IL-2受容体解析に基づいた新しい免疫抑制療法の開発

IL-2受容体解析に基づいた新しい免疫抑制療法の開

発

著者

里見 進

IL-2受容体解析に基づいた新しい免疫抑制療法の開発

ヰ九g阜及-平成6年度科学研究費補助金(-般研究C)研究成果報告書

平成10年3月

研究代表者 里見 進

(東北大学医学部)

【研究組織】

(研究代表者)里見 進(東北大学医学部)

(研究分担者)菅村 和夫(東北大学医学部)

加藤 博孝(東北大学医学部附属病院)

土井 秀之(東北大学医学部附属病院)

片山 正文(東北大学医学部附属病院)

【研究経費】

平成7年度 1､ 500千円

平成8年度   600千円

L  --K. Yasuda et al･ 1

The transmlttlng editor: Dr. Toshiyuki Hamaoka

Title: Prolongation of Allograft Survival by Administration of Monoclonal

Antibodies Specific for the Three Subunits of IL-2 Receptor

Koji Yasudal･ 2, Tadayoshi Nemotol･ 2, Youichi Ohashi2, susumu satomi2, Kazuko

Muratal , Naoto Ishii', Toshikazu Takeshital , and Kazuo Sugamural

lDepartment of Microbiologyand Immunology,and 2TheSecond Department of Surgery, Tohoku Umiversity School of Medicine, Sendai, Japan･

Running title: Suppressive effects ofanti-礼-2R mAbs onallograft

Totalnumber of pages: 22

Totalnumber of table: 1

Totalnumber of figures: 4

Correspondence: b･ Kazuo Sugamura, Deparhent of Microbiologyand Immunology,

Tohoku Umiversity School of Medicine, Seiry0-machi, Aoba-ku, Sendai980-77, Japan TEL: 81-22-717-8096, FAX: 8 1-22-273-2787,

K･ Yasuda et al･ 2

Abstract

The IL-2 receptor (礼-2R) Y chain, so-Called common γ (Yc) chain, which is shared with

multiple cytokine receptors, plays important rolesinthe immune system. We here assessedthe immunosuppressive abihtyof mAb specificforthe Yc chainininduction of

1

CTLsandallograft rejecdonincombinationwithmAbs specific forthe αand β chains

of IL2R. CB〟N (Hl2k)mice wereinjected intraperitoneal1y withauogeneic

splenocytesfrom BALB/C (H-2d)mice,andthen administered withcombinations of anti-IL-2Rα,anti-IL-2Rβ andanti-yc tnAbs or a control mAb. Addition ofanti-†c mAb

together withanti-I.-2Rαandanti｣112Rβ mAbsinddeea a complete inhibition of CTL response･ The numbersand populations of CDl+cD8-and CD4ーCD8'T cens were not

significandy affected by admhistration of the threeanti-IL-2R mAbs, whereas NK cens

were completely depletedinspleens of mice treated withtheanti-a-2R mAbs･

Furthermore, skinallograftsurvivalwasalso significandy prolonged by administradon

of the three anti-A-2R mAbs. These results suggestthatthe anti-†c mAb in

combination withanti-Ⅱ′2Rαandanti-礼-2Rβ TnAbs is capable of suppressing

K･ Yasuda et al･ 3

Introduction

ILl2 is a T cell cytokine仇atinduces growth, differentiation,and activation of various

types of hematopoietic cells such as T, B, NK cellsand monocyte/macrophages via

interaction with 礼-2 receptors on their surfaces (1). The IL-2R consists of three

1

distinct subunits,the α (ml2Rα), β (IL-2Rβ)and † (A.-2Ry) chains,and IL-2Rβand

nl2Ry are essential for formation offunctionalhigh-andintemediate-affinityreceptors

