Overcoming Immune Tolerance Against Multiple Myeloma With Lentiviral Calnexin-engineered Dendritic Cells

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1 The Amerin Soiety of Gene Therpy originl rtile Overoming Immune Tolerne Aginst Multiple Myelom With Lentivirl Clnexin-engineered Dendriti Cells Shuhong Hn 1, Bei Wng 1, Mtthew J Cotter 1, Li-Jun Yng 2, Jmes Zuli 3, Jn S More 3 nd Lung-Ji Chng 1 1 Deprtment of Moleulr Genetis nd Miroiology, University of Florid College of Mediine, Ginesville, Florid, USA; 2 Deprtment of Pthology, University of Florid College of Mediine, Ginesville, Florid, USA; 3 Deprtment of Mediine, University of Florid College of Mediine, Ginesville, Florid, USA The key to suessful ner immunotherpy is to indue n effetive ntiner immunity tht will overome the quired ner-speifi immune tolerne. In this study, we found tht dendriti ells (DCs) from multiple myelom () ptients suppressed rther thn indued ner ell-speifi immune response. We demonstrted tht CD4 + CD25 high T ells from ptients suppressed the prolifertion of tivted peripherl lood lymphoytes. Further nlysis illustrted tht ell lystes or -speifi idiotype immunogloulins ( Id-Ig) speifilly indued the expnsion of peripherl CD4 + CD25 high FoxP3 high T regultory (Treg) ells in vitro. Suprphysiologil expression of lnexin (CNX) using lentivirl (LV) vetors in DCs of ptients overme the immune suppression nd enhned -speifi CD4 nd CD8 T-ell responses. However, overexpression of CNX did not ffet the peripherl expnsion of Treg ells stimulted y ntigens. Thus, the immune suppression effet of Treg ells in ner ptients my e overome y improving ntigen proessing in DCs, whih in turn my lower the tivtion threshold of the immune effetor ells. This onept of modulting ntiner immunity y genetilly engineering ner ptients DCs my improve immunotherpeuti regimens in ner tretment. Reeived 14 Mrh 27; epted 29 Otoer 27; pulished online 11 Deemer 27. doi:1.138/sj.mt introdution Multiple myelom (), n inurle B-ell neoplsm, ounts for ~1% of ll ners ut it is the seond most ommon hemtologi mlignny fter lymphom. The stndrd high-dose hemotherpy nd utologous trnsplnt used in tretment only offer limited ontrol of the disese. 1,2 A potentilly effetive strtegy for uring is llogenei stem ell trnsplnt. However, llogenei stem ell trnsplnt is ssoited with high tretmentrelted mortlity nd limited to individuls with HLA-mthed donors. Furthermore, myelom ells my espe from the grft versus myelom effet in lrge numer of ptients undergoing this tretment. Consequently, most ptients eventully die from reurrent disese. 3 5 Further studies re urgently needed to overome these ostles nd led to suessful nd effetive immunotherpy for myelom. Immunotherpy is onsidered n lterntive mens for treting nd hs een the fous of multiple studies. 6 8 Dendriti ells (DCs) re key plyer in estlishing n effetive ntiner immunity. 9,1 The DC immuniztion pproh for ner therpy hs gined limited suess minly euse mny ptients hve estlished strong immune tolerne towrd their ner ells. Muh reserh is now foused on reking the tolerne estlished in ner ptients. 7,11 Reent evidene suggests tht DCs in ner ptients re tolerized erly in their development Therefore, it my e diffiult to lter the funtions of DCs or their preursors y mens of extrellulr djuvnts suh s ytokines nd inflmmtory meditors. 14 However, DCs or their preursors my e genetilly engineered to lter their T-ell stimultory funtions nd re-diret immune retivity. We hve previously reported tht DCs, modified y mens of lentivirl (LV) vetors to express exogenous ytokines, ostimultory moleules or smll interfering RNAs, n enhne T-ell responses. 15 Others hve shown tht hperones in the ytosol suh s het-shok proteins, lretiulin, gp96, tpsin, ER6 nd lnexin (CNX) ould enhne ntigen-speifi ellulr immunity Overexpression of CNX in DCs upregultes lss I mjor histoomptiility omplex (MHC-I) nd promotes entrl memory T-ell response (L.-J. Chng nd B. Wng, unpulished results). Thus, the tivtion of speifi ntiner immunity using genetilly modified DCs from ner ptients is promising new immunotherpeuti modlity ut hs not yet een linilly tested. A growing ody of evidene now indites tht ner ells promote T regultory (Treg) ell trffiking, differentition, nd expnsion, nd the Treg ells my suppress the tivities of ner-speifi T ells nd nturl killer ells In ptients, the numer of CD4 +, CD25 high, FoxP3 + Treg ells is signifintly inresed nd oinides with the progression of mlignnt trnsformtion. 24 On the other Correspondene: Lung-Ji Chng, 16 SW Arher Rod, ARB, R1-252, University of Florid, Ginesville, Florid , USA. E-mil: lhng@mgm.ufl.edu nd Jn S More, 16 SW Arher Rod, University of Florid, Ginesville, Florid , USA. E-mil: morejs@mediine.ufl.edu Moleulr Therpy vol. 16 no. 2, fe

