originl rtile See pge 1923 Cpsi-speifi T-ell Responses to Nturl Infetions With Aeno-ssoite Viruses in Humns Differ From Those of Nonhumn Primtes Hu Li 1, Mrio O Lsro 1, Bei Ji 1,2, Shih Wen Lin 1,3, Lriss H Hut 1, Ktherine A High 4 n Hilegun CJ Ertl 1 1 Immunology Progrm, Wistr Institute, Philelphi, Pennsylvni, USA; 2 Deprtment of Infetious Disese, The first ffilite hospitl of Chongqing Meil University, Chongqing, Chin; 3 Ntionl Cner Institute, Ntionl Institutes of Helth, Division of Cner Epiemiology n Genetis, Rokville, Mryln, USA; 4 Deprtment of Peitris, the Chilren s Hospitl of Philelphi n Howr Hughes Meil Institute, Philelphi, Pennsylvni, USA Hepti eno-ssoite virus serotype 2 (AAV2)-meite gene trnsfer file to hieve sustine trnsgene prout expression in humn sujets. We formulte the hypothesis tht rejetion of AAV-trnsue heptoytes is use y AAV psi-speifi CD8 + T ells tht eome retivte upon gene trnsfer. Although this hypothesis ws omptile with linil t, whih showe rise in irulting AAV psi-speifi T ells following injetion of AAV vetors, it i not explin tht AAV vetors hieve long-term trnsgene expression in rhesus mques, whih re nturlly infete with AAV serotypes losely relte to those of humns. To ress this pprent ontrition, we teste humn n rhesus mque smples for AAV psi-speifi T ells y intrellulr ytokine stining omine with stining for T-ell suset n ifferentition mrkers. This highly sensitive metho, whih oul provie tool to monitor verse T-ell responses in gene trnsfer trils, showe tht AAV psi-speifi CD8 + n CD4 + T ells n e etete in loo of nturlly infete humns n rhesus mques. They re present t higher frequenies in rhesus mques. Furthermore, T ells from humns n rhesus mques exhiit striking ifferenes in their ifferentition sttus n in their funtions, whih my explin the isprte urtion of AAV-meite gene trnsfer in these two speies. Reeive 5 Jnury 211; epte 2 April 211; pulishe online 17 My 211. oi:1.138/mt.211.81 INTRODUCTION In phse 1 linil tril, 1 humn sujets with severe hemophili B were infuse with reominnt eno-ssoite virus (AAV) vetors erive from the humn serotype 2 (AAV2) expressing ftor IX (AAV2-F.IX) for intrhepti expression. At the highest ose of vetor, one of the ptients evelope therpeuti levels of F.IX y week 2, ut y 4 weeks fter vetor infusion, levels of F.IX strte to erese n within few weeks returne to pregene therpy levels. At the time when F.IX levels strte to erese, the ptient showe n symptomti inrese in trnsminses, whih eventully resolve fter F.IX h erese to seline. 1 The tril ws ontinue with reue ose of vetor. The next ptient i not evelop etetle levels of F.IX upon gene trnsfer. Nevertheless, he lso presente with trnsminitis fter gene trnsfer with time ourse reltive to therpy ientil to tht seen in the other ptient. In the 2n ptient, T-ell responses to the psi ntigen of the AAV2 vetor n the trnsgene prout were ssesse y ELISpot ssys for interferon- (IFN- ) efore n fter gene trnsfer. 2 He h no etetle AAV2 psi-speifi T ells in loo efore gene trnsfer. Suh response evelope fter gene trnsfer n then eventully susie. Responses to F.IX oul not e etete efore or fter gene trnsfer. Overll these finings were omptile with the hypothesis tht humns ue to nturl infetions rry pre-existing T ells to ntigens of AAV psi, whih re retivte upon AAV-meite gene trnsfer n eliminte the trnsue ells. 2 The linil results were in ontrst to prelinil t generte in numer of speies inluing mie, 3 5 ogs, 3,6,7 n nonhumn primtes (NHPs) 8 1 where the AAV2 vetor h resulte in sustine gene trnsfer n in orretion of isese in niml moels of hemophili B. It ws postulte tht rejetion ws not enountere in prelinil moels with speies tht o not eome infete with primte AAVs suh s mie or nines n thus hve no immunologil memory to ntigens of AAV psi. Nevertheless, ttempts to mimi the linil finings in roents expose to ntigens of AAV psi prior to AAV gene trnsfer hve file thus fr. 11,12 Furthermore, the hypothesis of immunemeite estrution of AAV-trnsue heptoytes in iniviuls with immunologil memory to AAV oul not explin why sustine AAV-meite gene trnsfer oul e hieve in rhesus mques, whih re nturlly infete with AAVs tht show suffiient homology with strins tht infet humns to llow for rossreognition y T ells. 2 As ws shown previously, AAVs pper to Corresponene: Hilegun CJ Ertl, Immunology Progrm, Wistr Institute, 361 Sprue Street, Philelphi, Pennsylvni 1914, USA. E-mil: ertl@wistr.upenn.eu Moleulr Therpy vol. 19 no. 11, 221 23 nov. 211 221