(2). IL-2Ry, so-cauedthe common y (yc) chain, is shared as a common subunitamong

receptors for at least IL-2, 礼-4, ILl7, 礼-9, and 礼-15 (3)･ The †c chain is expressed

on almostall of the hematopoietic cell populations inT伽manand mouse (4, 5),and

mutations of the yc gene cause humanX-linked severe combined irrmunodeficiency

(XSCID) characterized by profound depression of Tand NK cell development (6, 7)･

Inmice deficient of the yc gene, development of mature Tand B ceus is profoundly

suppressedand NK ceus are absentinperipherallymphoid 也ssues (8-10)･ Recent

reports suggestthat signals throughthe Yc chain contribute to prevention of T ceu

anergyinduced by defect of costimulationthroughinteraction between CD28and B7

(1 I),and regulate activated T cell apoptosis (12). Moreover,the yc chainisinvolvedin

not onlythymocyte development butalso maintenanceand expansion of peripheralT

cells (13). Allthe cellularresponses throughthe yc chainarethought to be related tothe

In organtransplantation, T ceusinrecIPlentS are activated by donor auoanhgens

to become Cn･Sand helper T cells producing a variety of cytokinesincluding IL-2

( 14),and the activated T cells expressthe inducible IL-2Rα together with m12Rβ and Yc

chains to be responsive to nl2 for proliferationandfunctionalactivation (1, 3). Therefore, cytokine-secreting helper T cells and cytokine-responsive CTLs are

prerequisites fortheallograftrejection (15)A In addition to auoantigen-specific CTLs, NK ceusalso contribute to graft destruction (16). Not only CTLs butalso NK cells are known to be responsive to n･-2 for proliferationandfunctionalactivation (17-18),

K･ YasLLda etal･ 4

suggestlngthat suppression of IL-2 function resultsinsuccessful transplantation･ indeed, so far,anti-IL-2Rαandanti-臥-2Rβ mAbs have been used as effective

immunosuppressantsinanimalexperiments (19-23)and c血icalstudies (24, 25),

althoughthese agents could not achievethe complete inl1ibition of graft rejection. We

1

have estabhshed monoclonalantibodies (mAbs) specificforthe mouse yc chairl, One Of

which, TUGm2, is able to block cell growth signaltransduction mediated by 2,

IL-4, IL-7, a-9and n115 (26-29)･ Using TUGm2, we successively achieved a complete

inhibition ofinvivo CTLinduction and a significant suppression of skinallograft

-,号

K･ Yasuda etal･ 5

Methods Mice

Six to seven week old CBA伽(H-2k), BALB/C (H-2d),and C57BIJ6 (H-2b) male

mice, which were purchased from Japan SLC Inc･, were used･

Monoclonal antibodies

The mAbs used are PC615.3 (anti-mouse 礼-2Rα, rat-IgGl ) obtained fromAmeric'an

Type Culture Conection, TUm122 (anti一mouse IL-2Rβ, rat-IgG2b) (30), TUGm2

(anti-mouse Yc, rat-IgG2b ) (26),and REY7 (anti-HでLV-I gp46, rat-IgG2b) (31).

¶leSe mAbs were purified withprotein-A column (AmpureW,Amersham). S4B6, a

mAb specific for mouse m12, was obtained frbmAmericanTissue Type Culture

Couection,and purified from culture supematants by protein-G Sepharose 4 Fast Flow

(Pbamacia Biotech ).

Mixed lymphocyte reaction

For mixed lymphocyte cultures, responder cens were prepared from spleens of mice by

removing erythrocytes upon treatment with TriS-NH.Cl･ 4x 105 responder spleen cells

were coICultured for 5 days with2×105 sthulator cens irradiated with3000

radin96-well round-bottomedmicrotiter plates contamlng RPMI 1640 medium supplemented

with10% horse serum, amibiodcs, 2 mMLglutanhe, 25 mM HEPES, 0. 1 InM non-essemialamino acidsand 50 LLM 2-ME･ The weusintriplicate were pulsed withl LLCi of l3H]thymidineand radioac也vitiesinCorporatedinto cells were quandtated on a liquid

scintillation c ounter.