2 Overoming Cner Immune Tolerne The Amerin Soiety of Gene Therpy hnd, the Treg ells in hve een reported to e dysfuntionl nd unle to suppress nti-cd3 tivted T-ell prolifertion. 25 The speifi ntigens tht medite the Treg ell inrese hve not een hrterized. It is lso still ontroversil whether the Treg ells re immunosuppressive, nd whether the expnsion of the Treg ells is entrl or peripherl lymphoid event in ptients. 26,27 In this report, we nlyzed CD4 + CD25 + Treg nd DC funtions in ptients nd provided evidene tht CD4 + CD25 + Treg hd immunosuppressive pity nd tht oth ell lystes nd -speifi idiotype immunogloulin ( Id-Ig) loded DCs triggered suppressive ntimyelom immune response exemplified y the expnsion of peripherl Treg ells. These tolerizing DC funtions, however, ould e overome y LV-medited expression of the CNX gene in the ptient s DCs, whih, in turn, led to n nti- response y the effetor T ells. We elieve tht this is the first study to report tht engineered DCs from ptients n overome peripherl Treg ell-indued immune tolerne. Results DC phenotype nlysis in helthy donors nd ptients For immune nlysis, we pulsed ptients monoyte-derived DCs with ell lystes nd oultured them with utologous non-dherent peripherl lood mononuler ells (PBMCs) s illustrted in Figure 1. The ells were highly enrihed (96 98%) s shown y using ntiody (A)-onjugted mgneti eds speifi to phenotype: CD38 + CD138 + CD56 + CD45 (Figure 1). To investigte whether DCs from ptients were immunologilly different, the phenotypes of mture DCs from five helthy donors (s) nd five ptients () were ompred. Figure 1 illustrtes the results from CD11 + DC phenotypes from nd ptients. Although minor vritions existed, there ws no signifint differene in the surfe phenotype etween these two groups. Expnsion of CD4 + CD25 high FoxP3 high Treg ells y ell lyste pulsed DCs DCs of ptients were pulsed with lystes derived from utologous ells (), utologous norml PBMCs (DC/PBMC) or n llogenei Epstein-Brr virus trnsformed B-ell line (DC/BCL). Antigen internliztion ws onfirmed y exposure of immture DCs to B-ell lymphom (BCL) nd doule-stining for CD11 nd immunogloulin light hins. The κ or λ ntigens of BCL were effiiently internlized y DCs (4 13.1%) s deteted y flow ytometry (Figure 2 nd Supplementry Figure S4). We trnsdued immture DCs PBMC ( ptient) BM ells ( ptient) ells ell lyste DC Negtive seletion Freeze/thw Pulse (4 h) HLA-DR 3 CD1 CD86 CD4 CD83 CD14 HLA-I CD PBMC minus DC 2:1 Coulture 14 dys Funtionl nd phenotype nlysis Isotype Before seletion HLA-DR CD1 CD86 CD4 CD83 CD14 HLA-I CD8 CD56 CD38 98 CD CD45 After seletion ± ± ±.6 76 ± ± ± ± ± ± ± ±.6.6 ± ± ± ± ±1 Figure 1 Anlysis of funtionl immunity nd surfe phenotype of dendriti ells (DCs). () Experimentl digrm of immunity nlysis. () Flow ytometry nlysis of purified CD38 + CD138 + CD56 + CD45 multiple myelom () ells. The purified ells were nlyzed y surfe stining for CD38, CD138, CD56, nd CD45, efore nd fter mgneti ed seletion. The numers denote perentges of the speifi ell popultion. The results re representtive of five ptients. () Comprison of DC surfe phenotype etween helthy donors () nd ptients (). The surfe phenotype of mture DCs ws nlyzed with fluorohrome-onjugted ntiodies ginst CD11, CD1, CD83, CD8, HLA-ABC, CD86, CD4 nd HLA-DR s indited. The perentges of positive ells ginst isotype A ontrols re summrized in the ottom. PBMC, peripherl lood mononuler ell vol. 16 no. 2 fe. 28

3 The Amerin Soiety of Gene Therpy Overoming Cner Immune Tolerne Mok BCL1 BCL Mok BCL3 BCL4 BCL CD11 CD Isotype CD d CD4 + CD25 high % / CD4 + CD8 + DC lone CD25 CD4 DC/PBMC DC/BCL DC/BCL DC/PBMC IFN + /CD4 + T ells (%) IFN + /CD8 + T ells (%) FOXP3 DC/BCL / DC/BCL DC/PBMC DC/PBMC DC lone IFN + /CD4 + T ells (%) IFN + /CD8 + T ells (%) FOXP3 high /CD4 + CD25 high T ells (%) Figure 2 Multiple myelom () ell lyste pulsed dendriti ells (DCs) impir T ell response nd inrese the frequeny of CD4 + CD25 high FoxP3 high Treg ells. () Internliztion of ner ell lyste immunogloulins y DCs. Immture DCs were pulsed with ner ell lystes from five different donors (BCL-1 to 5) for 3 hours. Antigen internliztion in DCs ws evluted y doule ntiody (A) stining for CD11 nd ytoplsmi BCL-speifi immunogloulins (κ nd λ light hins). () Intrellulr ytokine stining (ICCS) of DC-tivted T ells. After restimultion with phorol myristte ette nd ionomyin, CD4 + or CD8 + T ells sereting tumor nerosis ftor-α (TNF-α) nd interferon-γ (IFN-γ) ws deteted y ICCS. () Reltive levels of peripherl lood CD4 + CD25 high T ells in ptients versus helthy donors (). Peripherl lood mononuler ells (PBMCs) were inuted with nti-cd4 nd nti-cd25 As nd nlyzed y flow ytometry. Lymphoytes were gted ording to forwrd stter nd side stter, nd CD4 + T ells were gted for further nlysis. Representtive flow ytometry nlyses of two ptients nd two re shown nd totl smple distriutions presented underneth. (d) Expnsion of Treg ells upon exposure to ell lystes. After 13 dys in oulture, the frequenies of CD4 + CD25 high FoxP3 high Treg ells were nlyzed with As ginst CD4, CD25, nd FoxP3. The CD4 + CD25 high T ells were gted for FoxP3 nlysis. Representtive triplite ssys from four ptients speimens re shown (P <.5 nd P <.1). BCL, B-ell lymphom. with LV-LZ expressing highly immunogeni teril β-gltosidse protein for hours nd indued them into mturtion (). Similrly treted DCs were exposed to ell lystes (/). The DCs were oultured with non-dherent utologous PBMCs t rtio of 1:2 for 14 dys nd TNF-α nd IFN-γ produing CD4 nd CD8 T ells were deteted y intrellulr ytokine stining (ICCS) fter stimultion with phorol myristte ette nd ionomyin. We noted tht the ell lystes () were not s immunogeni s the BCL lystes (DC/BCL), nd furthermore, T ell tivtion y DC/LV-LZ ells ws signifintly suppressed fter exposure to ell lystes (Figure 2, DC/LV-LZ versus /). It is known tht during ner progression, ptients my develop strong Treg tivities. We exmined Treg-relted CD4 + CD25 high lymphoytes in the peripherl lood of ptients nd. ptients showed elevted levels of CD4 + CD25 high lymphoytes (men ± 1.716%) in the peripherl lood thn did (men 6.57 ± 1.82%; P =.3, Figure 2 nd Supplementry Figure S2). To further exmine Treg ells, the expression of FoxP3, n importnt trnsription ftor ssoited with Treg ells, ws nlyzed. Figure 2d illustrtes signifint inrese in gted CD4 + T ells with CD25 high FoxP3 high phenotype when the ells enountered ell lystes (, men fluoresene index 8.9 versus 2.64, 2.83, nd 2.95 of DC lone, DC/PBMC nd DC/BCL, respetively). Thus, Moleulr Therpy vol. 16 no. 2 fe