persist more ommonly n t higher opy numers in NHPs suh s rhesus mques thn in humns. 13,14 These stuies lso showe eviene of ross-speies AAV trnsmission with n ientil le eing isolte from humns n rhesus mques. 14 Here, we teste humn n rhesus mque smples for AAV psi-speifi T ells. Speifilly we teste for T ells to psi ntigens of AAV serotypes tht hve een isolte from either speies, i.e., AAV2 15 n AAV8, 13 respetively, lthough it shoul e pointe out tht these two viruses shre 83% homology t the nuleotie level n therefore T-ell epitopes. 16 Our results show tht oth AAV psi-speifi CD8 + n CD4 + T ells n reily e etete in loo of oth speies ut re present t higher frequenies in rhesus mques. Our results lso ientify ifferenes in AAV psi-speifi T-ell ifferentition sttus n funtions etween humns n NHPs, whih my ontriute to the isprte urtion of AAV-meite gene trnsfer in these two speies. RESULTS Mgnitue n ifferentition sttus of humn AAV psi-speifi T-ell responses Frequenies of AAV psi-speifi T ells isolte from peripherl loo mononuler ells (PBMCs) of 17 helthy ults from the Philelphi re were etermine y multiolor stining with ntioies to ifferentition mrkers n ell funtions inluing IFN-, interleukin-2 (IL-2), tumor nerosis ftor- (TNF- ), perforin n mrophge inflmmtory protein-1 (MIP-1 ) followe y flow ytometri nlyses (Figure 1). Of the humn sujets 8 out of 17 h etetle AAV psi-speifi CD8 + T ells with frequenies of ove.5% in loo. The sum of frequenies g 1 5 FSC-H 25K 2K 15K 1K 5K 81.7 5K 1K 15K 2K 25K FSC-A SSC-A 25K 2K 15K 1K 5K 7.9 5K 1K 15K 2K 25K FSC-A <PE-Texs Re-A>: CD8 1 5 1 4 1 3 96.7 1 3 1 4 1 5 <AmCyn-A>: Live/De <APC-A>: IL-2 1 4 1 3 15 <APC-A>: IL-2 1 3 1 4 1 5 <APC-Cy5-5-A>: IFN- 5 1 1 5 12.7 4 1 1 4 1 3 3 1 16.5 1 3 1 4 1 5 <PE-Cy7-A>: TNF- f <Qot 655-A>: CD27 39.5 35.4 7.61 8.78 1 3 1 4 1 5 <Qot 75-A>: CD45RO e <PE-Cy5-5-A>: CD4 1 5 1 4 1 3 45.6 <PE-Texs Re-A>: CD8 <PE-Texs Re-A>: CD8 <APC-Cy7-A>: CD14/CD19 1 5 1 4 1 3 32.6 92.8 1 3 1 4 1 5 1 3 1 4 1 5 1 5 <FITC-A>: MIP-1 1 4 1 3 1 2 6.83 1.92 1 2 1 3 1 4 1 5 <Pifi Blue-A>: Perforin Figure 1 Gting for the humn ells. Cells were first gte onto () single ells n then on () lymphoytes n () AmCyn negtive live ells. () APC-Cy7 ws use s ump gte for CD14 n CD19. (e) Cells were then gte onto CD8 + or CD4 + ells, whih were then gte further oring to (f) expression of CD45RO n CD27. (g) CD45RO hi CD27 hi ells (entrl memory ells, TCM), CD45RO hi CD27 low ells (effetor memory ells, TEM) n CD45RO low CD27 low ells (effetor ells, TEff) were then gte onto interferon- (IFN- ), or interleukin-2 (IL-2) n tumor nerosis ftor- (TNF- ), perforin n mrophge inflmmtory protein-1 (MIP-1 ) s shown. 222 www.moleulrtherpy.org vol. 19 no. 11 nov. 211
of CD8 + T ells prouing ny of the possile omintions of ftors ws lulte (Figure 2). To etermine suset istriution the sum of frequenies ws then normlize to numers of ells present within 1 7 live PBMCs (Figure 2). Using numers of ells s re out showe tht pproximtely hlf of the AAV psi-speifi CD8 + T ells elonge to the entrl memory T ells (TCM) suset while the more tivte effetor T ells (TEff) n effetor memory T ells (TEM) ells represente out qurter of AAV psi-speifi CD8 + T ells eh (Figure 2). AAV psi-speifi CD4 + T ells oul e etete in 9 of the 17 smples (Figure 2). Cell frequenies were normlize n numers of speifi ells with 1 7 live PBMCs were etermine (Figure 2). The AAV psi-speifi CD4 + T-ell response in humns ws mrkely ominte y TCM ells, whih ontriute over 8% of AAV psi-speifi CD4 + T ells; the rest % Of ytokines + T ells/totl T ells in the sme suset Cytokines + T ell #/1 7 live PBMCs.2.15.1.5. 4,5 4, 3,5 3, 2,5 2, 1,5 1, 5 CD8-totl CD4-totl PBMC H1 H2 H3 H4 H5 H6 H7 H8 H9 H1 H11 H12 H13 H14 H15 H16 H17 Humn # CD4-TCM CD4-TEff CD4-TEM CD8-TCM CD8-TEff CD8-TEM CD8-TCM CD8-TEff CD8-TEM CD4-TCM CD4-TEff CD4-TEM Figure 2 Frequenies n ell numers of eno-ssoite virus serotype 2 (AAV2) psi-speifi CD8 + or CD4 + peripherl loo mononuler ells (PBMCs) in humns. Lymphoytes isolte from 17 humn loo smples were teste y intrellulr ytokine stining for the proution of interferon-, interleukin-2, tumor nerosis ftor-, perforin, n mrophge inflmmtory protein-1 upon stimultion with the AAV2 psi peptie pool. () The sum of the responses in totl CD8 + n CD4 + T ells in ifferent people re shown. () The numers of AAV2 psi-speifi ells of eh suset per 1 7 live PBMCs re shown. The verge reltive istriutions of the () AAV2 psi-speifi ifferent CD8 + n () CD4 + T-ell susets were lulte. Bkgroun responses (responses without pepties) were sutrte prior to plotting of responses. TCM, entrl memory ells; TEff, effetor ells; TEM, effetor memory ells. minly elonge to the TEM suset. Numers of highly tivte CD4 + TEff ells were negligile (Figure 2). Mgnitue n ifferentition sttus of NHP AAV psi-speifi T-ell responses T-ell responses to AAV8 psi were first teste from PBMCs of 21 young rhesus mques using ELISpot ssys for oth IFN- (Figure 3) n IL-2 (Figure 3), metho tht h een use to ssess T-ell responses to AAV gene trnsfer in humns. 