K･ Yasuda etal･ 6

P8 15 (H-2d), a mastcytoma cellline derived from DBjV2J strainofmice, ELl4 (H-2b), a T lymphoma cell he derived from C57BU6N strain,and B16 (H-2b), a melanoma cell line derived from C57BU6 strain, were used as target ceusin51cr release CTL assays as described previously (32)･ To induce CTLs, recipientmice were

1

intraperitoneany immunizedwith50×106 irradiated anogeneic splenocytes, andthen

intraperitoneaLy administered with anti-Ⅰし2R mAbs or a control mAb on day 0, 2,and

4･ On day 5, spleens were removedand the splenocytes were used as effTector cells in

51cr release assays･Altematively'the splenocytes were incubated withirradiated

-･せ

stimulator ceusinvitro for 5 days,andthen used as effector cellsin51cr release assays･

Target ceus (2xlO6) were labeled with0. I mCi Na251cro. (Amersham) at 3rC for 1 h

inRPMI 1640 medium containing lO% FCS,and washed threetimes with RPMI 1640

medium. 51cr-labeled target cells (5× 104) were added to seriald血tions of effector ceus

ina totalvolume of 200 plin96-weu round-bottomedmicroplates, centrifuged to estabhsh ceu-cen contact･andincubated･ After 4 hincubation, the plates were centrifugedandthe supernatants were haⅣested･ Radioactivities of the supematants

were countedina gamma counter･ Spontaneous release was lessthan10% of the

maximum release･ Results were expressed as follows･

% specific lysis =

Releaseintest - Spontaneous release

---一一---I--- X 1 00

Maximum release - Spontaneous release

Tail skin grafting

Skingr姐ing was performed on anesthetized recipients as described by Bauey with

some modifications (33, 34)･ Briefly, a piece of donor tail skinabout 0.5 cm2and most

of the demis were removed witha scalpel,and transferred ontothe side of the recIPlent

K･ Yasuda etall  7

monitored daily･ A graft was scored as rejected when 50% of the area of the graft had

undergone necrotic degeneration.

FIow cytometry

1

Immunofluorescence now cytomehicanalyses were performed as described elsewhere (5)I Monoclonalantibodies used fortheflow cytometric analyses were

FITC-conjugated anti-CD4 (H 129･ 19) and PE-coupledanti-CD8a (53-6.7) purchased from

GIBCO BRL,and PE-coupledanti-NKl. I (PK136), FITC-Conjugatedanti-mouse H-2b (KH95), PE-coupled anti一mouse H-2d (34-2-12 ), Fifc-Conjugatedanti-CD3e

(145-2Cl 1),and FITC-Conjugatedanti-αβTCR (H57-597) purchased from PharMhgen.

Ceus (lxlO6) wereincubatedwith 20 LLl of blocking reagents (culture supematant

containing 2･4G2,and-FcyRⅡ mAb,in50% normalrat serum 【Cedarlane]and 0.02%

NaN3)andallowed to stand on ice for 30min. They were then stained withthe mAbs on ice for 30min, andanalyzed with aflow cytometer (FACScan, Becton Dicknson Immunocytometry Systems) a氏er washing.

Statistical analysis

The statistical significance of the meansurvivalhe (MST) for skingraft was assessed

bythe Mann-W加tney U test･ The sigmificance of the other data was determined by

K･ Yasuda etal･ 8

Results

Inhibition of mixed lymphocyte reaction by administration of anti･ILl2R mAbs

Effects ofanti-IL-2R mAbs on immune responses were first aSSeSSed in anospecific

I

mied lymphocyte reaction (MLR)invitro. Responderand stimulator cells were

splenocytes from C57BIJ6 (H12b)miceand BAIB/C (H-2d)mice, respectively.

Addition of a finalconcentration of 5 LLg/ml ofanti-A-2Rα rnAb, PC615.3into the

mked lymphocyte culture induced a 55% decreaseinPH】thymidine uptake as

compared withthat of the control mAb, REY-7 (Fig.- ･1チ.Anti-Ⅱ′2 mAb, S4B6also

inhibitedthe MLR equany weu toanti一m-2Rα mAb (Fig. 1), In contrast,

eitheranti-m-2Rβ rnAb, TUm122 or anti-yc mAb, TUGm2 hardly suppressedthe MLR at the

same concentration asanti-a-2Rα mAb,although morethan10times higher

concentrations ofanti-IL-2Rβ andanti-yc mAbs induced obvious suppression of l3H】

thymidine uptakeinthe MLR (data not shown). Chthe other hand,the combination of

anti-IL2Rβandanti-Yc mAbs significantly blocked l3H】thymidine uptakeinthe MLR,

butthe blocking level did not exceedthat withanti-IL2Rα血Abalone (p<0.05) (Fig.