4 Overoming Cner Immune Tolerne The Amerin Soiety of Gene Therpy these results suggest tht the ells seletively indued CD4 + CD25 high FoxP3 high Treg ell expnsion. Isoltion nd expression of Id-Ig gene The tivtion of Treg ells y tumor ell lyste pulsed DCs, my e indued y multiple ner-relted ntigens. To see if this effet n e indued y Id-Ig ntigens, we loned Id-Ig genes for further investigtion. The ells of κ-hin speifi ptient (3) were fluoresene-tivted ell sorting (FACS)-sorted (CD38 + CD138 + CD56 + CD45, right pnel, Figure 3), nd the RNA ws hrvested for omplementry DNA (DNA) synthesis. We used speifi 5 V-region nd ommon 3 C-region primers to mplify the κ gene (Figure 3). After polymerse hin retion mplifition, one of the six primer pirs (Figure 3, irled) reveled disordnt pttern etween + (purified ells) nd DNAs (-minus BM ells). DNA nlysis reveled onsensus CDR3 sequene s underlined in Figure 3, resulting from lonl plsm ells. To onfirm this, n oligo-primer, speifi for the CDR3 sequene of the 3 ptient, ws used to mplify DNAs of different ells (Figure 4). In seprte studies, we hve noted tht ptients BM stroml ells ontinue to express high level of -speifi surfe mrkers (unpulished results). Therefore, we used oth BM ells nd BM stroml ells for this nlysis. A positive nd ws mplified from the orresponding 3 ptient s BM DNA (3 BM ells, L1, Figure 4) s well s the orresponding BM stroml ell DNA (3 stroml ells, L2), ut not from different ptient s (4) BM stroml ell DNA (4 stroml ells, L3). To express myelom-speifi Id-Ig, we loned the κ DNA into n LV vetor (ptyf-ef) under the ontrol of strong elongtion ftor 1α promoter. The DNA ws fused with n N-terminl Flg tg (ptyf-ef-κ-flg), nd the expression onfirmed y Western nlysis using n nti-flg A (Figure 4, with n internl expression ontrol of α-tuulin). Effiient trnsdution of DCs with LV vetors is illustrted in Figure 4d; up to 4% of DCs were trnsdued with reporter LV-eGFP t multipliity of infetion of 1. BM ells () ptient FACS sort SSC FSC ells (CD38 + CD138 + CD56 + CD45 ) BM ells (minus ells) mrna CD138 CD38 RT-PCR 5 FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 C CD56 CD45 K Purified ells (CD38 + CD138 + CD56 + CD45 ) BM ells (minus ells) GAPDH Figure 3 The loning strtegy nd sequening of multiple myelom ()-speifi Id-Ig omplementry DNA (DNA). () The strtegy of loning Id-Ig DNA. In this representtive ptient, one mrrow (BM) ells were sorted y flow ytometry sed on speifi surfe mrkers. The RNA ws isolted nd reverse trnsried into DNA, nd mplified with six pirs of primers speifi for the known κ light hin fmilies ssoited with the speifi ptients. () PCR identifition of -speifi κ hin DNA. () The κ hin DNA sequene (7 se pir with estimted MW of 23 kd). The CDR3 region of the κ gene is underlined. FACS, fluoresene-tivted ell sorting; FSC, forwrd stter; Id, idiotype; Ig, immunogloulin; mrna, messenger RNA; RT-PCR, reverse trnsriptse polymerse hin retion; SSC, side stter vol. 16 no. 2 fe. 28

5 The Amerin Soiety of Gene Therpy Overoming Cner Immune Tolerne BM ells (3) Stroml ells from BM ells (3) Stroml ells from BM ells (4) mrna ptyf-ef- -flg CMV-TAR EF1 -flg PPT 293T - -Flg 293T SIN-LTR 27 kd Anti-flg RT-PCR 54 kd Anti-tuulin 5 FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 C 3 L1 L2 L3 d MOI: GFP Trnsdution effiieny of DC with LV vetor GADPH CD11 Figure 4 Expression of multiple myelom () Id-Ig gene with lentivirl (LV) vetors. () Polymerse hin retion (PCR) onfirmtion of Id-Ig gene. The Id-Ig gene isolted from n ptient (3) ws onfirmed y PCR using sequene-speifi CDR3 primer. A different ptient s one mrrow (BM) ells were inluded s ontrol (4). () Speifi PCR mplifition of the Id-Ig gene. L1, DNA from 3 ptient s BM ells; L2, DNA from 3 ptient s BM-derived stroml ells; L3, DNA from the ontrol ptient s (4) BM-derived stroml ells. () LV expression of the κ-flg fusion protein. The self-intivting (SIN) LV vetor onstrut (ptyf-ef-k-flg) ws used to trnsdue 293T ells. The expression of the κ-flg fusion protein (7 se pir omplimentry DNA with estimted MW of 27 kd) ws onfirmed y Western nlysis using n nti-flg ntiody (A). Anti-α tuulin A ws inluded s ontrol. (d) LV-eGFP trnsdution of dendriti ells (DCs) nd nlysis of green fluoresent protein (GFP) expression. On Dy 5 immture DCs were trnsdued with LV-eGFP t different multipliity of infetions (MOIs of 1, 2, 4, nd 8) nd 48 hours lter, the expression of GFP ws nlyzed y flow ytometry. PPT, entrl polypurine trt; EF1α, elongtion ftor 1α; egfp, enhned green fluoresent protein; GADPH, glyerldehyde-3-phosphte