Dissection of the pre-germinal center B-cell maturation pathway in common variable immunodeficiency based on standardized flow cytometric EuroFlow tools

Dissec ion o he P e-Ge minal Cen e B-Cell Ma u a ion Pa hway in Common Va iable Immunodeficiency Based on S anda dized Flow Cy ome ic Eu oFlow Tools Lucı a del Pino-Molina 1 , Edua do Lo pez-G anados 1 *, Quen in Lec e isse 2,3 , Juan To es Canizales 1 , Ma ı nPe  ez-And e s 2,3 , Elena Blanco 2,3 , Ma jolein Wen ink 4 , Ca olien Bon oy 5 , Jana Nech a alo a 6 , Tomas Milo a 7 , Anne-Ka h in Kienzle 8 , Jan Philippe  5 , Ana E. Sousa 9 , Mi jam an de Bu g 10 , Tomas Kalina 11 , Jacques J.M. an Dongen 12 *and Albe o O ao 2,3 on behal o he Eu oFlow PID Conso ium 1 Clinical Immunology Depa men , La Paz Uni e si y Hospi al and Lymphocy e Pa hophysiology in Immunodeficiencies G oup, La Paz Ins i u e o Heal h Resea ch (IdiPAZ) and Cen e o Biomedical Ne wo k Resea ch on Ra e Diseases (CIBERER U767), Mad id, Spain, 2 Clinical and T ansla ion Resea ch P og am, Cance Resea ch Cen e (IBMCC, USAL- CSIC), Depa men o Medicine, Cy ome y Se ice (NUCLEUS), Uni e si y o Salamanca (USAL), Ins i u e o Biomedical Resea ch o Salamanca (IBSAL), Salamanca, Spain, 3 Biomedical Resea ch Ne wo king Cen e Conso ium o Oncology (CIBERONC) Ins i u o de salud Ca los III, Mad id, Spain, 4 Depa men o Immunology, E asmus Uni e si y Medical Cen e (E asmus MC), Ro e dam, Ne he lands, 5 Depa men o Labo a o y Medicine, Uni e si y Hospi al Ghen , Ghen , Belgium, 6 Depa men o Alle gology and Clinical Immunology, Facul y o Medicine, Masa yk Uni e si y and S Anne’s Uni e si y Hospi al in B no, B no, Czechia, 7 Depa men o Immunology, Second Facul y o Medicine, Cha les Uni e si y and Mo ol Uni e si y Hospi al, P ague, Czechia, 8 Nu field Depa men o Medicine, Expe imen al Medicine Di ision, Uni e si y o Ox o d, Ox o d, Uni ed Kingdom, 9 Ins i u o de Medicina Molecula , Faculdade de Medicina, Uni e sidade de Lisboa, Lisboa, Po ugal, 10 Depa men o Pedia ics, Labo a o y o Immunology, Leiden Uni e si y Medical Cen e , Leiden, Ne he lands, 11 CLIP - Childhood Leukemia In es iga ion P ague, Depa men o Pedia ic Hema ology and Oncology, 2nd Facul y o Medicine, Cha les Uni e si y and Uni e si y Hospi al Mo ol, P ague, Czechia, 12 Depa men o Immunohema ology and Blood T ans usion, Leiden Uni e si y Medical Cen e (LUMC), Leiden, Ne he lands In oduc ion: Common Va iable Immunodeficiency (CVID) is cha ac e ized by de ec i e an ibody p oduc ion and hypogammaglobulinemia. Flow cy ome y immunopheno yping o blood lymphocy es has become o g ea ele ance o he diagnosis and classifica ion o CVID, due o an impai ed di e en ia ion o ma u e pos -ge minal-cen e (GC) class- swi ched memo y B-cells (MBC) and se e ely dec eased plasmablas /plasma cell (Pb) coun s. He e, we in es iga ed in de ail he p e-GC B-cell ma u a ion compa men in blood o CVID pa ien s. Me hods: In his collabo a i e mul icen ic s udy he Eu oFlow PID 8-colo P e-GC B-cell ube, s anda dized sample p epa a ion p ocedu es (SOPs) and inno a i e da a analysis ools, we e used o cha ac e ize he ma u a ion p ofile o p e-GC B-cells in 100 CVID pa ien s, s 62 age-ma ched heal hy dono s (HD). Resul s: The P e-GC B-cell ube allowed iden ifica ion wi hin p e-GC B-cells o h ee subse s o ma u a ion associa ed imma u e B-cells and h ee subpopula ions o ma u e F on ie s in Immunology | www. on ie sin.o g Feb ua y 2021 | Volume 11 | A icle 6039721 Edi ed by: Sudhi Gup a, Uni e si y o Cali o nia, I ine, Uni ed S a es Re iewed by: Hans-Ha mu Pe e , Uni e si y o F eibu g Medical Cen e , Ge many Neil Rombe g, Child en’s Hospi al o Philadelphia, Uni ed S a es Manisha Rajan Madkaika , Na ional Ins i u e o Immunohaema ology (ICMR), India *Co espondence: Jacques J.M. an Dongen [email p o ec ed] Edua do Lo pez-G anados [email p o ec ed] Special y sec ion: This a icle was submi ed o P ima y Immunodeficiencies, a sec ion o he jou nal F on ie s in Immunology Recei ed: 08 Sep embe 2020 Accep ed: 29 Decembe 2020 Published: 17 Feb ua y 2021 Ci a ion: del Pino-Molina L, Lo pez-G anados E, Lec e isse Q, To es Canizales J, Pe  ez-And e sM,Blanco E, Wen ink M, Bon oy C, Nech a alo a J, Milo a T, Kienzle A-K, Philippe J, Sousa AE, an de Bu g M, Kalina T, an Dongen JJM and O ao A (2021) Dissec ion o he P e-Ge minal Cen e B-Cell Ma u a ion Pa hway in Common Va iable Immunodeficiency Based on S anda dized Flow Cy ome ic Eu oFlow Tools. F on . Immunol. 