u ct io n o f th e lym ph o c y t e popu l a t io n an d w eak er abil ity to d efe nd a gai nst vi ral in fect i on [ 26]. In o ur c orr elati on a nalys is , w e did not o bse rv e a s i gn if i c an t cor re la t io n be tw e e n ly mp ho c yt e p r opo r t io n s , e f fe c t or f ea tu r es, and a ge. O u r dat a s ug g est th at the sp eci fic p opul atio ns o f T and B c ells f or S AR S-C oV -2 a r e r ese rv e d in ag ed i ndivi du a ls, w h i ch nee d t o b e p rove n b y fu r th er re per toir e s e qu enci ng ana ly sis of T cel l - a nd B c ell -re cep tor s. In su m mar y, o ur s tud y s h o w s t hat S AR S-C o V-2 co u ld i nd uce rel ativ el y no r mal ada ptiv e imm u ne r e s po ns e. M os t pe opl e ac ros s diffe re nt a ge gr ou ps a re c ap abl e of m o bil izing t h e ada pti v e i m mu ne c ells, activ atin g c ell ul ar an d h um or al i m mu nity to d ef en d aga i nst t he vi r us w i th s uffici ent medic al ca re an d a nti-v i ral t re atmen t. M at eri al s and M eth od s E t hics s tat em en t This st ud y w as a pp rov ed by the Res e arch E t hi cs Com mi ssio n of t he E i g hth Hospi t al o f X i ' an ( 20 19 0730 - 1346 ) w i th a w a i ve r o f i n f o rm ed co ns e nt d ue t o a pu b l ic he a l th o u tb re ak inv esti gati o n. All ca s es wer e tak en fro m th e Ei ght h H os pi tal of Xi' a n ( X i 'a n, S haa nx i Pr ovinc e) , a de si gn ate d h os pi tal f or th e C O VI D - 19 by l ocal au t ho r i ty. P a tie nts Fro m Feb ru ar y 18 to Ma rc h 4, 2 02 0 , 18 he al thy c ont rols an d 38 c o nfi rm ed COVI D - 2 0 19 patie n ts wer e inc lu de d in t he st udy. P at ients w ere dia gn os e d an d ad mitt ed i n a ccor dan c e wit h th e gui del in e of t he na ti onal he al th co mmis s ion o f Chi na. All of 3 8 p at ient s w ere c o n f i r me d a s SAR S -C oV - 2 inf e c t io n us i ng t he R T -PC R tes t on th ro a t swa b sp e c im en s. Th e medi an a ge o f th e p atie nts was 3 9.0 6±4. 26 ye ars (Ta bl e 1). 2 3 pati ents (6 0.5 3 % ) w er e m en, an d 15 pati en ts (3 9. 47 %) w ere wo me n ( Ta ble 1 ). F low cyt om etr y a n aly si s Th e A bs use d i n t he fl o w c yto met ry analy s is we re as f ollo ws: FI T C anti -h uma n CD 3 ( UC H T 1), FI T C a nti -hu m a n TC R γ / δ (B 1), AP C/ C y an i ne 7 an ti-h um an C D4 (O K T4 ), Pe r CP/ C y a ni ne5 .5 an ti -hu ma n C D8 ( S K 1), A PC anti - h u m an C D19 ( H IB1 9), APC a n t i -h uman C D2 5 ( B C9 6) , P E an t i -h uma n C D 69 ( FN5 0) , P E an t i- h uman C D 185 (C XC R5 ) (J25 2 D4 ), P E a nti -hu ma n CD 183 (C X CR 3) (G0 25 H 7), A PC an t i-h um an C D 279 ( PD- 1) (E H12 .2 H 7), P E a nt i -hu ma n CD 95 ( Fas ) ( DX 2), PE anti -hu ma n CD 12 7 ( A 01 9 D5 ), A PC/ C y ani ne 7 anti -h um a n C D 4 5 RA (H I10 0), P E / Cy 5 anti -h um an C D 4 5 RO ( U C HL1 ), P E anti- hu m a n CD 9 5 (F as) (D X 2), FIT C a nti- mo use/ hu ma n GL 7 Anti g en ( GL7 ), w er e purc has ed f ro m B i ol eg en d. Bl ood c el ls we re stai ne d wi th A bs in th e dar k at ro o m tem pe rat ure fo r 15 m i n, a nd an aly z ed on a F A CS Can to II fl o w cyto met er (B D B i o sci e nc e s) . F l ow Jo 8 w a s u sed fo r da t a a na l y si s . S t atisti c al an aly sis Th e co nti n uo us vari abl e o f n o rma l d istrib uti o n is re pr ese nte d by me a n ± s ta nda rd devi a tion , t he non -n or mal dist rib u tion i s re pr ese nte d by m edi a n [I Q R ], a nd t h e cla s sifie d varia b le is r ep res ent ed b y co unt ( per ce nta ge) . The st u de nt’s t t est w as pe rfo r med fo r t w o gro up an al ysis u sing SP S S 22. 