dehydrogense; Id, idiotype; Ig, immunogloulin. Id-Ig displys low immunogeniity ut indues speifi Treg-ell response To ssess the immunogeniity of the Id-Ig, we trnsdued utologous immture DCs with the Id-Ig LV-κ vetor (DC- LV-κ) or ontrol LV-LZ vetor (). As positive ontrol, immture DCs were pulsed with memory ntigen tetnus toxoid (TT) for 4 hours efore mturtion (DC/TT). The DCs were oultured with utologous T ells for 14 dys nd immune effetor funtion ws exmined. Bkground response to the nontrnsdued DCs ws sutrted. For there ws no differene etween DC-LV-κ nd DC-LV-lZ in the CD4 nd CD8 T-ell response (Figure 5). In ontrst, DC-LV-κ indued less CD4 nd CD8 T-ell response thn did s illustrted y intrellulr nlysis of TNF-α nd IFN-γ (Figure 5). Further nlysis of CD4 + CD25 + nd FoxP3 Treg ells showed tht DC- LV-κ, ut not or DC/TT, mrkedly upregulted FoxP3 expression in CD4 + CD25 high T ells in the orresponding ptients (men fluoresene index of for DC-LV-κ, versus 8.23 nd 8.21 for nd DC/TT, respetively, P <.1). By ontrst, no signifint differene ws found for (Figure 5 nd ). It hs een reported tht CD4 + CD25 + Treg ells of ptients re dysfuntionl. 27 We evluted CD4 + CD25 + versus CD4 + CD25 T ells of ptients nd in PBMC prolifertion ssy. Autologous CD4 + CD25 + or CD4 + CD25 T ells were oultured with 5-(nd 6)-roxyfluoresein diette suinimidyl ester ()-leled PBMCs t different rtios nd tivted with phytohemgglutinin. The intensity of in the ulture dereses with inresed ell prolifertion. The results showed tht CD4 + CD25 + T ells from ptients suppressed PBMC prolifertion in dose dependent mnner (Figure 5d nd e), similr to tht of (not shown). The ontrol CD4 + CD25 T ells, y ontrst, did not show suh n effet. LV-CNX-trnsdued DCs enhne the -speifi CD4 nd CD8 T-ell response Both ell lystes nd the speifi Id-Ig ntigens filed to indue n immune effetor response ut insted, promoted strong Treg-ell response. To overome this -speifi immune suppression, we ttempted to modify the ntigen presenttion funtions of ptients DCs. CNX is hperone in the endoplsmi retiulum ritil to the proessing of glyoproteins nd hs een shown to promote ntigen presenttion in immune ells. 28,29 We explored the effet of CNX on ptients DCs nd T ells y LV-medited overexpression of CNX in ptients DCs. The humn CNX DNA ws loned into LV vetor (LV- CNX) ehind strong elongtion ftor 1α promoter. Western nlysis deteted high expression of CNX in CEM-NKR ells ( CNX-defetive ell line) fter LV-CNX trnsdution (Figure 6). Upregultion of CNX expression ws lso onfirmed in DCs when trnsdued with LV-CNX (not shown). To ssess the effet of CNX, immture DCs from ptients were trnsdued with LV-CNX (DC-LV-CNX/), nd ontrol DCs were trnsdued with LV-LZ (/), nd then pulsed with lystes. After mturtion, the DCs were oultured with utologous PBMCs t rtio of 1:2 for 14 dys. The IFN-γ nd TNF-α response of the T ells ws nlyzed upon re-stimultion Moleulr Therpy vol. 16 no. 2 fe

6 Overoming Cner Immune Tolerne The Amerin Soiety of Gene Therpy DC/TT DC-LV- CD25 CD4 DC/TT DC-LV % TNF + CD4 T ells % TNF + CD8 T ells DC-LV DC/TT % FOXP3 high /CD4 + CD25 high T ells d CD % IFN + CD4 T ells % IFN + CD8 T ells FOXP3 Rtio of PBMC:CD4 + CD25 + (or CD4 + CD25 ) T ells PBMC CD4 + CD CD CD4 + CD25 + 1:4 1:2 1:1 1:.5 1: M M e 1:4 1:2 CD4 + CD25 1:1 1:.5 CD4 + CD % Suppression Figure 5 Dendriti ells (DCs) trnsdued with LV- Id-Ig suppress CD4 nd CD8 T-ell response nd upregulte Treg response. () Anlysis of Ag-speifi effetor ell response. The immture DCs from multiple myelom () ptients (s) nd helthy donors (s) were trnsdued with LV-LZ (), LV-κ (DC-LV-κ) or pulsed with tetnus toxoid (TT) (DC/TT), nd oultured with utologous non-dherent peripherl lood mononuler ells (PBMCs) for 14 dys. For speifi response, the T ells were re-stimulted with the sme ntigen-treted DCs. CD4 nd CD8 T-ell sereting tumor nerosis ftor-α (TNF-α) nd interferon-γ (IFN-γ) were nlyzed y intrellulr ytokine stining. (, ) Flow ytometry nlysis of Treg ells. On dy 13, the oultured ells were stined with fluorohrome-leled CD4, CD25, nd Foxp3 ntiodies. Representtive of three repeted experiments is shown (P <.5 nd P <.1). (d) Dose dependent suppression of PBMC prolifertion y CD4 + CD25 + T ells. 5-(nd 6)-roxyfluoresein diette suinimidyl ester ()-leled PBMCs were tivted with phytohemgglutinin nd inuted with CD4 + CD25 + or CD4 + CD25 T ells t inresing rtios s indited. After 3 dys, the prolifertion of PBMCs ws mesured sed on intensity. Representtive profile from one ptient is shown (n = 3, plus three helthy donors); M1 represents the perentge of dividing ells. (e) Br grph nlysis of suppression of PBMC prolifertion (one of three ssys). with phorol myristte ette nd ionomyin. The lyste pulsed DCs gin illustrted immune suppression of oth CD4 nd CD8 T ells (/ versus, Figure 6). Importntly, LV-CNX effetively reversed this trend (Figure 6, DC-LV-CNX/ versus ). However, nlysis of the LV- CNX effet on Treg ells reveled no redution in the numer of CD4 + CD25 high FoxP3 high T ells in oulture with DC-LV-CNX/ (dt not shown). LV-CNX enhnes the Id-Ig-speifi DC immunity The ove study demonstrtes tht DCs from ptients, when trnsdued with LV-CNX n effetively upregulte speifi CD4 nd CD8 T-ell responses. We hve oserved n enhned immune tivtion effet of CNX in seprted study (Supplementry Figure S3). T ells were rpidly expnded within 3 dys when LV-CNX-DCs were inluded in the oulture (Figure 7, lusters of expnded ells). This ws verified y prolifertion nlysis. We pre-stined the T ells with efore DC oulture. After 3 dys, the intensity of the LV-CNX DC oulture group mrkedly deresed s ompred with tht of the other three groups, inditing n inresed T-ell prolifertion indued y LV-CNX (Figure 7, right pnel). To investigte whether CNX ould overome the tolerne effet of -speifi ntigens, the Id-Ig gene (LV-κ) ws trnsdued into utologous immture DCs; lso inluded were LV-CNX (DC-LV-κ + LV-CNX), nd LV-LZ lone s ontrol (). The mture DCs were oultured with utologous non-dherent PBMCs, nd ntigen-speifi T-ell response ws exmined y ICCS for IFN-γ nd TNF-α. The result showed n enhned response of CD4 nd CD8 effetor T ells when DCs were o-trnsdued with LV-CNX (Figure 7). The numer of CD4 + CD25 high FoxP3 high Treg ells etween the DC-LV-κ + LV- CNX nd the DC-LV-κ lone, however, ws not signifintly hnged (not shown). To diretly orrelte the immune effetor funtion with ntiner immunity, we exmined the ytotoxi tivity of the speifi T ells, using non-rdiotive trget ell killing ssy. The T ells were re-stimulted with the orresponding ntigentreted DCs for 5 dys nd hrvested s effetor ells. Autologous stroml ells trnsdued with LV-κ (Stroml ells-lv-κ) or LV- LZ (Stroml ells-lv-lz), were used s trget ells. T ells derived from the DC-LV-κ + LV-CNX oulture killed trget stroml ells-lv-κ with inresed tivity nd speifiity, s ompred to T ells from DC-LV-κ t effetor/trget rtio of 5:1 or 25:1 (P <.3, Figure 7) vol. 16 no. 2 fe. 28

7 The Amerin Soiety of Gene Therpy Overoming Cner Immune Tolerne DC-LV-CNX/ DC-LV-CNX CD4 + CD8 + CEM-NKR CEM-NKR-LV-hCNX / IFNγ + /CD4 + T ells (%) IFNγ + /CD8 + T ells (%) 9 kd 54 kd hcnx α-tuulin DC-LV-CNX/ DC-LV-CNX / TNFα + /CD4 + T ells (%) TNFα + /CD8 + T ells (%) Figure 6 Exogenous lnexin (CNX) expression in dendriti ells (DCs) upregultes multiple myelom ()-speifi T-ell response. () Expression of LV-CNX. LV-CNX ws used to trnsdue CEM-NKR ells, CNX-defiient ell line. After 96 hours, the expression of CNX ws verified y Western nlysis. CNX nd ontrol α-tuulin were deteted using speifi monolonl ntiodies. () LV-CNX-trnsdued DCs enhne -speifi T-ell immunity. ptients immture DCs were trnsdued with LV-LZ or LV-CNX ( nd DC-LV-CNX), nd pulsed with ell lystes (/ nd DC-LV-CNX/). After oulture with utologous non-dherent peripherl lood mononuler ells, the T ells were stimulted with phorol myristte ette nd inomyin nd nlyzed y intrellulr ytokine stining. Representtive result of triplites of four ptients speimens is shown (P <.5 nd P <.1). IFN-γ, interferon-α; TNF-γ, tumor nerosis ftor-α. To see if this ytotoxi tivity ould trget primry ells, utologous ells were isolted from BM ( ells), nd utologous BCL ells were used s ontrol ells. T ells from the oulture of DC-LV-κ + LV-CNX killed the trget ells with inresed tivity (>4% of speifi ell lysis t n effetor/trget rtio of 3:1) s ompred to the T ells from the DC-LV-κ oulture (<2%, P <.2, Figure 7 ottom pnel). Together, these results illustrte tht the suprphysiologil expression of CNX in DCs enhnes ytokine prodution nd ytotoxi funtion of oultured immune ells with speifiity towrd the primry ells. Disussion Immune tolerne to ner ntigens my rise from immune ignorne, deletion or funtionl intivtion (nergy) of nerspeifi T ells. 13,3 A growing ody of evidene now supports the notion tht indution nd expnsion of regultory T ells my e responsile for the lk of linilly-suffiient ntiner immune response. 23 The ltter hs een verified in niml ner models nd in mny studies where evidene of the expnsion of CD4 + CD25 high FoxP3 + Treg ells hs een reported in different mlignnies, inluding tht of. 24 Even though the immune system of ner ptients is onstntly exposed to ner ntigens, these ntigens often fil to deliver dnger signl ; insted, they tend to estlish tolerne. ells produe numer of immune modultors tht my suppress the ntiner immune response, suh s TGF-β, interleukin-1 (IL-1), vsulr endothelil growth ftor nd muin These ftors ould lok DC differentition nd funtion, resulting in immture nd/or prtilly differentited DCs. 12 These dysfuntionl DCs my proess nd present ner ntigens nd stimulte Treg-ell differentition nd expnsion insted of promoting n nti- immune response. Among the CD4 + Treg ells, Treg type 1 (Tr1) ells hve een shown to downregulte immune responses through the tion of immunosuppressive ytokines IL-1 nd TGF-β. Tr1 ells mintin peripherl tolerne, ontrol utoimmunity, nd prevent llogrft rejetion nd grft versus host disese. Fiore et l. found tht DCs pulsed with neroti primry myelom ells fvor the indution of Tr1-like ells in vitro; whether these Tr1 ells express FoxP3 hs not een nlyzed. 