11:603972. doi: 10.3389/ immu.2020.603972 ORIGINAL RESEARCH published: 17 Feb ua y 2021 doi: 10.3389/ immu.2020.603972 naï e B-lymphocy es. CVID pa ien s showed o e all educed median absolu e coun s ( s HD) o he wo mo e ad anced s ages o ma u a ion o bo h CD5 + CD38 +/++ CD21 he CD24 ++ (2.7 s 5.6 cells/µl, p=0.0004) and CD5 + CD38 he CD21 + CD24 + (6.5 s 17 cells/ µl, p<0.0001) imma u e B cells (below no mal HD le els in 22% and 37% o CVID pa ien s). This was associa ed wi h an expansion o CD21 - CD24 - (6.1 s 0.74 cells/µl, p<0.0001) and CD21 - CD24 ++ (1.8 s 0.4 cells/µl, p<0.0001) naï e B-cell coun s abo e no mal alues in 73% and 94% cases, espec i ely. Addi ionally, educed IgMD + (21 s 32 cells/µl, p=0.03) and IgMD - (4 s 35 cells/µl, p<0.0001) MBC coun s we e ound o be below no mal alues in 25% and 77% o CVID pa ien s, espec i ely, always oge he wi h se e ely educed/unde ec able ci cula ing blood pb. Compa ison o he ma u a ion pa hway p ofile o p e-GC B cells in blood o CVID pa ien s s HD using Eu oFlow so wa e ools showed sys ema ically al e ed pa e ns in CVID. These consis ed o : i) a no mally-appea ing ma u a ion pa hway wi h al e ed le els o exp ession o >1 (CD38, CD5, CD19, CD21, CD24, and/o smIgM) pheno ypic ma ke (57/88 pa ien s; 65%) o a o al o 3 dis inc CVID pa ien p ofiles (g oup 1: 42/88 pa ien s, 48%; g oup 2: 8/88, 9%; and g oup 3: 7/88, 8%) and ii) CVID pa ien s wi h a clea ly al e ed p e-GC B cell ma u a ion pa hway in blood (g oup 4: 31/88 cases, 35%). Conclusion: Ou esul s show ha ma u a ion o p e-GC B-cells in blood o CVID is sys ema ically al e ed wi h up o ou dis inc ly al e ed ma u a ion p ofiles. Fu he s udies, a e necessa y o be e unde s and he impac o such al e a ions on he pos -GC de ec s and he clinical he e ogenei y o CVID. Keywo ds: CVID, P e-GC B-cell ube, p e-GC ma u a ion pa hway, exp ession ma ke s, Eu oFlow s anda diza ion INTRODUCTION Common Va iable Immunodeficiency (CVID) is he mos p e alen symp oma ic p ima y immunodeficiency (PID). I is cha ac e ized by de ec i e an ibody p oduc ion ha leads o hypogammaglobulinemia (1–3) wi h an inc eased suscep ibili y o in ec ions, associa ed in some CVID pa ien s wi h en e opa hy, au oimmuni y, lymphop oli e a ion, and/o isk o lymphoid malignancy due o mo e p o ound immunological dys egula ion (4,5). Despi e dis inc monogenic de ec s a e p esen in a mino ac ion (<20%) o cases, and o he complex oligo o polygenic gene ic p edisposi ion (6), and epigene ic al e a ions (e.g., impai ed deme hyla ion in genes ele an o he B cell unc ions) ha e been associa ed wi h he de elopmen o CVID (7), a well-defined pa hogenic mechanism s ill emains o be iden ified in mos CVID pa ien s. Thus, assessmen o he dis ibu ion o lymphocy es, pa icula ly pos -ge minal cen e (GC) B-cells and plasmablas s/plasma cells in blood o suspicious pa ien s by flow cy ome y has become o g ea ele ance o he diagnosis and classifica ion o CVID (8,9). Impai ed pos -GC B cell ma u a ion in he pe iphe y (i.e., in blood and seconda y lymphoid issues) is a hallma k o CVID. Howe e , CVID is a a he he e ogeneous disease om he clinical, gene ic and immunologic poin o iew. Thus, se e al classifica ion algo i hms ha e been p oposed o CVID, which a e based on he specific al e a ions encoun e ed o he majo B cell popula ions in blood (8), hei p oli e a ion his o y and soma ic hype mu a ion le els (9), in combina ion o no wi h he clinical mani es a ions o he disease and/o mo e sophis ica ed compu a ional (i.e., hie a chical clus e ing) app oaches (10). O e all, impai ed di e en ia ion o ma u e pos -GC B-cells, consis ing o se e ely educed ci cula ing class-swi ched memo y B-cells (MBC) and s ongly dec eased (i.e., unde ec able) plasmablas /plasma cell p oduc ion, a e he mos consis en de ec s in CVID. Because o his, demons a ion o educed class-swi ched MBC is now used among he diagnos ic c i