1). 60%and 57% reductionsinthe MLR were obtained withthe addition

ofanti-m-2Rβ or a血-yc mAb together withanti-臥-2Rα mAb (p<0.05), respectively. Moreover,

the combination ofthethree mAbsalso showed 60%inhibition of the MLR. These

results suggestthatandJLl2Rα mAb, PC615･ 3 is capable of suppresslng

alloantigen-specific MLR,andalthough anti一m-2Rβ hlAb, TUm122 andanti-γc mAb, TUGm2,

themselves, have litde suppressive effect onthe MLR,they shghtly enhancethe

suppressive effect ofanti一m-2Rα mAb.

Effects of ants-IL･2R mAbs on T and NK cell populations in vivo

Prior to examination ofinvivo effects of the and一m12R mAbs on immune responses,

K･ Yasuda etal･ 9

mAbs･ CBA伽(H-2k)mice wereintraperitoneal1y hnmunized withirradiated

splenocytes of BALB/C (H12d)mice, and administeredintraperitoneally witha finalof

300 lLg Ofanti-a-2R mAbs consisting of 100 pg each ofanti-IL-2Rα, anti-礼-2Rβand

antl-YC mAbs, or 300リ.g Of a control mAb, REY-7, on day 0, 2and 4 after 1

irrmunization･ Their splenocytes were subjected to flow cytomeb･ic analyses. Authe

micethat had receivedthe combination of the threeanti-ml2R mAbs or REY-7, were weu, withno sign of pnoerection, diarrhea or hypomotilityduringtheir treatment with

the mAbs･ Numbers of whole splenocytes were shghtly higher inmice treated with REY-7 control mAb as compared withmice untreated-arid treated withthe combination

ofthethreeanti-臥-2R mAbs (data not shown). In spleen, CD4+cD81 and CD4 CD8+ T

cells wereincreasedinthemice treated withREY-7 mAb as compared withthemice untreatedand treated withthe combination of the three mAbs (CD4+cD8 : 29.4±1.7%

vs 26･7±3･8%, p<0･005, CD4lCD8': 18･7±1･9% vs 16･7±1.5%, p<0.05)昨ig. 2).

NKl I 1+TCRαβ NK cells were comp.letely depletedinspleens ofthemice treated with

thethreeanti一m-2R mAbs, butNKl･ 1+ TCRαβ+ T cens were unaffected (Fig. 2). On

the other hand･ T cell subpopulationsinthymus were notalteredanongal1the groups

of mice (data not shown)･ A血ost dl of the censinspleenand thymus were stained by

anti一mouse H12kantibody'confirrning thatallogeneic stimulator cells were elimhated in

vivo (data not shown). These resultsindicatethatinvivo admhistration of REY_7

control mAb resultsina shghtincrease of mature T cell subsets, whereas administradon

ofthethree anti-nl2R mAbs does not lead to such increase.

Effects of anti･ILl2R mAbs on induction ofalIospecific CTLs in vivo

Sincethe addition ofanti-礼-2Rβand anti-yc mAbsinduced a sight enhancement of

MLR suppression by anti-臥-2Rα mAb, we examined effTects of combinations

K･ Yasudaetal･ 10

mice wereintraperitoneally immunized withirradiated splenocytes of BAIB/C (H-2d)

donormice,and administered either REY-7 control mAb or combinations of the three

anti-IL-2R mAbs intraperitoneally on day 0, 2and 4 after immunization. CTL

responses to donoralloanhgen were measured 5 days after immunization by a

51cr-1

release assaywith P815 (H-2d) target cells･ There was no statistically sigmificant difference in CTL responses between themice treated with300 LLg each of REY-7