32 Our results indite tht ell lystes, or more speifilly, the Id-Igs indue Treg-ell response insted of n nti- response. ptients DCs pulsed with ell lystes or expressing Id-Ig, effetively upregulte CD4 + CD25 high FoxP3 + Treg ells, whih is omined with redued immune effetor funtions. To our knowledge this is the first report tht lerly demonstrtes tht -speifi ntigens n seletively expnd peripherl Treg ells. We hve not yet determined if ny speifi epitope(s) or immunogloulin domin(s) re immunosuppressive in ptients. Bsed on previous report tht nïve, entrl, nd effetor memory Treg ells re signifintly expnded in ptients, 24 it is oneivle tht this in vitro expnsion of Treg ells is representtive of the in vivo response. Understnding how Treg ells expnd in ner ptients will help ddress importnt ner tolerne nd therpy issues. T ells my develop from the thymus into nturlly ourring or dptive Treg ells. 33 The norml FoxP3-negtive nïve T ells my differentite into FoxP3-positive Treg ells upon low dose or systemi ntigen stimultion in the presene of TGF-β. Cner ntigen-speifi CD4 + CD25 + Treg ells hve een isolted from tumor-infiltrting lymphoytes nd shown to suppress the funtion of CD4 T-effetor ells. 34 In trnsgeni utoimmune disese mouse model of multiple slerosis, it ws found tht myelin si protein-speifi Treg ells, ompred to Treg ells speifi for other ntigens, re superior in disese protetion. 35 Cner-speifi Treg Moleulr Therpy vol. 16 no. 2 fe

8 Overoming Cner Immune Tolerne The Amerin Soiety of Gene Therpy DC DC + LV-κ DC + LV-lZ DC + LV-κ + LV-CNX Dy 3 DC:T oulture DC DC + LV-κ DC + LV-lZ DC + LV-κ + LV-CNX Speifi ell lysis (%) Effetor killing ssy Stroml ells-lv-κ ells LV-κ + LV-CNX LV-κ IFNγ + /T ells (%) Cytokine prodution nlysis 9 LV-LZ LV-κ LV-κ + LV-CNX 6 3 Speifi ell lysis (%) LV-κ LV-κ + LV-CNX BCL TNFα + /T ells (%) CD4 + T ells CD8 + T ells CD4 + T ells CD8 + T ells Figure 7 Suprphysiologil expression of lnexin (CNX) in dendriti ells (DCs) promotes effetor ell funtions nd overomes multiple myelom ()-speifi immune tolerne. () LV-CNX-DCs promote T-ell prolifertion. Immture DCs were trnsdued with LV-LZ (DC-LV- LZ), LV-κ (DC-LV-κ) or LV-κ plus LV-CNX (DC-LV-κ + LV-CNX), or without LV (DC lone) efore eing oultured with utologous non-dherent peripherl lood mononuler ells. The ells were photogrphed in 96-well ulture plte under n inverted mirosope (5 15). The proliferting low ells re shown in the fluoresene-tivted ell sorting grph. () LV-CNX trnsdued DCs upregulte effetor T-ell funtions. The oultured T ells were re-stimulted with the pproprite ntigen-treted DCs nd nlyzed y intrellulr ytokine stining. The response to nd to DC/TT is onsidered s the primry nd memory response, respetively. Representtion of three ssys is shown (P <.5 nd P <.1). () LV-CNX trnsdued DCs indue inresed ytotoxi T-lymphoyte tivity trgeting ells. DC-LV-κ nd DC-LV-κ + LV-CNX oultured T ells were re-stimulted with DC-LV-κ for 5 dys. The effetor ells were inuted with trget ells inluding primry ells isolted with mgneti eds, utologous stroml ells infeted with LV-κ (stroml ells-lv-κ) or ontrol trget ells. For primry ells nd stroml ells-lv-κ, the ontrol trget ells were utologous B-ell lymphom (BCL) nd stroml ells trnsdued with LV-LZ (stroml ells-lv-lz), respetively. The ytotoxi effetor funtion ws mesured s desried in Mterils nd Methods. The perentge speifi lysis = perentge lysis of trget ells with speifi ntigen perentge lysis of ontrol trget ells. Representtion of three ssys is shown (P <.5)., 5-(nd 6)-roxyfluoresein diette suinimidyl ester; TT, tetnus toxoid. expnsion hs een reported in different mouse ner models. 34,36 Treg ells my intert with ntigen-speifi effetor ells nd ntigen presenting ells nd it is likely tht multiple immune suppression mehnisms re opertive. Control of Treg-ell expnsion my filitte the development of ntiner immunity. For exmple, seletive elimintion of CD4 + CD25 + Treg ells using IL-2-diphtheri toxin onjugte in renl ell rinom ner ptients hs een shown to enhne vine-medited ntiner immunity. 37 We hve demonstrted tht the immune tolerne n e overome y modifying DCs to express CNX, n essory protein tht enhnes ntigen proessing nd promotes DC nd T-ell intertions. Overexpression of CNX my modulte the tivtion threshold of ner-speifi effetor T ells. CNX plys key role in oth mjor histoomptiility omplex-lss I nd II ntigen proessing pthwys nd my lso e involved in CD1d lipid ntigen presenttion. 28,38 CNX my lso enhne ross-presenttion of exogenous ntigens through the lss I mjor histoomptiility omplex pthwy. 39 ptients DCs presenting ner ntigens do not indue n effetive T helper or ytotoxi T-lymphoyte response ginst ner ells. LV-CNX-modified DCs, y ontrst, effetively oosted ytokine prodution in oth CD4 nd CD8 T ells oupled with inresed ner ell killing tivity. In seprte study, we hve oserved tht DCs modified with LV-CNX n indue effetor T-ell tivtion exhiiting inresed T-ell reeptor ffinity nd redued ntigeni threshold, nd tht the tivted T ells disply entrl memory phenotype (B. Wng, S. Hn, nd L.-J. Chng, mnusript in preprtion). These findings indite tht the tolerogeni DCs in ner ptients my e engineered into retive DCs to promote ntiner immunity with potentil linil enefit. Although our results suggest tht DCs modified with CNX do not lter the tivtion potentil of the FoxP3 + Treg ells speifi to ner ntigens, one nnot rule out the possiility tht the tivted Treg ells my hve ltered funtions vol. 16 no. 2 fe. 28