e ia p oposed by he Eu opean Socie y o Immunodeficiencies (ESID) o CVID (11). In u n, depending on he specificde ec s encoun e ed in he pos -GC MBC and Pb compa men s in blood, and he se e i y o such de ec s, dis inc CVID pa ien subg oups, associa ed wi h dis inc clinical p ofiles, ha e also been iden ified (12). Apa om he al e a ions in pos -GC B-cells and plasmablas s/plasma cells, an inc easing numbe o e idences Abb e ia ions: APS, au oma ed popula ions sepa a o ; BCR, B-cell ecep o ; BM, bone ma ow; CVID, common a iable immunodeficiency; ESID, Eu opean Socie y o Immunodeficiencies; FCS, flow cy ome y s anda d; FSC, o wa d ligh sca e ; GC, ge minal cen e ; HD, heal hy dono ; Ig, immunoglobulin; IUIS, In e na ional Union o Immunological Socie ies; MBC, memo y B-cell; MFI, mean fluo escence in ensi y; Pb, plasmablas /plasma cell; PC, p incipal componen ; PCA, p incipal componen analysis; PID, p ima y immune deficiency; sm, su ace memb ane; SOP, s anda d ope a ing p ocedu e; SSC, sidewa d ligh sca e ; WBC, whi e blood cell. del Pino-Molina e al. Dissec ion o he P e-GC B-Cell Ma u a ion Pa hway in CVID F on ie s in Immunology | www. on ie sin.o g Feb ua y 2021 | Volume 11 | A icle 6039722 indica e ha he p oduc ion and ma u a ion o B-cells in bone ma ow (BM) is also al e ed in a leas a ac ion (e.g., a ound one hi d) o all CVID pa ien s due o ei he a ma u a ion blockade (13) and/o an al e ed bone ma ow en i onmen which is non-pe missi e o B-cell ma u a ion (14). Thus, a significan p opo ion o CVID pa ien s display educed absolu e B-cell coun s in blood (5) and an ea ly B-cell ma u a ion a es in BM (15). In addi ion, expansion o ansi ional/imma u e B cells, and CD21 low B-cells has also been epo ed in a subse o CVID pa ien s (8,16). Al oge he , hese findings u he suppo an impai ed ma u a ion o p e-GC B-cells in CVID. He ein, we in es iga ed in de ail he p e-GC B-cell ma u a ion compa men in blood o 100 CVID pa ien s, aking ad an age o he no el and s anda dized flow cy ome ic app oaches de eloped by Eu oFlow o his pu pose (17–19), e.g., he ecen ly p oposed Eu oFlow PID 8-colo an ibody panels (20) ha can be easily implemen ed in mos diagnos ic labo a o ies wo ldwide, oge he wi h he Eu oFlow s anda d ope a ing p ocedu es (SOPs) o sample p epa a ion, da a acquisi ion and analysis, including inno a i e da a analysis ools ecen ly de eloped by Eu oFlow o assess no mal s al e ed p e-GC B-cell ma u a ion p ofiles in blood (17,20–23). MATERIAL AND METHODS Pa ien s, Con ols, and Samples O e all, 100 adul CVID pa ien s -50 men and 50 women; median age: 41 yea s (y); ange: 16–82y- and 62 heal hy dono s (HD) no ela ed o he pa ien s (33 men and 29 women; median age: 34y; ange: 19–67y) we e s udied in pa allel, a eigh di e en Eu oFlow-PID cen e s. CVID was diagnosed locally a each cen e , acco ding o he ESID c i e ia (3,24). Rele an clinical da a on CVID pa ien s was ob ained om he pa ien s’heal h elec onic eco ds o om na ional pa ien egis ies and collec ed a each o he 8 pa icipa ing cen e s, including da a on: pa ien age, gende , immunoglobulin (Ig) le els and esponse o accina ion a diagnosis, oge he wi h da a on p io in ec ions and ype o in ec ions (e.g., uppe and lowe bac e ial espi a o y in ec ions, i al and ungal in ec ions), au oimmuni y (e.g., cy openias, o gan-based and sys emic au o-immuni y), lymphop oli e a ion, lymphoid in e s i ial pneumoni is (LIP), g anulomas, splenomegaly, hepa omegaly, b onchiec asias, en e opa hy, and malignancy, as well as p io he apy, including Ig eplacemen he apy. Blood samples we e ob ained, p ocessed and measu ed by flow cy ome y a each o he 8 pa icipa ing cen e s a e in o med consen had been gi en by each indi idual pa icipan , acco ding o he p inciples o he Decla a ion o Helsinki. The s udy was app o ed by he local E hics Commi ees o he pa icipa ing cen e s: Hospi al Uni e si a io La Paz, Mad id, Spain (PI-2833 and 2009/3348/I); Cha les Uni e si y, P ague, Czech Republic (15-28541A); E asmus MC, Ro e dam, The Ne he lands (MEC-2013-026); S Anne´s Uni e si y, B no, Czech Republic (METC 1G2015); BRC-T ansla ional Immunology Lab, Uni e si y o Ox o d, Ox o d, Uni ed