2 In ition, plsm from the sme set of NHPs were nlyze for neutrlizing ntioies to AAV8 (Figure 3). Eleven nimls h neutrlizing ntioies to AAV8 n nine nimls h irulting T ells tht proue IFN- upon in vitro stimultion with AAV8 peptie pools with ut-off for positive responses of 55 spots/1 6 PBMCs. None of the smples were positive for T ells prouing IL-2 to the AAV8 peptie pools. IFN- responses were low n Spot forming ells/million ells Spot forming ells/million ells Reiprol titers of neutrlizing ntioies 18 16 14 12 1 8 6 4 2 18 16 14 12 1 8 6 4 2 175 15 125 1 75 5 25 PBMCs stimulte with AAV8 peptie pools - IFN Pool 4 Pool 3 Pool 2 Pool 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 NHP # PBMCs stimulte with AAV8 peptie pools - IL-2 Pool 4 Pool 3 Pool 2 Pool 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 NHP # 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 NHP # Figure 3 ELISPOTs for interferon- (IFN- ) n interleukin-2 (IL-2) responses n neutrlizing ntioies to eno-ssoite virus serotype 8 (AAV8) in nonhumn primtes (NHPs). Freshly isolte peripherl loo mononuler ells (PBMCs) from nive rhesus mques were test y n ELISpot ssy for IFN- n IL-2 seretion upon stimultion with four ifferent peptie pools ontining overlpping pepties of the AAV8 psi. The grphs showe responses of 21 nimls. Responses to meium (negtive ontrol) were sutrte from responses to eh pool. (,) Our ut-off for positive responses ws 55 spots/1 6 PBMCs. Error r showe the SD of two ifferent wells. () Plsms from these rhesus mques were lso ollete n titers of neutrlizing ntioies to AAV8 were teste. Moleulr Therpy vol. 19 no. 11 nov. 211 223
there ws no orreltion etween mgnitue of T-ell responses n neutrlizing ntioy titers. We teste PBMCs from six itionl rhesus mques for T-ell responses to AAV8 psi y intrellulr ytokine stining (ICS) n multiolor flow ytometry for proution of IL-2, IFN- n TNF-. AAV8 psi-speifi CD8 + T ells oul e etete in five out of six smples (Figure 4). The AAV psispeifi CD8 + T-ell response ws strongly ominte y TEff ells, whih ontriute more thn 8% of the responing ells. TCM n TEM CD8 + ells oul e etete t pproximtely equl numers (Figure 4,). AAV psi-speifi CD4 + T ells oul e etete in ll smples (Figure 4); numers of speifi CD4 + TCM n TEff ells were similr while those of TEM ells were slightly lower (Figure 4,). % Of ytokines + T ells/totl T ells in the sme suset Cytokines ell #/1 7 live PBMCs.8.6.4.2. 12, 1, 8, 6, 4, 2, PBMC CD8-totl CD4-totl Rts 9 Rvv 7 Rwk 8 Rzu 1 Rvf 1 Rvt 9 CD8-TCM CD8-TEff CD8-TEM NHP # CD4-TCM CD4-TEff CD4-TEM CD8-TCM CD8-TEff CD8-TEM CD4-TCM CD4-TEff CD4-TEM Figure 4 Frequenies n ell numers of eno-ssoite virus serotype 8 (AAV8) psi-speifi CD8 + or CD4 + peripherl loo mononuler ells (PBMCs) in nonhumn primtes (NHPs). PBMCs from six nive rhesus mques were teste y intrellulr ytokine stining for proution of interferon-, interleukin-2 n tumor nerosis ftor- upon stimultion with the AAV8 psi peptie pool. () The sum of the responses in totl CD8 + n CD4 + T ells in ifferent NHP smples re shown. () The numers of AAV8 psi-speifi ells of eh suset per 1 7 live PBMCs re shown. The verge reltive istriutions of the () AAV8 psi-speifi ifferent CD8 + n () CD4 + T-ell susets were lulte. Bkgroun responses (responses without pepties) were sutrte efore plotting of responses. TCM, entrl memory ells; TEff, effetor ells; TEM, effetor memory ells. Funtionlity of humn T ells Assessing five ifferent funtions for eh T-ell suset llows in theory for the etetion of 31 popultions in eh of the six T-ell susets tht proue either only one ftor or ifferent omintions of two, three, four or ll five ftors. Perforin is ommonly present in humn T ells tht re not stimulte y ntigen in vitro n hs to e proue in omintion with nother ftor to llow for ientifition of n ntigen-responsive popultion. T ells tht upon stimultion with AAV pepties exhiite proution of perforin only were thus not tken into ount in the nlyses (hene 31 n not 32 popultions). Results shown in the figures only inlue funtions exhiite y ny of the AAVspeifi T-ell susets uner interrogtion. In loo AAV psi-speifi CD8 + TEff were rre n the lrgest popultion tht oul e etete proue multiple ftors, i.e., IFN- (g), IL-2 (l), n perforin (p). Those prouing IFN- only ominte CD8 + TCM ells, lthough smller popultions prouing vriety of ftors or omintions of ftors oul lso e etete. The response profile ws similr for AAV psi-speifi CD8 + TEM ells for whih seon ominnt pek of ells prouing IFN- together with perforin oul e etete (Figure 5). Upon normliztion for CD8 + T ells prouing