Control mAbandtheanti-IL-2Rα mAb, PC615･3 (Fig･ 3A), suggestingthat 300 LIE Of

anti-IL-2Rα mAb hardly providesaninhibitory effect on auoantlgen-specific CTL

inducdoninvivo･ In contrast,the combination of 150- pgeach

ofanti-IL2Rαandanti-IL-2Rβ mAbs significandyinhibited CTLinduction as compared withREY-7 Control

mAbalone oranti-礼-2Rα mAbalone orthe combination of 150帽eaCh ofanti-A-2Rα

andanti-yc mAbs (Fig･ 3A)･ Moreover, administration of 100 LLg Ofanti-yc mAb

together with100 LLg each ofanti-Ⅱ-2Rαand anti-Ⅱ′2RP mAbs resultedinalmost

complete suppression of CTLinduction (Fig･ 3A)･ Administration of 300 pg of

a血-IL-2Rβ mAb oranti-yc mAb, or a totalof300 pg mAbs containing 150 pg each of

a血一m-2Rβandanti-Yc mAbs showed litde suppressive effect on CTLinduction (data not

shown)･ Usingthe reciprocalanogeneic combinadon of recipient BAL玉/C (H-2d)mice

and donor C57BU6 (H-2b)mice, effects ofanti-IL2R mAbs on CTLinduction was

also examined. The combined admistration bfthe three anti-Ⅱ′2R mAbsinduced the

most dramadc suppression of CTLinduction,and a significant suppression of CTL

induction wasinduced bythe combination of anti一m-2Rαand anti-IL-2Rβ mAbs, but

anti-nl2Rα mAbalone or its combination with ant1-YC mAb showed no effect on CTL

inducdon (Fig･ 3B). ne suppressive effect of combination ofthethree mAbs or anti_

臥-2Rαandanti-IL-2Rβ mAbs on CTLinduction was confirmed to be stadsticany

significant (Table 1)･ neallospecificities of CTLsinduced werealso con血med

reciprocally withP815 (H-2d)and B 16 (H-2b) (data not shown). These resultsindicate

K･ Yasudaetal･ ll

induction ofalloantlgen SPeCific CTLs･ Since such CTLinduction was htde affected by

each mAbalone or the combination of the a山一IL-2Rβ and anti-Yc mAbs,and partially

inhibited by the combination of the anti-臥-2Rαandanti-IL-2Rβ mAbs, suggestingan

importance oftheanti-yc mAb, TUGm2, for complete suppression ofinvivo CTL

1

reSpOnSeS･

Surrvivalprolongation of MIIC-incompatible skin allografts inmice

treated with anti-IL･2R mAbs

Since skinallograftsareknown to be generally rejected血sterthanorganauografts, We

assessedtheinvivo immunosuppressive effect ofthethreeanti-nl2R mAbsinskin

auogTaftS･ Tan skingrafts of BALB/Cmice were trazISPlanted onto tans of recIPlent

CBAがmice on day 0, andthe recIPlentmice were adm血steredintraperitoneanywith a

totalof 300 pg of rr止bsinthe stipulated combinations ofanti-IL-2Rα,anti-IL-2Rβ,

anti-yc and REY-7 control mAbs, on day 0, 2, 4, 6and 8 after transplantation･ The

recIPlentmice treated withanti-ml2Rα mAb or REY-7 control mAb showed 8･ 6 days

or 6･4 days of meansurvivaltimes (MSTs) of skinanografts, whilethe combination of

anti-IL-2Rtxandanti-礼-2Rβ mAbs prolongedallograft survivalup to ll.4 days of

MST (Fig･ 4)･ Furthermore,the combination ofthethreeanti一m-2R

IIIAbs,anti-IL-2Rα,anti-IL-2Rβandanti-yc mAbs,induced bore significant prolongation of the skin

graft survivalup to 22･2 days of MST･ These results fairly correlated withthose of

K･ Yasudaetall 12

Discussion

ne immunosuppressive effTects of anti一m-2Rαandanti-礼-2Rβ mAbs have been

previously reportedintransplantationsinhumanSandmice (20-25)･ Sincethefully

functionalm-2 receptorincludesthe Yc chain asanindispensable receptor subunit in

1

addition to IL-2R(x and ml2RP, coupledwiththe factthat it is sharedwithreceptors for other cytokines such as IL-4, IL-7, m19and 臥-15 (3), we hypothesizedthat antトγc

mAb contributes to culmination ofinhibitory effects of anti-礼-2Rαandanti-礼-2Rβ

mAbs on 礼-2 function･and to suppression offunctions of the other cytokines sharing

the yc chain, which would be beneficialto immune suppression. We here demonstrated