Kingdom; Uni e si y o Salamanca, Salamanca, Spain (USAL/ CSIC 20-02-2013); Uni e si y Hospi al o Ghen , Belgium (B670201523515); and Faculdade de Medicina da Uni e sidade de Lisboa and Cen o Hospi ala Uni e si a io Lisboa No e, Lisbon, Po ugal (937/13). Flow Cy ome ic Iden ifica ion o B-Cells, Plasmablas s/Plasma Cells and Thei Subse s in Blood Blood samples om bo h CVID pa ien s and HD we e p ocessed and s ained a each cen e wi h he Eu oFlow 8-colo PIDOT and P e-GC B-cell ubes, ollowing he Eu oFlow SOPs o s aining o cell su ace memb ane (sm) ma ke s only, as p e iously desc ibed (20–22). De ails abou he specific an ibody clones and fluo och ome-conjuga ed eagen s used a e p o ided in Supplemen a y Table 1. Ins umen se -up and calib a ion we e pe o med p io o da a acquisi ion on ≥1x10 6 cells ( ange: 1 x 10 6 -5 x 10 6 cells) in FACSCan o II flow cy ome e s −Bec on/Dickinson Biosciences (BD), San Jose, CA-, ollowing he Eu oFlow SOPs a ailable a www.Eu oFlow.o g (21). Da a analysis was pe o med cen ally on pseudoanonymazed flow cy ome y s anda d (FCS) da a files deposi ed in he Eu oFlow da a eposi o y, using he Infinicy so wa e (Cy ognos SL, Salamanca, Spain). Fo da a analysis, a s anda dized ga ing s a egy was used o iden ifica ion o all p e-GC (defined as CD19 + CD27 - sIgM + B- lymphocy es) and pos -GC B-cell subse s (defined as CD19 + CD27 + o CD19 + CD27 - smIgM - B-cells) p esen in blood, based on he Eu oFlow-PID P e-GC B cell ube as illus a ed in Supplemen a y Figu e 1.B iefly, CD19 + B-cells and plasmablas s/plasma cells we e bo h iden ified by hei low- o- in e media e o wa d (FSC) and sidewa d (SSC) ligh sca e p ope ies a e excluding deb is and cell double s. Subsequen ly, bo h cell subse s we e sub-classified in o 11 di e en subse s based on hei s aining p ofile o CD19, CD38, CD24, CD21, CD27, CD5, smIgM, and smIgD: a) CD27 - CD38 hi CD24 hi CD5 + smIgM ++ D + imma u e/ ansi ional B cells; b) CD27 - CD38 - CD24 he CD5 he smIgM + IgD ++ ma u e nai e B lymphocy es; c) CD27 + CD38 lo CD5 - CD24 he smIgM ++ D + (MD + ) unswi ched MBCs; d) CD27 +/- CD38 lo CD5 - CD24 he smIgM - D - (MD - ) swi ched MBCs; and, e) CD27 ++ CD38 hi CD5 - CD21 - CD24 - plasmablas s/PCs. Imma u e/ ansi ional B cells we e u he sub-classified acco ding o he pa e n o exp ession o CD38, CD5, CD21, and CD24 in o h ee subse s o inc easingly mo e ma u e B-lymphocy es: a1) CD5 - CD38 ++ CD21 he CD24 ++ ; a2) CD5 + CD38 +/++ CD21 he CD24 ++ , and a3) CD5 + CD38 he CD21 + CD24 + imma u e/ ansi ional B lymphocy es. In u n, ma u e nai e B-lymphocy es and unswi ched MBCs we e also u he sub-classified in o h ee subse s each, based on he exp ession p ofile o CD21 and CD24, in o CD21 + CD24 + , CD21 - CD24 ++ , and CD21 - CD24 - ma u e naï e B cells and unswi ched MBC, espec i ely. Fo each B cell popula ion, absolu e coun s we e calcula ed using a dual pla o m assay based on he whi e blood cell (WBC) coun , as assessed in a con en ional hema ological cell coun e , and he pe cen age o o al B cells ob ained wi h he PIDOT ube del Pino-Molina e al. Dissec ion o he P e-GC B-Cell Ma u a ion Pa hway in CVID F on ie s in Immunology | www. on ie sin.o g Feb ua y 2021 | Volume 11 | A icle 6039723 o he same sample, as p e iously epo ed (22). No mal e e ence anges we e defined by he 5 h and 95 h pe cen ile alues obse ed in blood o 62 (age- and sex-ma ched) HD analyzed in pa allel o he CVID pa ien s (Supplemen a y Table 2). P e-GC B-Cell Ma u a ion Pa hway in Blood A da abase eflec ing he no mal B-cell ma u a ion pa hway o p e-GC B-lymphocy es in blood was buil by me ging da a files om 18 ep esen a i e HD, using he Infinicy so wa e (Cy ognos SL) and p e iously desc ibed p ocedu es (25). Fo his pu pose, p e-ga ed da a files which specifically con ained ga ed da a exclusi ely on he h ee di e en subse s o imma u e blood B cells (CD5 - CD38 ++ CD21 he CD24 ++ , CD5 + CD38 +/++ CD21 he CD24 ++ and CD5 + CD38 he CD21 + CD24 + imma u e B cells), oge he wi h bo h he CD21 + CD24 + and CD21 - CD24 - ma u e nai e B cell subse s, om blood o 18 HD s ained wi h he P e-GC B-cell ube we e me ged in o a single da a file. Ma u e nai e CD21 - CD24 ++ B cells