9 The Amerin Soiety of Gene Therpy Overoming Cner Immune Tolerne In summry, ner ptients inluding ptients grdully develop tolerne to their ner ells with inresed Treg tivities. We hve shown tht this tilted lne of ner immunity my e ltered y properly engineering DCs using LV-CNX. Comining this pproh of using modified DC vine with other wys to modulte the numer nd/or the funtions of Treg my eome n effetive ntiner immunotherpy pproh. The results of our study support the potentil pplition of CNX-sed immunotherpy in nd possily other mlignnies. Mterils And Methods Ptients nd donors. Bone mrrow nd peripherl lood were otined from ptients with newly dignosed or relpsed/refrtory who signed informed onsent pproved y the Institutionl Review Bord t the University of Florid. Peripherl lood of nonymous s ws otined from LifeSouth Blood Center, Ginesville, FL. Genertion of monoyte-derived DCs nd one mrrow derived stroml ells. PBMC from s or ptients with were isolted from uffy ots y grdient density entrifugtion in Fioll-Hypque (Sigm-Aldrih, St. Louis, MO) s previously desried. 15 DCs were prepred ording to the method of Thurner et l., 4 with the following modifitions: on Dy, the PBMCs were inuted t 37 C for 2 hours nd the dherent monoyti ells were ultured in AIM-V medium (Invitrogen, Sn Diego, CA). On dy 1, one hlf of the AIM-V medium ws supplemented with 5 ng/ml of reominnt humn grnuloyte-mrophge olony-stimulting ftor nd 25 ng/ml of IL-4 (Biosoure Interntionl, Cmrillo, CA). On dy 3, fresh AIM-V medium ontining 1 ng/ml of grnuloyte-mrophge olony-stimulting ftor nd 5 ng/ml of IL-4 ws dded to the ulture. On dy 5, the non-dherent ells were hrvested y gentle pipetting. The purity of immture DCs ws routinely exmined using fluorohrome-onjugted nti-cd11 A (BD Phrmingen, Sn Diego, CA). Immture DCs were indued into mturtion with TNF-α (2 U/ml, Biosoure Interntionl) nd lipopolyshride (1 µg/ml, Sigm-Aldrih). The phenotype of the mture DCs ws verified with fluorohrome-onjugted ntiodies ginst different DC mturtion mrkers inluding CD1, CD83, CD8, CD86, CD4 (BD Phrmingen), HLA-I (HLA-ABC) nd HLA-DR (Cltg, Invitrogen, Sn Diego, CA). These DCs re funtionl in stimulting n ntigen-speifi T-ell response s illustrted in Supplementry Figure S1 nd Supplementry Figure S5. BM-derived stroml ells were generted y plting BM ells in α-minimum essentil medium, supplemented with peniillin nd streptomyin nd 2% fetl ovine serum, nd the tthed ells were propgted s stroml ell ulture. Isoltion of ells nd preprtion nd pulsing of ell lystes. ells were enrihed from one mrrow mononuler ells y negtive seletion with mgneti eds ording to the mnufturer s instrutions (Stem Cell Tehnologies, Vnouver, BC) or enrihed y FACS with the following mixture of ntiodies: fluoresein isothioynte leled ntihumn CD38, phyoerythrin (PE)-leled ntihumn CD138, PE-Cy7 leled nti-cd56 nd llophyoynin-leled nti-cd45 monolonl A (BD Phrmingen, Sn Diego, CA). These ells nd the Epstein-Brr virus trnsformed B-ell lines from different donors were lysed y mens of five rounds of freeze-nd-thw rried out etween liquid nitrogen nd 37 C wter th. Cell deris ws disrded y entrifugtion (2,8g, 2 minutes) nd the superntnts were stored frozen t 8 C until use. The ell lystes were used to pulse immture DCs t rtio of 1:1 (ell numer) for 4 hours. TT (intivted tetnus toxin) ws used to pulse the DCs t onentrtion of 5 U/ml for 4 hours. Susequently, DCs were mtured with lipopolyshride (1 µg/ml) nd TNF-α (2 U/ml) for 24 hours. Tle 1 PCR primers used for Id-Ig gene mplifition V region-speifi primers: V1: 5 -CTC GCA ACT GCC TGC AGG GAC ATC CAG ATG ACC V2: 5 -CTC GCA ACT GCC TGC AGG GAT GTT GTG ATG ACT V3: 5 -CTC GCA ACT GCC TGC AGG GAA ATT GTG TTG ACG V4: 5 -CTC GCA ACT GCC TGC AGG GAC ATC GTG ATG ACC V5: 5 -CTC GCA ACT GCC TGC AGG GAA ACG ACA CTC ACG V6: 5 -CTC GCA ACT GCC TGC AGG GAA ATT GTG CTG ACT Cκ1 (k onstnt region): 5 -TCA TCC TCG ACT TGG CCG CCT CGG CCC TAA CAC TCT CCC CTG TTG AAG CTC TTT GTG ACG GGC GAT CTC A-3 κ hin speifi primers ( ptient): 5 -primer: 5 -AA GGA TCC ACC ATG CTC GCA ACT GCC-3 3 -primer: 5 -AAA CTA GTC ACT AAC ACT CTC CCC TGT TGA AGC κ-flg fusion primer: 5 -AAA CTA GTC TAC TTG TCG TCA TCG TCT TTG TAG TCA CAC TCT CCC CTG TTG-3 κ CDR3 speifi primer: 5 -AGT ATG ATG TTC TCC CAT AC-3 Isoltion nd expression of Id-Ig gene. The V H nd V L genes of FACS-sorted ells were polymerse hin retion mplified using primers speifi for H hin nd L hin, followed y loning nd sequening. The reverse primers of H hin nd L hin were omplementry to the onstnt (C) region. The forwrd primers for H hin nd L hin were omplementry to the V-region of different sufmilies. The sequenes of these primers re listed in Tle 1. LV vetor preprtion nd trnsdution of DCs. LVs were onstruted s desried previously The self-intivting ptyf vetors expressing CNX, κ hin, κ-flg fusion, nlz nd enhned green fluoresent protein genes were under the elongtion ftor 1α promoter ontrol. The dy 5 immture DCs, plted t per well in 24-well plte ontining 2 µl of medium supplemented with grnuloyte-mrophge olonystimulting ftor (5 ng/ml) nd IL-4 (25 ng/ml), were trnsdued with onentrted LVs t multipliity of infetion of 4. The infeted ells were inuted t 37 C for 2 hours with gentle shking every 3 minutes, followed y dding 1 ml of medium nd inuted for n dditionl 12 hours. DC mturtion ws indued y dding lipopolyshride (1 µg/ml) nd TNF-α (2 µ/ml) nd inuted for 24 hours. FACS sort of CD4 + CD25 + lymphoyte susets. CD4 + CD25 + peripherl lood lymphoytes of s nd ptients were isolted with FACSAri high-speed ell sorter (BD Biosiene). Briefly, PBMC were inuted with PE-nti-humn CD25 A (BD Phrmingen) nd llophyoyninnti-humn CD4 A (Cltg) for 3 minutes on ie in the drk. The ells were wshed three times efore sorting. Lymphoytes were gted sed on forwrd nd side stter for further nlysis of CD4 nd CD25 expression. CD4 + CD25 + nd CD4 + CD25 T-ell popultions were sorted ording to fluoresene of PE (CD25) nd llophyoynin (CD4). The men purity of the sorted CD4 + CD25 + nd CD4 + CD25 ells ws in the rnge of 98%. Moleulr Therpy vol. 16 no. 2 fe