IFN-, IL-2 or TNF-, numers n perentges of ells prouing IFN- or IL-2 were similr while only ~1% proue TNF- (Figure 5). CD4 + TEff in loo proue wie vriety of omintions of ftors. TCM ells minly proue IFN-. Most CD4 + TEM ells were positive for single funtions, i.e., IFN-, MIP-1 (M) or TNF- (T) (Figure 5). Upon normliztion to live PBMCs out hlf of the CD4 + T ells proue IFN- lone or with other funtion, 4% proue IL-2 n the rest proue TNF- (Figure 5). Funtionlity of T ells from NHPs AAV psi-speifi T ells from NHPs were nlyze for only three funtions, i.e., IFN- (g), IL-2 (l), n TNF- (T) (Figure 6,), euse suitle ntioies were not ville for perforin n MIP-1. Cells prouing only IL-2 ominte responses of ll CD8 + n CD4 + T-ell susets (Figure 6,), whih ontrste to the responses of humn PBMCs. A summry of the three funtions, i.e., IFN-, IL-2 n TNF- for ifferent CD8 + T-ell susets from humn n NHP smples is shown in Supplementry Tle S1. Phenotypes of humn n NHP AAV psi-speifi T ells CD8 + n CD4 + T ells from humn n NHPs tht serete IFN- n IL-2, the two most ommon funtions oserve in humn n NHP AAV psi-speifi T ells, were nlyze for expression of PD1, n immunoinhiitory mrker tht is highly expresse on reently tivte s well s on so-lle exhuste T ells. In ition, ells were nlyze for intrellulr expression of Ki-67, moleule tht is ssoite with ell prolifertion (Figure 7). Nonytokine sereting ells of the sme suset s well s nive ells (CD27 hi CD45RO low ) were nlyze in prllel. Levels of expression of these mrkers on AAV psi-speifi CD4 + or CD8 + T ells were omprle to those on non-aav-speifi T ells from the sme susets in oth 224 www.moleulrtherpy.org vol. 19 no. 11 nov. 211
% Of ytokines T ells/ totl CD8 T ells % Of ytokines T ells/ totl CD8 effetor ells.2.15.1.1.8.6.4.2..2.15.1.1.8.6.4.2 CD8-totl CD8-effetor..2.15.1.1.8.6.4.2..2.15.1.1.8.6.4.2. % Of ytokines T ells/ totl CD4 T ells % Of ytokines T ells/ totl CD4 effetor ells.2.15.1.1.8.6.4.2..2.15.1.1.8.6.4.2 CD4-totl CD4-effetor % Of ytokines T ells/ totl CD8 CM ells % Of ytokines T ells/ totl CD8 EM ells CD8-CM CD8-EM % Of ytokines T ells/ totl CD4 CM ells % Of ytokines T ells/ totl CD4 EM ells..2.15.1.1.8.6.4.2..2.15.1.1.8.6.4.2. CD4-CM CD4-EM Cytokines Cytokines CD8-totl CD8-IFN- CD8-IL-2 CD8-TNF- CD4-totl CD4-IFN- CD4-IL-2 CD4-TNF- Figure 5 Funtionl properties of CD8 + or CD4 + T ells from humn loo. The sme 17 humn peripherl loo mononuler ells (PBMCs) shown in Figure 2 were teste for seretion of interferon- (IFN- ) (g), interleukin-2 (IL-2) (l), tumor nerosis ftor- (TNF- ) (T), perforin (p) n mrophge inflmmtory protein-1 (MIP-1 ) (M) y intrellulr ytokine stining of CD8 + or CD4 + T ells tht h een stimulte with the eno-ssoite virus serotype 2 (AAV2) peptie pool. (,)The rs show the frequenies of ifferent susets of ells tht stine for the ifferent funtion either lone or in omintions. Frequenies <.5% over ll ells of the suset n frequenies of T ells prouing perforin only re not shown. The error r shows the stnr error of men of 17 humn PBMCs. (,)The verge reltive istriution of IFN-, IL-2 or TNF- sereting AAV2 psi-speifi CD8 + n CD4 + T ells re shown. Bkgroun responses (responses without pepties) were sutrte. humns n NHPs n lerly istint from those of nive T ells, with the exeption tht PD1 ws ownregulte on AAV psispeifi CD8 + TEM n TEff ells from NHPs, while Ki67 ws upregulte in the AAV psi-speifi CD8 + TEff n CD4 + TEM ells. Nevertheless, ifferenes were sutle (Figure 7 e). T ells were in ition stine for ntioies to CD16, nother immunoinhiitory moleule n CD57, mrker of terminl T-ell ifferentition. Antioies tht ret with these moleules from NHPs re not ville n this nlysis ws therefore restrite to humn smples. AAV psi-speifi CD8 + T ells of the TCM n TEM Moleulr Therpy vol. 19 no. 11 nov. 211 225
% Of ytokine T ells/ totl CD8 T ells.6.5.4.3.2.1 CD8-totl % Of ytokine T ells/ totl CD4 T ells.6.5.4.3.2.1 CD4-totl.. % Of yrtokine T ells/ totl CD8 effetor ells.6.5.4.3.2.1 CD8-effetor % Of ytokine T ells/ totl CD4 effetor ells.6.5.4.3.2.1 CD4-effetor % Of ytokine T ells/ totl CD8 CM ells..6.5.4.3.2.1 CD8-CM % Of ytokine T ells/ totl CD4 CM ells..6.5.4.3.2.1 CD4-CM % Of ytokine T ells/ totl CD8 EM ells..6.5.4.3.2.1 CD8-EM % Of ytokine T ells/ totl CD4 EM ells..6.5.4.3.2.1 CD4-EM.. Cytokines Cytokines CD8-totl CD8-IFN- CD8-IL-2 CD4-totl CD4-IFN- CD4-IL-2 CD8-TNF- CD4-TNF- Figure 6 Funtionl properties of CD8 + or CD4 + T ells from nonhumn primtes (NHPs). The sme NHP peripherl loo mononuler ells (PBMCs) shown in Figure 4 were teste for seretion of interferon- (IFN- ) (g), interleukin-2 (IL-2) (l) n tumor nerosis ftor- (TNF- ) (T) y intrellulr ytokine stining of CD8 + or CD4 + T ells tht h een stimulte