we e no included in he p e-GC B- cell da abase since his subse is ba ely de ec able in no mal blood om HD (26). Then, he me ged da a file was used o define he ma u a ion pa hway o p e-GC B-cells using he ma u a ion ool de eloped by Eu oFlow and implemen ed in Infinicy ( 2.0-4b o Eu oFlow membe s only). This ool allows o au oma ic i) defini ion o ec o s ha eflec ma u a ion pa hways based on cu e analysis algo i hms, ii) classifica ion o e en s in o di e en ma u a ion s ages a bi a ily se a equal dis ances, iii) calcula ion o desc ip i e s a is ics o all e en s classified wi hin each ma u a ion s age, ollowed by di ec isualiza ion in a (balanced) 3-dimension (3D) APS (au oma ed popula ion sepa a o ) diag am, cons uc ed using he fi s h ee p incipal componen s (PC1 o PC3) de i ed om PC analysis (PCA) pe o med wi h he Infinicy so wa e (Figu e 1A). Thus, based on he ma u a ion ool o he Infinicy so wa e, 10 dis inc p e-GC B-cell ma u a ion s ages we e (a bi a ily) defined and he no mal mean fluo escence in ensi y (MFI) ange (2SD) pe ma u a ion s age was calcula ed o each indi idual A B FIGURE 1 | Illus a ing example o he no mal ( e e ence) p e-ge minal-cen e (GC) ma u a ion pa hway. O e all pheno ypes (A) and exp ession le els o indi idual ma ke s pe s age o ma u a ion (B) a e shown o each indi idual dono (n=18) included in he no mal e e ence p e-GC B-cell ma u a ion pa hway used o define e e ence no mal alues, depic ed as colo -coded lines pe ma ke /pa ame e . del Pino-Molina e al. Dissec ion o he P e-GC B-Cell Ma u a ion Pa hway in CVID F on ie s in Immunology | www. on ie sin.o g Feb ua y 2021 | Volume 11 | A icle 6039724 cell su ace ma ke . A e wa d, decon olu ion o he ma u a ion ec o defined by he median MFI alues pe ma u a ion s age (n=10) da a was plo ed o he whole se o pheno ypic ma ke s included in he Eu oFlow P e-GC B-cell ube (Figu e 1A). Subsequen ly, median ( ange) and mean (+2 SD) fluo escence in ensi y (MFI) alues pe ma ke in HD blood p e-GC B-cells oge he wi h he co esponding B cell pe cen age, we e plo ed along he di e en ma u a ion s ages and used as no mal e e ence ange (Figu e 1B). Subsequen ly, ga ed p e-GC B- cells om e e y indi idual CVID case we e plo ed agains he no mal e e ence ma u a ion da abase and di e ences in MFI alues s. he no mal blood ange we e eco ded pe ma ke o each o he 10 p e-es ablished p e-GC B-cell ma u a ion s ages. MFI alues below 2SD o abo e 2SD o he no mal e e ence ange (pe ma u a ion s age) o a leas wo consecu i e s ages o ma u a ion o p e-GC B-cells, we e conside ed o be al e ed. In pa allel, a g aphical display o he alues pe ma ke along he p e-GC B-cell ma u a ion pa hway, in which each pa ien is ep esen ed agains he da abase, was ob ained. S a is ical Analyses S a is ical analyses we e pe o med wi h G aphPad P ism So wa e e sion 6.0 (G aph- Pad so wa e, San Diego, CA). To define no mal anges o each B cell subse iden ified in blood, 5 h and 95 h pe cen ile alues om he 62 adul HD we e used. G oup compa isons we e pe o med using he Mann-Whi ney U and K uskal-Wallis es s ( o con inuous a iables) o he Fishe exac and X 2 es s ( o ca ego ical a iables). Clus e ing analysis based on K-means was pe o med using he JMP so wa e ( ee ial e sion 14; SAS Ins i u e Inc., Ca y, NC) based on he immunopheno ypic p ofiles and ela i e dis ibu ions o p e-GC B-cells along he p e-GC ma u a ion pa hway, pe ma u a ion s age. Clus e analysis was pe o med by simul aneously compa ing he MFI alues o each su ace ma ke and he pe cen age o e en s pe s age o ma u a ion pe CVID pa ien agains he ma u a ion e e ence da abase. P- alues<0.05 we e conside ed o be associa ed wi h s a is ical significance and coded he ea e as ollows: *p- alue<0.05; ** p- alue<0.01; *** p- alue<0.001; and, **** p- alue<0.0001. RESULTS Dis ibu ion o P e-GC B-Cell Subse s in Blood o CVID Pa ien s Based on he da a p o ided by he PID-o ien a ion ube (PIDOT) (22), and he P e-GC B-cell ube, de ailed cha ac e iza ion o B cells in pe iphe al blood was achie ed o a o al o 11 dis inc B cell subse s: i) imma u e/ ansi ional B-cells (including h ee ma u a ion-associa ed popula ions o CD5 - CD38 ++ CD21 he CD24 ++ ,CD5 + CD38 +/++ CD21 he CD24 ++ ,andCD5 + CD38 he CD21 + CD24 + imma u e B-cells); ii) ma u e naï e B- cells (and hei h ee CD21 + CD24 + , CD21 - CD24 ++ , and CD21 - CD24 - subse s); iii) unswi ched IgMD + , IgM + -only, and IgD + -only MBC (and hei CD21 + CD24 + , CD21 - CD24 - , and CD21 - CD24 ++ subse s); i ) swi ched IgMD - MBC; and ) plasmablas s/ plasma cells (Figu es 2A–C). O e all, he o al B-cell coun in blood o CVID was significan ly educed s. age-ma ched HD (median: 149 s 206 cells/µl; p=0.04). Suchdec easewasmos lydue oasignifican educ iono imma u e B-cells (11 s 27 cells/µl, p<0.0001), IgMD + MBC (21 s 32 cells/µl, p=0.03) and pa icula ly, IgMD - MBCcoun s(4 s35cells/µl, p<0.0001), in he absence o i ually no plasmablas s/plasma cells (Figu e 3A). Despi e he o e all educed median B-cell coun s obse ed in CVID, a significan o e lap wi h HD was s ill obse ed wi h a iable equencies and pa e ns o al e a ion among CVID pa ien s. Thus, dec eased coun s below no mal alues (<5 h pe cen ile o age-ma ched HD) o IgMD - MBC we e de ec ed in 77% o he CVID pa ien s in es iga ed, oge he wi h unde ec able plasmablas s/plasma cells in 100% o cases. In con as , educed imma u e/ ansi ional B cells and IgMD + MBC coun s we e only ound in 29% and 25% o cases, espec i ely (Figu e 3A). In u n, only a small pe cen age o all CVID pa ien s showed educed ma u e naï e B-cell coun s (11%). Al oge he , he dec eased numbe s o he dis inc B-cell subse s led o o e all low o al B- cell coun s in blood o 21% o all CVID pa ien s (Figu e 3A). Mo e de ailed analysis o he p e-GC B-cell compa men also showed dis inc pa e ns o al e a ion o di e en subse s o imma u e B-cells and ma u e nai e B-cells. Thus, educed coun s ( s age-ma ched HD) o he mo e ad anced s ages o ma u a ion o CD5 + CD38 +/++ CD21 he CD24 ++ (2.7 s 5.6 cells/µl, p=0.0004) and CD5 + CD38 he CD21 + CD24 + (6.5 s 17 cells/ µl, p<0.0001) imma u e/ ansi ional B cells was de ec ed in 22% and 37% o CVID pa ien s. In con as , he less di e en ia ed CD5 - CD38 ++ CD21 he CD24 ++ imma u e/ ansi ional B lymphocy es (0.79 s 0.89, p>0.05) we e dec eased in blood in only 7% o CVID pa ien s (Figu e 3B). Rega ding ma u e nai e B-cells, an inc ease in CD21 - CD24 - (6.1 s 0.74 cells/µl, p<0.0001) and CD21 - CD24 ++ (1.8 s 0.4 cells/µl, p<0.0001) nai e B cell coun s was obse ed in CVID s. HD, wi h a clea bimodal dis ibu ion (Figu e 3B) due o he p esence o a majo subg oup o pa ien s (73% and 94%, espec i ely) p esen ing a significan expansion o hese wo nai e B-cell subse s, in associa ion o no wi h low CD21 + CD24 + naï e B-cell coun s, which we e ound o be educed in only 21% o CVID pa ien s (Figu e 3B). Rega ding pos -GC MBC, CVID pa ien s displayed a significan educ ion ( s. HD) o CD21 + CD24 + IgMD + MBC (16 s 31 cells/µl, p=0.0006), associa ed wi h no mal o sligh ly inc eased (p>0.05) CD21 - CD24 - and CD21 - CD24 ++ IgMD + MBC numbe s (Supplemen a y Figu e 2). Plasmablas s/ plasma cells, we e ei he no de ec ed o se e ely educed in 100% o CVID pa ien s. Al oge he , hese esul s sugges he exis ence o di e en ma u a ion blockades and p ofiles in CVID, which equen ly also in ol e p e-GC B-cells, in addi ion o pos - GC MBC and plasmablas s/plasma cells. P e-GC B-Cell Ma u a ion P ofile in No mal Blood Based on he inno a i e ma u a ion ools de eloped by Eu oFlow (18,25), a no mal p e-GC B-cell e e ence del Pino-Molina e al. Dissec ion o he P e-GC B-Cell Ma u a ion Pa hway in CVID F on ie s in Immunology | www. on ie sin.o g Feb ua y 2021 | Volume 11 | A icle 6039725 ma u a ion pa hway was buil as desc ibed abo e in he ma e ial an me hods sec ion and illus a ed in Figu e 1A. De ailed analysis o he no mal p e-GC B-cell ma u a ion p ofile showed down egula ion o CD38 om s age 3 on, associa ed wi h s ong CD5 and CD24 exp ession a ea ly s ages (s ages 2–4 and s ages 1–3, espec i ely), CD5 becoming nega i e om s age 7 onwa ds while CD24 p og essi ely dec eased om s age 4 onwa d; in u n, CD21 was s ongly exp essed om s age 1 un il he las s ages o ma u a ion (s ages 9 and 10), when i was down egula ed. Among he o he ma ke s in es iga ed, smIgM showed sligh ly highe exp ession le