thatanti-yc mAb c0-Operates withanti-礼-2Rαandanti-IL2Rβ mAbsinimmune

suppression of in vivoalloantigen-specific CTL responsesand skin allografts･

We f:lrSt COnfirmedthe suppressive activity ofanti-礼-2Rα mAb for MLR

representlng PrOliferadon of T cellsinresponse to anoandgen in vitro･Althoughthe

combination betweenanti-臥-2Rα mAbandanti一m-2Rβ mAb oranti-yc mAb,and

combination ofthethreeanti-臥-2R mAbs showed shghdy enhanced levels of inhibition

of MLRincomparison withanti-ILl2Rα mAbalone, eitheranti-ml2Rβ oranti-Yc rnAb

alone had no effect on MLR･ These results are consistent withthe previous

observationsthatthe IL12R complex consisting of the 臥-2Rα, Ⅱ′2Rβand †c but not

the complex consisting of the H･-2Rβand †c isfunctionalinthe murine system unlike

the humansystem,andtherebyanti-礼-2Rα mAb completely blocks IL-2-mediated cell

growth, while each ofanti-礼-2Rβ oranti-Yc mAb reduces a-2 bin'ding affinity of the

high-affhityIL-2R but is unable to blockthe T cen growthmediated by 礼-2 (26, 30,

35)･ Sincethe 2Rα chain is not shared with other cytokine receptors apart from

IL-2R,the significantinhibition of MLR bythe anti-臥-2Rα mAbalone suggeststhat 礼-2

is a major humoralfactor mediating MLRinvitro.

Notwithstandingthe significant suppressive activity ofantiJL-2Rα mAb onin

K･ Yasudaetal･ 13

similar toanti-ml2Rβandanti-Yc mAbs, whereas the combination ofantLm-2Rα and

anti-礼-2Rβ mAbs but notthe combination of anti-礼-2Rαandanti-†c mAbs showed

significantinhibition of CTLinduction. Furthermore,the combination of the threeand一

礼-2R mAbsinducedalmost completeinhibition of Cn responseinthe combination

I

between recIPlent CB〟Nand donor BALB/Cmice･ In considering why anti-Ⅱ′2Rα

mAbalone has no suppressive effect oninvivo CTL response,although itinduces a powerful suppressive effect oninvitro MLR, one could attribute this disparityto a differenceintheinvivoandinvitro environmentalcircumstances, affecting dilution, inactivation and possibly degradation of a血-Ⅱ′2R蝕 mAb. We also observed

significant suppression of skinallograft by a combined admimistradon of a山一Ⅱ√2Rα

andanti-Ⅱ′2Rβ mAbs orthethree and-a-2R mAbs, butanti-Ⅱ′2Rα InAbalonelitde

induced suppression of skinauograft. However, administration ofanti-礼-2Rα mAb

alone was previously reported to be significantly effective for suppression of skin

anograftrejectionincertainstrains of mice but not generauy (23). These observations taken together withour present results suggestthattheinvivo immunosuppressive effect of and-IL-2Rα mAbalone may beinfluenced by irrmunogenetic basis of mouse

strainS･ These results flrSt SuggeStthe possibihtythatanti-ycandanti-IL-2Rβ mAbs

engendersthe significant suppressive activity ofanti-礼-2Rα mAb forinvivo IL2

funcdon which is critiCallyinvolved inin■vivo immune responses such as CTL

inductionand auograft reJection･ However, it has been reportedthat islet ceu anografts

canbe rejected eveninn-21deficientmice, suggesting the possible existence of a-2-independent mechanismforallograftrejectiOn probablyincludinginduction of CTLand other effector ceus such as NK cells (36). Hence, it isalSo possiblethatanti-ycand an也一m-2Rβ mAbs contribute to suppression of funcdons of other cytokines sharing the

ycand a-2RP chains as receptor subunits, which would beinvolvedininvivo immune

responsesI In fact, ILl4, 臥-7, 礼-9and 礼-15, au of whichinteract withthe γc chain,

(37-K･ Yasudaetal･ 14

41);the aJlti-YC mAb, TUGm2, used here isknown to significandyinhibitinvitro

functions of 臥-4, 臥-7, IL-9and IL-15 (26-29),andinvivo admhistration of

anti-IL-2Rβ mAb isalsoknown to deplete NK ceus (42). nerefore, NK ceus as wen as T

ceus are target ceus of anti-臥-2Rβandanti-Yc mAbs for suppression of induction of I

CTLandallograft rejection.