0 sof t w are . * an d * * s tra nds fo r P <0. 05 a n d P <0 .0 1, resp ec tiv ely . A C KN O WL ED G EM EN T This work w as s u pp ort ed by g ra nts f rom Nat ural S c i enc e F ou nd a tio n of C hina ( No . 81 8 20 1 08 0 17), N at ur al Scie nce F ou n datio n o f Chi na ( No. 8177167 3), an d CO V I D- 1 9 spec i al p roje c t o f Xi’ an Jiao to ng U nive r si ty F ou nda tion (xz y03 20 20 002 ). We th ank all th e doct o r s , n urs es , pu bl ic he alth w o rk ers , an d pati ent s f or thei r c ont rib uti on ag ains t S AR S-C o V-2 i nf ecti on. D e cla rati on of i nt er ests W e de c l ar e n o co mp et i n g in te r es t s . A u th or c on tri buti o ns Xia ofe ng Ya ng, Xi aob o Z ho u, L ei Lei , Xin g zh e Z ha ng, Dan Z ha ng a na ly zed t he d at a a nd w r ot e t he m an u scri pt. To ng xin Dai, Hong bo Qia n, Rui Gu o and Yalin g Gu o c oll ec t ed sam p les a nd inf o rma ti on. Li n S hi an d Y anbi n C he ng dis cuss e d d ata a nalys i s. Ji nso ng H u per for me d t h e F CA S anal y sis. Baoj un Zh ang g ene ra ted th e id ea, d es igne d t he expe ri m en t a n d w rot e th e m an us cri pt. All a uth ors ag re e to be res po nsi bl e fo r thei r o w n par t of th e wo rk. Fi gu r e l eg end s F ig 1. Th e p erc en ta ge c ha ng es o f T a nd B c ells be twe en H D a nd C O VID -1 9 p ati e nts (A ) T he pe rc en ta ge of ly m pho cyte s in to tal b lo od c ell s. ( B) T he p e r c ent ag e of C D4 + T c e l ls in ly m ph ocyt e p opul atio n. ( C ) Th e p erc ent age of CD 8 + T cell s i n ly mp hoc y te pop ulati on. ( D ) Th e per cen ta g e of B cell s i n ly mp hocyt e pop ula ti on . Eac h dot re pr ese n ts a si ngl e patie n t of CO VI D-19 or h e alth y d ono r. * P< 0.0 5 w a s co nsi d ere d statis ti cally sig nif ica nt. F ig 2. A sli gh t inc re as e of acti va t ed CD 4+ T c ells i n CO VID - 19 pa tien ts (A ) T he p erc en t ag e o f C D6 9 + c e l l s i n C D 4 + T c el ls. ( B ) T he p erc ent ag e of CD 25 + c ells i n CD 4 + T c el ls. ( C) Th e pe r c e nta ge of PD -1 + c e l l s i n C D 4 + T c e l ls. ( D ) T he pe rc en ta ge o f CD 4 5RA + CD 45 R O - c e l l s i n C D 4 + T c ell s. ( E) T h e p e r c e nta ge o f CD4 5 RA - CD 45R O + c e l l s in C D 4 + T c ells. ( F ) Th e p e r c e nta ge of Tr e g c ells in C D 4 + T c e l l s . E a c h d o t r e p r e s e n t s a si ngle pa tien t o f C O V I D -19 o r h ealt h y do no r. * P <0. 05 an d P<0 .01 w as consi der ed stati stic al ly si gnific a n t a nd extr em ely si gni fic ant, r esp ec tivel y . F ig 3. A s tr ong in c re ase of acti vat ed C D8 + T cel ls in C O VID -1 9 p ati e nts (A ) T he p erc en t ag e o f C D6 9 + c e l l s i n C D 8 + T c el ls. ( B ) T he p erc ent ag e of CD 25 + c ells i n CD 8 + T c el ls. ( C) Th e pe r c e nta ge of PD -1 + c e l l s i n C D 8 + T c e l ls. ( D ) T he pe rc en ta ge o f CD 4 5RA + CD 45 R O - c e l l s i n C D 8 + T c ell s. ( E) T h e p e r c e nta ge o f CD4 5 RA - CD 45R O + c e l l s in CD 8 + T c ells . Eac h d ot r