els a ea ly (s ages 1–3) s la e s ages wi h s able le els om s age 4 o 10, while IgD and CD19 showed simila ly s able exp ession le els along all s ages o ma u a ion o p e-GC B-cells. By defini ion CD27 was no exp essed in p e-GC B-cells (Figu e 1B). Al e ed Ma u a ion P ofiles o Blood P e-GC B-Cells in CVID Di ec compa ison o he pheno ype o ma u a ion-associa ed blood p e-GC B-cell subse s om CVID pa ien s s. he no mal ma u a ion was pe o med in 88 pa ien s by plo ing pheno ypic da a om each CVID agains he no mal e e ence ma u a ion da abase (Figu es 4 and 5). Fo be e isualiza ion o he pheno ypic de ia ions om no mal, a no malized scale (Supplemen a y Figu e 3) was used. O e all, dis inc pa e ns o al e a ion (cell coun s and/o MFI alues pe ma ke in ≥2/10 consecu i e s ages o ma u a ion o p e-GC B-cells alling ≥2SD apa om he no mal dis ibu ion) o he p e-GC B-cell ma u a ion we e de ec ed in e e y case (88/88; 100%). Thus, ou dis inc pa e ns o al e a ions we e iden ified: a) CVID wi h no mally-appea ing ma u a ion pa hways (g oups 1 o 3) bu AB C FIGURE 2 | Dis ibu ion o he majo subse s o imma u e, nai e and memo y B cells (MBC) and plasmablas s/plasma cells (A), including he di e en subse s o p e-ge minal-cen e (GC) B-cells (B), in blood o a ep esen a i e adul heal hy dono (HD) and hei ma u a ion-associa ed ela ionship (C). Th ee-dimensional p incipal componen (PC) analysis (PCA) plo s in (A, B) we e buil based on PC1 (A: mean fluo escence in ensi y o CD27, 29.25%; IgD, 22.56%; IgM, 20.45%; CD21, 11.16%; CD38, 9.63% and CD5, 6.95%; B: mean fluo escence in ensi y o CD21, 30.15%; CD24, 27.64%; CD38, 23.07% and CD5, 19.14%), PC2 (A: mean fluo escence in ensi y o IgM, 43.89%, CD27, 34.62%, CD38, 10.18%, IgD, 9.96%; CD21, 1.16% and CD5, 0.18%; B: mean fluo escence in ensi y o CD21, 43.66%, CD38, 23.62%, CD24, 17.09% and CD5, 15.63%) and PC3 (A: mean fluo escence in ensi y o CD38, 42.02%, CD21, 19.56% CD27, 12.19%, CD5, 12.03%, IgD, 11% and IgM, 3.20%; B: mean fluo escence in ensi y o CD5, 56.16%, CD24, 27.80%, CD38, 14.85% and CD21, 1.19%) ec o s using he (balanced) au oma ed popula ion sepa a o (APS1-2) 3-D iew o Infinicy . In bo h (A, B), he dis inc colo -coded cell popula ions displayed we e ga ed as desc ibed in Supplemen a y Figu e 1. del Pino-Molina e al. Dissec ion o he P e-GC B-Cell Ma u a ion Pa hway in CVID F on ie s in Immunology | www. on ie sin.o g Feb ua y 2021 | Volume 11 | A icle 6039726 dis inc pa e ns o al e a ion on he le els o exp ession o indi idual pheno ypic ma ke s (57/88, 65%) and b) CVID pa ien s wi h a clea ly al e ed p e-GC B-cell ma u a ion pa hway (31/88 cases, 35%; CVID g oup 4) (Figu es 4A and B). In u n, unsupe ised clus e ing analysis u he e ealed he p esence o h ee di e en p ofiles among he o me CVID pa ien g oups 1 o 3, depending on he pa e n o de ia ion in he numbe o cells pe ma u a ion s age and he le els o exp ession o indi idual ma ke s, om he no mal e e ence ma u a ion pa hway (Figu e 5 and Supplemen a y Figu e 3). In g oup 1 a la ge ac ion o CVID pa ien s (42/88, 48%) was included, who showed significan ly educed o al B-cell (128 s 206 cells/µl, p=0.008) and bo h CD5 + CD38 +/++ CD21 he CD24 ++ (2.6 s 5.6 cells/µl, p=0.0013) and CD5 + CD38 he CD21 + CD24 + (7.3 s 17 cells/µl, p<0.0001) imma u e/ ansi ional B-cell numbe s, in he absence o o e all ele an pheno ypic de ia ions om he no mal ma u a ion p ofile o blood p e- GC B-cells (Supplemen a y Figu e 3). G oup 2 consis ed o 8/88 (9%) CVID pa ien s ha showed o e exp ession o smIgM a in e media e s ages o ma u a ion o p e-GC B-cells and o CD38 a ea ly and la e s ages o ma u a ion o p e-GC B-cells; compa ed o HD, CVID cases classified in g oup 2 also showed significan ly highe coun s o he mos imma u e CD5 - CD38 ++ CD21 he CD24 ++ (7.6 s 0.89 cells/µl, p=0.0009) p e-GC B-cell subse , associa ed wi h educed numbe s o mo e di e en ia ed CD5 + CD38 he CD21 + CD24 + imma u e B lymphocy es (5.1 s 17 cells/µl, p=0.0183) and IgMD + (pa icula ly CD21 + CD24 + ) MBC (4 s 31 cells/µl, p<0.0001); in addi ion, g oup 2 pa ien s also showed dec eased le els o CD21 a s ages 7–8, eflec ing he pa allel inc ease in CD21 - A