The present study con血med血e entire depletion of NK1. 1十TCR岬NK cells

upon treatment of mice withtheantiJL-2R mAbs,although T cell subpopulations such

as CD4-CD8+and CD4+CD8- T cens were little affTected byinvivo admistradon of

anti-IL-2R mAbs･ Administration of REY7 Control mAb血duced a slightincrease of the

T cell subpopulations, which may be due to amigenic stimulation with REY7 mAb (rat IgG), whileantigenic stimulation withanti-a-2R mAbs (rat IgG) mayalso be

suppressed bythernselvesI The deletion of NK cells isthought to be derived from

the 礼-2Rβand Yc chainSare receptor subunits for IL-2and 礼-15 (29),and not only

IL-2 butalso 臥-15 areknown to beinvolvedindevelopmentand activation of NK

cells (44-46)･ Authese observations supportthe observationthat anti-†C mAb in

combination withanti-ml2Rαand anti-臥-2RP mAbs is more effective fTorinvivo

immunosuppressionthan礼-2Rα mAbalone or any other combinadon of the

K. Yasudaetal･ 15

Acknowiegements

we thank Dr. S･ Moffatt for critically readingthe manuscrlPt･ Ths work was supported

impart by a grant-in-aid for sciemific research on priorityareasfromthe Ministry of

Education, Science, Sports,and Culture of Japan, a grant-in-aidfromthe Minis吋of

1

Healthand Welfare of the Japanese Govemment forthe comprehensive 10-yearstrategy

for cancer control, a grant from specialcoordinationfunds of the Scienceand

Teclmology Agency of Japan, and a grant fromthe hamoriFoundation･

K･ Yasudaetal･ 16

_t 々

Abbreyiations

IL-2R, IL-2 receptor

礼-2Rα, 臥-2R α chain

IL-2Rβ, ml2R β chain I Yc, 臥-2R † chain / common Y chain

K･ Yasudaetal･ 17

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･･や

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K. Yasudaetal. 21

Legends to Figures

Figure 1: Effects of anti･IL-2R mAbs onmixed lymphocyte reaction･

Responder spleen ceus of C57BIJ6mice were co-cultured with irradiated stimulator

spleen ceus of BALB/Cmice for 5 daysin961Well roundbottomedmicrotiter plates in 1

the presence of purified mAbs: (a) 15 LLg Of REY7 Control mAb, (b)

5帽Ofanti-IL-2Rα rrlAb (PC 615.3)and 10 pg REY-7, (C) 5 pg ofanti｣し2Rβ mAb (TUm122)and

long ofREY-7, (d) 5帽Ofanti-yc mAb (TUG血2)and 10 LLg OfREY-7, (e) 5 pg of

anti-礼-2Rα mAb, 5 p･g ofanti-臥-2Rβ mAband 5 LLg Of REY7, (f).5 LLg Ofanti-IL-2Rα

mAb, 5 pg ofanti-Yc mAband 5 pg ofREY7, (g) 5腔Ofanti-礼-2Rβ mAb, 5 pg of

anti-Yc mAb and 5 pg of REY7, (h) 5 pg ofanti-A-2Rα mAb, 5帽Ofanti-Ⅱ√2Rβ

mAband 5一g ofanti-Yc mAb,and (i) 5 p･g ofanti-IL-2 rnAb (S4B6)and 10 llg Of

REY-7･ ne reponder cdls werealso stimulated withsyngeneic C57Bu6 spleen ceus o). Duringthe last 18 hr of culture, 1.0 mCi of l3H]thymidine was pulsed･

Radioactivities incorporatedintothe cens were counted･ Results are represented as

meanvaluesand standard deviations in triplicate assays･ b, e, f, g, hand i are p < 0･05･

Figure 2: Immunofluorescence analysis for T and NK cell poplllations in spleens of mice treatedwith anti･ILl2R mAbs･