10 Overoming Cner Immune Tolerne The Amerin Soiety of Gene Therpy leling sed lymphoyte prolifertion ssy. PBMC were suspended in phosphte-uffered sline ontining.1% ovine serum lumin t /ml nd inuted with (Moleulr Proes, Eugene, OR) t finl onentrtion of 1 µmol/l for 7 minutes t 37 C. Cells were wshed nd resuspended in ulture medium for 15 minutes to stilize the stining. The leled PBMC (responders) were ultured in 96-well U-shpe plte t ells/well with phytohemgglutinin (PHA-P, 1 µg/ml, Sigm-Aldrih, St. Louis, MO) in the presene of vrying mounts of CD4 + CD25 + T ells (Treg popultion) nd CD4 + CD25 T ells (ontrol). After 3 dys, ells were hrvested nd intensity of gted lymphoytes ws nlyzed y flow ytometry. The suppression effet of Treg ws expressed s the reltive derese of low ells [1 (1-% low PBMC in oulture/% totl low PBMC)]. utofluoresene of unleled CD4 + CD25 + nd CD4 + CD25 T ells ws sutrted using CellQuest softwre to exlude kground interferene with low ells. DC nd non-dherent PBMC oulture. Non-dherent PBMC were oultured with utologous mture DCs t rtio of 2:1 in serum-free AIM-V medium for 3 dys. On dy 3, IL-7 (1 ng/ml) nd IL-2 (12.5 U/ml) were dded nd fresh medium replenished every other dy for 14 dys. On dy 13, the T ells were olleted for Treg ell nlysis using fluorohromeonjugted As ginst CD4, CD25, nd FoxP3. On dy 14, the T ells were re-stimulted with the sme ntigen treted mture DCs. For myelom ell lyste pulsed DCs, the T ells were re-stimulted with phorol myristte ette (1 ng/ml or.162 µmol/l) nd ionomyin (1 µg/ml, Sigm-Aldrih) for 4 hours, with Brefeldin A (1.5 µg/ml) dded during the lst 2.5 hours of ulture. Then, the ells were fixed, permelized, nd stined with fluoresein isothioynte leled nti-ifn-γ-, PE-leled nti-cd8, PE-Cy7-leled nti-cd4 nd llophyoynin-leled nti- TNF-α monolonl As (BD Phrmingen). The ells were nlyzed using FACSCliur flow ytometer (BD Biosienes). Immune ell ytotoxiity ssy. The immune ell ytotoxiity ssy ws sed on non-rdiotive fluorometri nlysis of T-lymphoyte ntigen-speifi lysis ssy s desried y Sheehy et l., 44 with modifitions. 9 On dy 14 fter DC:T ell oulture, the T ells were re-stimulted nd 5 dys lter, hrvested s effetor ells. The trget ells inluded stroml ells infeted with LV-κ or LV-lZ, utologous ells or Epstein- Brr virus trnsformed B-ell line (BLCL). The trget ells were leled with PKH-26 (Sigm-Aldrih) nd (Moleulr Proes). The doule-leled trget ells were dispensed in duplite t ells/well into 96-well U-ottom pltes (BD Biosienes). Effetor ells were dded t vrious effetor:trget (E:T) rtios. After 5 hours of inution, the ells were hrvested nd fixed in 1% prformldehyde in phosphteuffered sline nd nlyzed using FACSCliur flow ytometer nd the CellQuest progrm (BD). PKH-26 positive ells were gted nd the sme ell numers were quired for eh smple. The perentge of trget ell lysis ws determined y the dispperne of the ntigen-speifi trgets from the high popultion ompred to the ontrol trgets in the high popultion. Western nlysis. Cell extrts were prepred in lysis uffer of Cell Signling Tehnology (Dnvers, MA) ontining proteinse inhiitors (Sigm-Aldrih). The protein smples were seprted on sodium dodeyl sulfte 4 12% grdient polyrylmide gels, eletro-lotted to polyvinylidene difluoride memrnes (PerkinElmer, Boston, MA), nd exposed to ntiodies ginst Flg or CNX (Snt Cruz Biotehnology, Snt Cruz, CA). The signls were deteted using horserdish peroxidse kit with enhned hemiluminesene (Amershm Biosienes, Pistwy, NJ). Sttistis. Dt were nlyzed using GrphPd Prism 4 nlysis softwre (GrphPd Softwre, Sn Diego, CA) nd student s t-test. A 2-sided P vlue of <.5 ws onsidered sttistilly signifint. AknowledgmentS We re grteful to the ner ptients nd lood donors who prtiipted in this study, nd thnk Wyne Chou, Qing Yng nd Liyin Chen for tehnil ssistne. We would like to dedite our work to der deesed friend Don Hinton nd his fmily who inspired this multiple myelom study. This work ws prtly supported y Multiple Myelom Reserh Foundtion nd Ntionl Institutes of Helth grnt HL Supplementry Mteril Figure S4. Phenotype omprisons of DCs pulsed with ell lystes from different soures. Figure S2. Proportions of CD4 + nd CD8 + ells in PBMCs of (multiple myelom) ptients (1 nd 2) nd helthy donor (). Figure S3. Clnexin (CNX) lone does not non-speifilly upregulte effetor ell funtions, ut suprphysiologil expression of CNX in DCs promotes T ell response ginst known tumor ntigens (HPV E6 nd E7). Figure S1. Development of ntigen-speifi T ell response in vitro. Figure S5. Anlyses of ytokine-produing CD4 nd CD8 T ells stimulted y llogenei mture DCs or monoytes. Referenes 1. Hideshim, T, Rihrdson, P nd Anderson, KC (23). Novel therpeuti pprohes for multiple myelom. Immunol Rev 194: Denz, U, Hs, PS, Wsh, R, Einsele, H nd Engelhrdt, M (26). 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