with eno-ssoite virus serotype 8 (AAV8) peptie pool. (,) The rs showe the frequenies of ifferent susets of ells tht serete one, two or three ytokines lone or in omintions. The error r showe the stnr error of men of six NHPs. (,) The verge reltive istriution of IFN-, IL-2 or TNF- sereting AAV8 psi-speifi CD8 + n CD4 + T ells re shown. Bkgroun responses (responses without pepties) were sutrte. susets expresse neither CD16 nor CD57, while ~59% of AAV psi-speifi CD8 + TEff ells n ~69% of other CD8 + TEff ells expresse CD57. CD4 + TEff ells expresse elevte levels of CD57 with frequenies of 5% in AAV psi-speifi T ells n 65% in ells with other speifiities. CD16 expression ws only inrese on frtion of CD8 + TEff ells (35% for AAV psi-speifi CD8 + TEff ells versus 46% of non-aav psi-speifi CD8 + TEff ells, t not shown). Overll phenotypi nlyses i not revel phenotypi ifferenes etween AAV psi-speifi T ells ompre to T ells irete to other ntigens in humns n NHPs. DISCUSSION Experiments esrie in this mnusript were esigne to ress two issues. First, we wnte to evelop metho tht is 226 www.moleulrtherpy.org vol. 19 no. 11 nov. 211
PD1 CD8 CD4 Ki67 PD1 Ki67 Nive NHP 1 3 1 4 1 5 1 3 1 4 1 5 1 3 1 4 1 5 1 3 1 4 1 5 <PE-Cy7-A>: PD1 <APC-Cy5-5-A>: Ki67 <PE-Cy7-A>: PD1 <APC-Cy5-5-A>: Ki67 Humn 1 3 1 4 1 5 1 3 1 4 1 5 1 3 1 4 1 5 1 3 1 4 1 5 <PE-Cy7-A>: PD1 <APC-Cy5-5-A>: Ki67 <PE-Cy7-A>: PD1 <APC-Cy5-5-A>: Ki67 Nive GMFI 8 7 6 5 4 3 2 1 Nive CD8-PD1 NHP Humn P <.5 P <.5 GMFI 1, 75 5 25 Nive CD8-Ki67 NHP Humn P <.5 1,5 1,25 1, NHP Humn CD4-PD1 e 1, 75 NHP Humn CD4-Ki67 P <.5 GMFI 75 5 25 GMFI 5 25 Nive Nive Figure 7 Phenotypes of eno-ssoite virus (AAV) psi-speifi ifferent CD8 + n CD4 + T-ell susets. The verge expression levels of PD1, Ki67 were etermine on interferon- (IFN- ) n/or interleukin-2 (IL-2) sereting (TCM +,TEM +,TEff + ) n IFN- n IL-2 oule negtive (TCM,TEM,TEff ) CD8 + n CD4 + T-ell susets in oth nonhumn primte (NHP) n humn peripherl loo mononuler ells (PBMCs). () The expression levels in nive ell popultion re shown for omprison. ( e) The geometri men fluoresene intensity (GMFI) of PD1, Ki67 expression in ifferent ell susets re shown. The error rs show the SD of three to four ifferent smples whih h suffiient numers of ells for the nlysis in eh group. suitle to monitor humn CD4 + n CD8 + T-ell responses to AAV psi ntigens to i AAV gene trnsfer trils. Previous trils teste for T ells using minly ELISpot ssys. Suh ssys only etete AAV psi-speifi T-ell responses in loo of 2 out of 46 or in spleens of 1 out of 28 humn sujets prior to gene trnsfer lthough most h ntioies to AAV initing prior infetions. Following gene trnsfer AAV psi-speifi T ells oul trnsiently e etete in loo of AAV vetor reipients enrolle into ifferent trils. 2,17,18 Till reently, ELISpot ssys were viewe s eing more sensitive thn ICS to etet memory T ells present t low frequenies. Nevertheless, vnes in flow ytometry tht now llow to etet up to 18 ifferent fluorohromes on iniviul ells, exlusion of e ells, testing for susets y stining for ifferentition mrkers, n use of ntioies for multiple funtions hve reue the kgroun of ICS n inrese its sensitivity to tht of or ove ELISpot ssys. 19,2 Results otine y ICS re lerly more informtive thn those gthere y ELISpots s the former ut not the ltter istinguishes etween responses y CD4 + or CD8 + T ells n llows for ientifition of ifferent T-ell susets. Furthermore ICS n monitor T-ell smples simultneously for proution of multiple ytokines, hemokines or other ftors Moleulr Therpy vol. 19 no. 11 nov. 211 227
tht re inue upon ntigeni stimultion while ELISpot ssys only mesure one funtion. Although it is tehnilly fesile to use ELISpot ssys for n rry of funtions, the numer of ells require for multiple prllel testing woul e prohiitive. Lst ut not lest, s we reporte previously, 21 ELISpot ssys my not e optiml to test for terminlly ifferentite T ells s those my ie uring the 18 24-hour inution perio of the ssy. Our t show tht multiolor ICS followe y flow ytometri nlysis my e etter suite thn ELISpot ssys to monitor immune responses in AAV gene trnsfer trils. The ICS ssy we use here for humn smples mesures five funtions proue y ifferent T-ell susets. Smples from NHPs were only teste for three funtions, s ntioies for the other two funtions, i.e., MIP-1 n perforin, were not ville. Frequenies of AAV-speifi T ells in NHPs re thus presumly unerestimte ompre to those of humns. Notwithstning, NHPs, whih o not rejet AAV upon gene trnsfer, hve on verge 4 5 times higher frequenies n numers of AAV psispeifi T ells irulting in loo ompre to humns, whih pper to eliminte AAV-trnsue ells. In ition to ifferenes in frequenies, there were lso mrke ifferenes in suset istriution of AAV psi-speifi CD4 + n CD8 + T ells. The humn response ws ominte y CD4 + T ells, whih minly elonge to the TCM suset while in NHPs TEff CD4 + T ells were more ommon followe y TCM ells. Approximtely 5% of AAV psi-speifi CD8 + T ells in humns elonge to the TCM suset, while in NHPs more thn 8% were tivte CD8 + TEff ells. Differenes in memory ell istriution my reflet ifferenes in the AAVs lifeyle in the two speies. As ws shown previously, AAVs pper to persist more ommonly n t higher opy numers in NHPs suh s rhesus mques thn in humns. 