epr es e nts a si ngl e pa tie nt of C O VI D - 19 o r h ealt h y do no r . * P<0 .05 a nd P< 0.0 1 wa s co n sid er ed sta tistic a lly s i gnific ant a nd ex trem ely si gnific a nt , resp ec tiv ely . F ig 4. An inc re as e of ge r minal c e nte r-li ke c ell s i n CO VID - 1 9 p ati ents ( A) Th e p er c enta g e o f P D- 1 + CX CR 5 + c e l l s i n C D 4 + T ce lls. ( B) T he pe r cen ta g e of Fas + GL 7 + c ells i n B c ells. E ach d ot r e p r e s en ts a s in gle pa tie nt of CO VI D- 19 or he alth y don or. * P<0. 05 a nd P<0 .0 1 was c ons id er ed st atis tic all y sig nifi ca nt a nd extre mel y si gnific an t, res p ectiv ely. F ig 5. C or r e la tio n a nal y sis b etw ee n f unc ti on al s ig na tu res an d pa ti ent a ges Th e c o rr e l atio n a naly si s b et we en pa ti ent ag e an d i mm une pa ra me ter s was per form e d usi ng P ea rso n’ s c orr elati on coe ffi cie nt. Th e p erc ent ag e of tot al lym p h ocyt es ( A) , B cells (B ) , C D8 + CD 2 5 + T c e l l s ( C ) , C D 8 + PD - 1 + T ce l ls (D ) , T f h- l i ke ce l ls (E ) and GC B - l ike ce l ls (F ) w er e c or rel ate d to a ge i n C O VI D - 19 pat ient g ro up. Ea ch d o t rep res ents a si n gl e pati en t of C OV ID -19 o r h ea l t h y d on or . P at ien t s un d er th e a ge o f 15 y ea r s we re e x c l uded . T a ble 1. C ha rac te rist ic an aly sis of C O VID -1 9 p ati ent s a nd he al th y d on ors Ref er enc e 1. Wang C , Horb y P W , Hayd e n F G, Gao GF : A nov e l coronavirus outb r e ak o f g lobal heal th concern . La n c et 20 2 0, 395 (1 0223 ):470 -473. 2. 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Fig1 H D P a tie n t 0 1 0 2 0 3 0 4 0 5 0 6 0 % C D 4 + c e lls H D P a tie n t 0 1 0 2 0 3 0 4 0 5 0 % C D 8 + c e lls H D P a tie n t 0 5 1 0 1 5 2 0 2 5 3 0 3 5 % L y m p h o c y te s H D P a tie n t 0 5 1 0 1 5 2 0 2 5 3 0 % B c e lls * A B C D Fig2 H D P a tie n t 0 5 1 0 1 5 2 0 2 5 3 0 % T re g in C D 4 + c e lls H D P a tie n t 0 5 1 0 1 5 2 0 2 5 % C D 2 5 + c e lls in C D 4 + c e lls H D P a tie n t 0 1 5 3 0 4 5 6 0 % C D 4 N a iv e c e lls * H D P a tie n t 0 2 0 4 0 6 0 8 0 % C D 4 + e ffe c to r/m e m o ry c e lls H D P a tie n t 0 3 6 9 1 2 1 5 % C D 6 9 + c e lls in C D  + T c e lls H D P a tie n t 0 1 0 2 0 3 0 4 0 5 0 % P D -1 + c e lls in C D 4 + c e lls * * A B C D E F Fig3 H D P a tie n t 0 2 0 4 0 6 0 8 0 1 0 0 % C D 8 N a iv e c e lls H D P a tie n t 0 1 0 2 0 3 0 4 0 % C D 8 + e ffe c to r/m e m o ry c e lls H D P a tie n t 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 % P D -1 + c e lls in C D 8 + c e lls * H D P a tie n t 0 5 1 0 1 5 2 0 2 5 % C D 2 5 + c e lls in C D 8 + T c e lls * * H D P a tie n t 0 1 2 3 4 5 6 % C D 6 9 + c e lls in C D 8 + T c e lls A B C D E Fig4 H D P a tie n t 0 3 6 9 1 2 1 5 % T fh c e lls * H D P a tie n t 0 5 1 0 1 5 2 0 2 5 3 0 % G C B c e lls * * A B Fig5 2 0 4 0 6 0 8 0 1 0 0 0 5 1 0 1 5 2 0 A g e % L y m p h o c y te s 2 0 4 0 6 0 8 0 1 0 0 0 5 1 0 1 5 2 0 2 5 A g e % C D 2 5 + c e lls in C D 8 + T c e lls 2 0 4 0 6 0 8 0 1 0 0 0 2 0 4 0 6 0 8 0 A g e % P D -1 + c e lls in C D 8 + c e lls 2 0 4 0 6 0 8 0 1 0 0 0 5 1 0 1 5 2 0 A g e % T fh c e lls r= 0 .4 0 7 7 2 0 4 0 6 0 8 0 1 0 0 0 5 1 0 1 5 2 0 2 5 A g e % G C B c e lls 2 0 4 0 6 0 8 0 1 0 0 0 5 1 0 1 5 2 0 2 5 A g e % B c e lls A B C D E F