C57BL/6 (H-2b)mice were administered with purified mAbs: no mAb (untreated), 300

pg of REY-7 (Control mAb),and 100 LLg each ofanti-IL-2Rα mAb,anti-_IL-2Rβ mAb

andanti-yc mAb (combination), on day 0, 2, and 4･ On day 5, splenocytes ofthemice

were subjected to flow cytomebicanalyses;they were incubated with20 ul of blocking

reagents (culture supematant containinganti-FcyRII mAb (2･4G2) in 50% normalrat

serum lCedarlane]and O･02% NaN3) on ice for 30min,then stained withanti-CD4 mAb (H129.19),anti-CD8a mAb (53-6.7)andanti-NKl･l mAb (PK136) on ice for 30

K. Yasudaetal. 22

positively stained cells is indicated bythe numberineach quadrant･ * : p < 0･005, ** :

p<0.05

Figure 3: Effects of anti-IL･2R mAbs on in vivo CTL induction.

cB〟N 2k)mice were intraperitoneally immumized withsplenocytes of BALB/C

(H-2d)mice (A), or vice verse (B). Themice werethen treated withmAbs: 300帽Of

REY7 Control mAb (○), 300 pg ofanti-IL-2Rα mAb (田), 150 LLg each ofanti-IL-2Rα

mAband a血- Yc mAb (△), 150 pg each ofantilLl2Rα mAbandanti-IL-2Rβ rnAb

･一や

(+ ),and 100 pg each ofanti-IL-2RαmAb, anti-ILl2Rβ mAbandanti-yc mAb (● ),

on day 0, 2and 4 after immunization. On day 5, spleens were removedand splenocytes

were used as effector ceusincytotoxicityassays withradiolabened target cells of P8 15 (H-2d) (A)and EL-4 (H-2b) p). Results were represented as meanValuesand standard

diviations in triplicates.

Figure 4: Effects of anti･IL-2R mAbs on MHC-incompatible skin

allograrts.

Tail skingrafts of BALB/C (H12d)mice were transplanted onto the tails of

CBA桝田-2k)mice. Recipientmice were admimisteredintraperitoneany withanti一m-2R mAbs on

day 0, 2, 4, 6and 8 after transplantation. Fivemice were administered with300 llg Of

REY7 Control mAb (○), fivemice were administered with300 LLg Ofanti-IL-2Rα

mAb, PC615.3 (● ), tenmice were administeredwith150 LLg each ofanti-IL12Rα

mAbandanti-IL-2Rβ mAb, TUm122 (A),and sixmice were administered with 100 LLg

each of anti一m-2Rα mAb,anti一m-2Rβ mAbandanti-†c mAb, TUGm2 (◆ ). Grafts

Table l : Effects of AntHL2R mAbs on in vivo AlIospecific CTL Induction

Allospecific CTL

Mice in vivo treatment Stimulator Target E/r Ratio

200:1     100:1       50:1 CBAノN control n=5      α α+γC α+β BALD/C 4° BALB/c B ALB/c B ALB/C α +β+γ c BALB/C

22=522iiif･_il13i';92 ,3] a:b 1125387g'66?87享i?lI.:.;24 ],: a:d

13.0±2.8 9.4±2.0 9.4±1.7 4.1±1.3 3.3±1.0 a ] ]a ]C ]a BALB/c COntrOl n=3      α α+γC α十β C57BU6   EL-4       59.6 ± 7.4 C57BU6   EL4 C5 7 BL/6  .EL_4 C57 BU6   EL-4 α +β +γ c c57BU6  EL-4 66.0±2.8

66･5- ]]

1) CBA /N(H-2k) mice were intraperitoneally immunized with splenocytes of BALB/C(H-2d)mice, or vice verse･ THemice were then

treated with mAbs: 300iL g REY7(control), 300FL g PC615.3( a ), 150FL g each ofPC615･3 and TUm122 ( a +P), and lOOFL g each ofPC615･3, TUm122 alld TUGm2( α + β + γ C ), on day 0, 2 and 4 a鮎r immunization. On day 5, spleens were removed and splenocytes were used as

5   5   5   5     5 1   1     1     1       1 円 関   門   関   門 a a b

untreated

Control mAb

combination

+

i

.

L

N

N

200:1 100:1  50:1 200:1  100:1   50:1 ( % )   ^ ) ! 3 ! X O t 0 7 ^ 3

10        20

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