13,14 These stuies lso showe eviene of ross-speies AAV trnsmission with n ientil le eing isolte from humns n rhesus mques. 14 AAV most ommonly infets together with enoviruses. There is mounting eviene tht infetions with enoviruses, whih provie helper funtions to AAVs tht s epenoviruses nnot replite y themselves, re more pervsive in NHPs thn in humns. 22 Most helthy NHPs she enoviruses while this is only oserve uring or shortly fter ute infetions of humns. 23,24 Uner the ssumption tht in simins helper funtions to AAVs re provie y enoviruses n tht AAVs o-persist with enoviruses in the sme ells, this my inite tht retivtion of enogenous AAVs might lso e more ommon in NHPs. This in turn oul ffet not only frequenies of irulting AAV-speifi T ells ut in ition their funtionlity, s ontinue exposure to high levels of ntigen, s ws shown upon hroni infetions of mie with ertin strins of lymphoyti horiomeningitis virus 25 or of humns with humn immunoefiieny 26 or heptitis virus, 27 n le to funtionl impirment of CD8 + T ells, whih ue to exhustion or terminl ifferentition oul lose the ility to expn upon ntigeni stimultion or to eliminte ntigen presenting trget ells. AAV psi-speifi T-ell responses in humns exhiite numer of funtions while those of NHPs were funtionlly more restrite. Compring funtions of AAV psi-speifi T ells etween the two speies showe tht humn CD8 + n CD4 + T ells proue minly IFN- or IL-2 lone or in omintion with other ftors suh s MIP-1 or perforin in ontrst to those from NHPs, whih proue nerly exlusively IL-2. This ws unexpete s in generl IL-2 is more ommonly proue upon retivtion of resting TCM ells while IFN- is proue y more tivte TEff or TEM ells. In ition results from the ELISpot nlyses for PBMCs from NHPs sore positive for T ells prouing IFN- in response to ntigen n negtive for those prouing IL-2 while whih is in ontrst to results from ICS gin stressing tht these two methos my not neessrily give onornt results s T ells, espeilly highly tivte T ells, my ie rpily upon in vitro ulture. 21 AAV psi-speifi T ells from humn n NHPs were further teste for some phenotypi mrkers tht re typilly use to ssess T-ell funtionlity. Agin the nlysis ws more omprehensive for humn smples ue to vilility of ntioies to more mrkers. Levels of expression of PD-1 n Ki-67, whih re use s initors for T-ell exhustion or for the T ells prolifertive potentil, were very similr on AAV psi-speifi T ells n non- AAV psi-speifi ntigen-experiene T ells in either speies. In the sme token, CD57, mrker for terminl T-ell ifferentition, n CD16, whih is lso mrker of exhustion, were similr on oth popultions in humns. Neither of these stins thus supporte the notion tht AAV psi-speifi T ells were impire or phenotypilly istint from other ntigen-experiene T ells. Overll, ifferenes in sustine gene trnsfer y AAV vetors in humns n NHP, orrelte well with the oserve ifferenes in T ells ifferentition sttus n funtionlity. TCM ells, whih re reltively more ommon in humns, proliferte opiously upon re-enounter of their ntigens, unlike the more tivte TEM or TEff ells. One woul thus expet tht upon AAV-meite gene trnsfer, expnsion of AAV psi-speifi TCM ells woul in the en result in higher numers of effetor ells in humns thn the more moest expnsion of tivte AAV psi-speifi T ells in NHPs. In ition, AAV psi-speifi T ells in humns proue IFN-, ytokine tht upregultes mjor histoomptiility omplex (MHC) lss I expression. MHC lss I moleules re ruil for CD8 + T-ell reognition n previous stuies showe tht in sene of IFN- hepti trnsfer of highly immunogeni enovirus vetors results in sustine gene trnsfer. 28 AAV psi-speifi CD8 + T ells from NHPs file to proue IFN- when teste y ICS. We ssume tht this my llow for the espe of AAV-trnsue ells in monkeys while in humns proution of IFN- y AAV psispeifi CD8 + T ells my le to upregultion of MHC lss I moleules on heptoytes n their reognition n estrution y AAV psi-speifi CD8 + T ells. One oul view this s irulr rgument, s T ells require reognition of their trget ntigen suh s pepties erive from the AAV psi in ssoition with MHC moleules to proue ytokines inluing IFN-. Nevertheless, the initil T-ell stimultion oul e provie y ells other thn heptoytes present in the liver resulting in IFN- proution, whih woul then trigger upregultion of moleules of the ntigen presenttion pthwy in heptoytes. While our first gol, to evelop more sensitive metho for monitoring of AAV-meite humn gene therpy trils ws esily met y multiolor flow ytometry. Our seon gol, to eluite why AAV gene trnsfer reily hieves sustine trnsgene 228 www.moleulrtherpy.org vol. 19 no. 11 nov. 211