B FIGURE 3 | Dis ibu ion o dis inc subse s o p e-ge minal-cen e (GC) and pos -GC B-cells in blood o Common Va iable Immunodeficiency (CVID) pa ien s (n=100) s age-ma ched HD (n=62). (A) absolu e coun s o majo p e-GC and pos -GC B-cell subse s a e shown using box and whiske s plo s sepa a ely o heal hy dono (HD) (n=62) and CVID pa ien s (n=100) whe e ho izon al lines and e ical lines ep esen he median and bo h 5 h and 95 h pe cen ile alues, espec i ely. Pe cen age o CVID pa ien s wi h dec eased o inc eased coun s below o abo e no mal alues (<5 h and >95 h pe cen ile) de ec ed in age-ma ched HD a e shown as pe cen alues. (B) absolu e p e-GC CD5 - CD38 ++ CD21 he CD24 +++ , CD5 + CD38 +/++ CD21 he CD24 ++ , and CD5 + CD38 he CD21 + CD24 + imma u e B-cell and CD21 + CD24 + , CD21 - CD24 - , and CD21 - CD24 ++ naï e B-cell subse coun s in CVID s HD. N.S. no s a is ically significan di e ences de ec ed, *p- alue<0.05; ***p- alue<0.001; ****p- alue<0.0001 (Mann Whi ney U es ). del Pino-Molina e al. Dissec ion o he P e-GC B-Cell Ma u a ion Pa hway in CVID F on ie s in Immunology | www. on ie sin.o g Feb ua y 2021 | Volume 11 | A icle 6039727 CD24 - naï e B cell coun s (8.8 s 0,74 cells/µl, p=0.02) (Table 1). Finally, g oup 3 included 7/88 (8%) CVID pa ien s cha ac e ized by showing unde -exp ession o CD38 oge he wi h highe le els o CD24 a he las s ages o ma u a ion o p e-GC B- cells, in line wi h he unde lying inc eased coun s o CD21 + CD24 + ma u e naï e B cells obse ed among hese cases s. he o he CVID pa ien g oups 1 o 3: 137 s 76, 42, and 59 cells/µl in CVID g oups 1, g oup 2, and g oup 4, espec i ely (Table 1). As e e ed abo e, g oup 4 CVID pa ien s p esen ed clea ly al e ed p e-GC B-cell ma u a ion p ofiles, he mos common al e a ion (31/31 cases) in hei blood p e-GC B-cells consis ing o absence/dec eased numbe o cells a he ea lies s ages o ma u a ion (s ages 1-3). In addi ion, g oup 4 CVID pa ien s showed abno mally lowe coun s in blood o CD5 - CD38 ++ CD21 he CD24 ++ -0.36 s 0.98, 7.6, 1.6, and 0.89 cells/µl in g oup 1 (p=0.015), g oup 2 (p<0.0001) g oup 3 (p=0.003) pa ien s and HD (p=0.004), espec i ely- and CD5 + CD38 +/++ CD21 he CD24 ++ -1.4 s 2.6, 12 and 5.6 cells/µl, g oup 1 (p=0.03), g oup 2, (p=0.0117) CVID pa ien s and HD (p<0.0001)-, imma u e/ ansi ional B cells, oge he wi h significan ly highe coun s o CD21 - CD24 - -9.9 s 4, 0.74 cells/µl in CVID g oup 1 A B FIGURE 4 | Illus a ing 2-dimensional and 3-dimensional do plo g aphical examples o he ma u a ion pa hway o p e-GC B-cells in ep esen a i e Common Va iable Immunodeficiency (CVID) pa ien s showing no mal-appea ing (A; CVID g oups 1–3) s se e ely dis u bed ma u a ion pa hways (B; CVID g oup 4). In all plo s, he e e ence p e-ge minal-cen e (GC) B-cell ma u a ion pa hway defined o 18 (indi idual) HD g een lines is shown oge he wi h a g ay/black line co esponding o he ma u a ion pa hway o he wo indi idual CVID pa ien s shown in (A, B), espec i ely. Colo ed do s co espond o he median alues ob ained o he 10 di e en ma u a ion s ages (colo code defined in he igh ) whe e s age 1 is colo ed as blue and s age 10 is depic ed in ed. del Pino-Molina e al. Dissec ion o he P e-GC B-Cell Ma u a ion Pa hway in CVID F on ie s in Immunology | www. on ie sin.o g Feb ua y 2021 | Volume 11 | A icle 6039728 FIGURE 5 | Ma u a ion diag ams pe ma ke o all Common Va iable Immunodeficiency (CVID)pa ien s included in each o he h ee CVID g oups o pa ien s displaying a no mal-appea ing p e-GC ma u a ion p ofile in blood (g oups 1–3). In each diag am he (no malized) e e ence ma u a ion pa hway is shown as a g ey a ea o he ±2 SD o no mal MFI alues pe ma ke . The p e-GC B-cell ma u a ion p ofile ound in CVID pa ien s om each g oup is displayed as ±2 SD ba s colo - coded by CVID pa ien g oup (CVID g oup 1 is shown in blue, g oup 2 in g een and g oup 3 in pink). Values below o abo e he no mal 2SD limi o ≥2 consecu i e ma u a ion s ages we e conside ed o eflec an al e ed ma ke exp ession p ofile. del Pino-Molina e al. Dissec ion o he P e-GC B-Cell Ma u a ion Pa hway in CVID F on ie s in Immunology | www. on ie sin.o g Feb ua y 2021 | Volume 11 | A icle 6039729