expression in mie n monkeys ut only trnsient expression in humns touhes the very omplex n not yet fully unerstoo pthwys tht govern CD8 + T-ell ifferentition. Monkeys n humns evelop AAV psi-speifi CD8 + T ells upon nturl infetions while suh T ells n reily e inue in mie y their immuniztion with n pproprite virl vetor. 11 In mie, AAV psi-speifi memory CD8 + T ells n reognize their ognte ntigen n mount proliferte response 29 ut they fil to reue liver-speifi expression of the AAV vetor s trnsgene. We speulte tht this reflets tht in mie AAV-trnsue heptoytes fil to express suffiient ensities of MHC lss I AAV psi epitope omplexes to llow for their lysis through CD8 + T ells. We ssume tht this pthwy ultimtely ontriutes to sustine trnsgene prout expression upon hepti AAV trnsfer into NHPs. The high pervsiveness of A viruses in this speies my llow for onomitnt synthesis of AAV proteins. Constnt proution of AAV psi ntigens woul result in reurrent T-ell tivtion n initite ifferentition progrm hrterize y reue funtionlity, i.e., loss of prolifertive potentil n reue proution of ytokine n lyti enzymes. In ontrst, in humns, mny of the AAV psi-speifi CD8 + T ells ifferentite into memory ells, whih provie iniret eviene tht proution of AAV ntigens might e of more trnsient urtion. Upon re-enounter of their ntigen suh memory ells n proliferte vigorously n eome fully ompetent effetor ells le to exhiit full spetrum of funtions inluing seretion of IFN-, whih inreses moleules nee for optiml ntigen presenttion, perforin n grnzyme, whih re le to lyse ells expressing the CD8 + T ells ntigen. While hepti gene trnsfer of single-strne AAV2 vetor for hf.ix i not result in sustine trnsgene expression, more reent stuies with self-omplementry AAV8 vetor yiele more promising results. 3 Whether this reflets ifferenes in T-ell responses, vetor preprtions or ifferentil proessing of psis from serologilly istint AAVs within heptoytes remins to e investigte. MATERIALS AND METHODS Humn smples. Humn loos were rwn from 18- to 55-yer-ol, norml helthy humns from the Philelphi re. NHPs smples. Smples from 2- to 3-yer-ol, helthy n simin immunoefiieny virus-uninfete Inin origin M multt tht h not yet een enrolle into ny experiments were use. Isoltion n preservtion of lymphoytes. PBMCs were isolte s esrie. 31 They were teste immeitely fter isoltion or frozen in 9% fetl ovine serum n 1% imethyl sulfoxie (Sigm, St Louis, MO) t 8 C. ELISpot ssys. The ELISpot ssys for IFN- n IL-2 were onute s esrie. 21 Experimentl wells were inute with pools of AAV psi pepties eh t finl onentrtion of 1 μg/ml, negtive ontrol wells were inute with meium n positive ontrol wells were inute with phorol 12-myristte 13-ette (PMA) (finl onentrtion.5 μg/ ml) n ionomyin (finl onentrtion 1 μg/ml). Bse on previous pulitions, 32,33 smples were sore positive if they h t lest 55 spots per 1 6 ells upon ntigeni stimultion, if numers of spots/well in presene of the peptie pools were >3 times higher thn numer of spots/well tht were ulture without pepties n if numers of spots in experimentl wells were t lest 3 SD ove those in ontrol wells. ICS Humn smples: The funtion of AAV psi-speifi CD8 + T ells ws ssesse y ICS fter stimultion with AAV2 psi peptie pool. All pepties were use t finl onentrtion of 2.5 μg of eh peptie per ml. Frozen ells were thwe n inute in RPMI 164 meium supplemente with 1% fetl ovine serum overnight t 37 C in 1% CO 2. Then ells were wshe with Hnk s Blne Slt Solution (HBSS) supplemente with 2 units/ml DNse I, resuspene with RPMI 164 meium n stimulte for 6 hours in the presene of nti-cd28 (lone CD28.2), nti-cd49 (lone 9F1), n Brefelin A. 31 Cells were stine with LIVE/DEAD Fixle Aqu e ell stining kit- AmCyn (Invitrogen, Crls, CA), nti-cd14-apc-cy7 (lone TüK4; Invitrogen), nti-cd19-apc-cy7 (lone SJ25-C1; Invitrogen), nti-cd8- PE-Texs Re (lone SFCI21Thy2D3; Bekmn Coulter, Fullerton, CA), nti-cd4-pe Cy5.5 (lone S3.5; Invitrogen), nti-cd27-qd 655 (lone CLB-27/1; Invitrogen), n nti-cd45ro-qd 75 (UPenn, Philelphi, PA) for 3 minutes t room temperture (RT) in the rk. After fixtion n permeiliztion with Cytofix/Cytoperm (BD Biosienes, Sn Jose, CA) for 2 minutes t RT, ells were then stine with nti-cd3-percp- Cy5.5 (lone SP34-2), nti-ifn- -APC-Cy5.5 (lone B27), nti-il-2-apc (lone MQ1-17H12), nti-tnf- -PE-Cy7 (lone MA11), nti-perforin- Pifi Blue (UPenn) n nti-mip1 -FITC (lone 93342; R&D Systems, Minnepolis, MN) for 45 minutes t RT in rk. Cells were wshe twie, fixe with BD Stilizing Fixtive (BD Biosienes), n then nlyze y FACS using LSRII (BD Biosienes) n DiV softwre. Post-quisition nlyses were performe with FlowJo (TreeStr, Ashln, OR). Single olor ontrols use CompBes Anti-Mouse Ig, k (BD Biosienes). Unless otherwise note, ntioies were purhse from BD (BD Biosienes). Smples were only sore positive if t lest.5% of given supopultion stine for ytokine or ftor; this perentge for most smples require more thn 1 positive events on the flow ytometry lots. For the phenotypi nlysis of AAV2 psi-speifi CD8 + n CD4 + T ells, ells were first stine with LIVE/DEAD Fixle Aqu e ell stining kit-amcyn, nti-cd14-apc-cy7, nti-cd19-apc-cy7, nti-cd8-pe-texs Re, nti-cd4-pe Cy5.5, nti-cd27-qd 655, nti- CD45RO-QD 75, n nti-pd1-pe-cy7 (lone EH12.2H7; BioLegen, Sn Diego, CA) for 3 minutes t RT in the rk. After fixtion n permeiliztion with Cytofix/Cytoperm for 2 minutes t RT, ells were then stine with nti-cd3-percp-cy5.5, nti-ifn- -PE (lone B27), nti-il-2-fitc (lone MQ1-17H12), nti-ki67-apc-cy5.5 (lone B56) for 45 minutes t RT in rk. The lst three ntioies were purhse from BD (BD Biosienes). Cells were gte s follows (Figure 1): Single ells (Figure 1) were first gte onto lymphoytes (Figure 1), n then gte onto the live ells (Figure 1) (AmCyn s e ell stining). APC-Cy7 use s ump gte for CD14 + n CD19 + ells (Figure 1). Live ells were then gte onto CD8 + ells or CD4 + ells (Figure 1e), whih were then gte onto CD45RO n CD27 (Figure 1f). CD45RO hi CD27 hi ells (TCM), CD45RO hi CD27 low ells (TEM) n CD45RO low CD27 low ells (TEff). Susets were then gte onto IFN-, or IL-2 n TNF-, Perforin n MIP-1 (Figure 1g) whih shows results for CD8 + T ells stimulte with polylonl tivtors. NHP smples: The funtion of AAV psi-speifi CD8 + T ells in NHPs ws teste y ICS fter stimultion with AAV8 psi peptie pool using the similr methos s esrie ove. Frozen ells were thwe n wshe, then immeitely use for the ICS. Cells were first stine with LIVE/DEAD Fixle Violet De Cell stin Kit-Pifi Blue (Invitrogen), nti-cd14-pifi Blue (lone M5E2), nti-cd16-pifi Blue (lone 3G8), nti-cd2-pifi Blue (lone 2H7, AD serote, Oxfor, UK), nti-cd8- APC-H7 (lone SK1), nti-cd4-alex7 (lone OKT4; ebiosiene, Sn Diego, CA), nti-cd95-pe-cy5 (lone DX2), n nti-cd28-pe-texs Re (lone CD28.2; Bekmn Coulter) for 3 minutes t 4 C. Aitionlly, ells were stine with nti-ccr7-pe (lone 1553; R&D Systems). After fixtion n permeiliztion, ells were stine with nti-ifn- -APC Moleulr Therpy vol. 19 no. 11 nov. 211 229
(lone B27), nti-il-2-fitc (lone MQ1-17H12), nti-tnf- -PE-Cy7 (lone MA11) n nti-cd3-percp-cy5.5 for 3 minutes t 4 C. Unless otherwise note, ntioies were purhse from BD (BD Biosienes). For the nlysis of phenotypes of AAV8 psi-speifi CD8 + n CD4 + T ells, ells were first stine with LIVE/DEAD Fixle Violet De Cell stin Kit-Pifi Blue, nti-cd14-pifi Blue, nti-cd16- Pifi Blue, nti-cd2-pifi Blue, nti-cd8-apc-h7, nti-cd4- QD65 (lone OKT4; ebiosiene), nti-cd95-pe-cy5, n nti-cd28- PE-Texs Re, nti-pd1-pe-cy7 for 3 minutes t 4 C. Aitionlly, ells were stine with nti-ccr7-pe. After fixtion n permeiliztion, ells were stine with nti-ifn- -APC, nti-il-2-fitc, nti-ki67-apc- Cy5.5 n nti-cd3-percp-cy5.5 for 3 minutes t 4 C in rk. Cells were gte s follows: single ells were first gte onto CD3 (PerCP-Cy5.5 n Pifi Blue use s ump gte for CD14 +, CD16 + n CD2 + ells n e ells). Live CD3 + ells were then gte onto CD8 + ells (or CD4 + ells), whih were then gte onto CD95 n CD28. CD95 hi CD28 hi ells (effetor ells) were gte onto IL-2 n IFN- or IL-2 n TNF-. CD95 int CD28 low ells (memory ells) were gte onto CCR7. CCR7 hi (TCM) n CCR7 low (TEM) ells were then gte onto the ifferent fluorohromes ientifying ntioies to ytokines. For oth humn n nonhumn primte smples ells inute in meium without pepties were use s negtive ontrols n ells inute in PMA n ionomyin (finl onentrtion 5 μg/ml) were use s positive ontrols. Titrtion of AAV neutrlizing ntioies. The AAV8 neutrlizing ntioy titers in plsm of NHP were teste s esrie efore 11 on 293 HEK ells infete with AAV8 vetor expressing green fluoresent protein. Sttistis. Signifine ws etermine y one-tile Stuent s t-tests ompring results otine from ytokines positive iniviul popultions to those otine from ytokine negtive iniviul popultions. Signifine ws set t P.5. SUPPLEMENTARY MATERIAL Tle S1. Perent of ytokine + T ells/ totl CD8 + T ells in the sme suset (verge ± SEM). 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