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1 Korean J Hematol Vol. 41, No. 3, September, 2006 Original Article 다발성골수종세포주로부가된단구 - 유래수지상세포를이용한골수종세포주 - 특이세포독성 T 림프구의유도 1 화순전남대학교병원암백신팀, 2 전남대학교의과대학혈액종양내과, 3 순천향대학교의과대학혈액종양내과, 4 공주대학교산업과학대학특수동물학과 박명숙 1,2 박정선 1,2 강현규 1,2 김상기 1,2,4 원종호 3 최보화 1,2 신시원 1,2 주효미 1,2 김춘희 1 니엔판탄냔 1,2 조덕 1 남종희 1 김영진 1 김여경 2 양덕환 2 정익주 1,2 김형준 2 이제중 1,2 Induction of Myeloma Cell Line-specific Cytotoxic T Lymphocytes using Monocyte-derived Dendritic Cells Pulsed with Myeloma Cell Line Lysates Myong-Suk Park 1,2, Jung-Sun Park 1,2, Hyun-Kyu Kang 1,2, Sang-Ki Kim, D.V.M. 1,2,4, Jong-Ho Won, M.D. 3, Bo-Hwa Choi 1,2, Shi-Won Shin 1,2, Xiao-Wei Zhu 1,2, Chun-Ji Jin 1, Thanh-Nhan Nguyen Pham 1,2, Duck Cho, M.D. 1, Jong-Hee Nam, M.D. 1, Young-Jin Kim, M.D. 1, Yeo-Kyeoung Kim, M.D. 2, Deok-Hwan Yang, M.D. 2, Ik-Joo Chung, M.D. 1,2, Hyeoung-Joon Kim, M.D. 2 and Je-Jung Lee, M.D. 1,2 1 Cancer Vaccine Team, Chonnam National University Hwasun Hospital, Jeonnam, 2 Department of Hematology-Oncology, Chonnam National University Medical School, Gwangju, 3 Department of Hematology-Oncology, Soon Chun Hyang University College of Medicine, Seoul, 4 Department of Companion Laboratory Animal, Science College of Industrial Science, Kongju National University, Gongju, Korea Background: In multiple myeloma (MM), the idiotype (ID) determinant of the paraprotein has been used for immunotherapy using dendritic cells (DCs). However, ID-specific immune responses showed limited clinical responses after the Id vaccination. Therefore, an alternative approach using DCs pulsed with other tumor antigens is required. Methods: We investigated the possibility of immunotherapy for MM using myeloma cell line-specific cytotoxic T lymphocytes (CTLs), that were stimulated in vitro by monocyte-derived DCs pulsed with the myeloma cell line ysates. CD14 + cells isolated from the peripheral blood of HLA-A healthy donors were cultured in the presence of GM-CSF and IL-4. On day 6, the immature DCs were pulsed with the myeloma cell line lysates (IM-9: HLA and ARH-77: HLA ), and then maturation of DCs was induced by the addition of TNF-α for 2 days. CTL lines were generated by a 2 time stimulation with DCs to the autologous CD3 + T cells. Results: DCs pulsed with myeloma cell lysates showed the production of IL-12p70, but less than that of unpulsed DCs. CTLs lines stimulated with the DCs pulsing, for the myeloma cell line lysates, showed potent cytotoxic activities against autologous target cells, but not against HLA-A2 - cell lines (RPMI-8226). Mature DCs pulsed with the myeloma cell line lysates showed a higher stimulatory capacity for autologous CTL when compared with mature non-pulsed DCs. 접수 :2006 년 1 월 31 일, 수정 :2006 년 7 월 23 일승인 :2006 년 9 월 12 일교신저자 : 이제중, 전남화순군화순읍일심리 160 번지 , 화순전남대학교병원혈액종양내과 Tel: , Fax: drjejung@chonnam.ac.kr 본연구는과학기술부 21 세기프론티어연구개발사업인세포응용연구사업단 (SC3290) 및산업자원부지방혁신사업 (RTI ) 의연구비지원에의해수행되었음. 186 Correspondence to:je-jung Lee, M.D., Ph.D. Department of Hematology-Oncology, Chonnam National University Hwasun Hospital 160, Ilshim-ri, Hwasun-eup, Hwasun-gun, Jeonnam , Korea Tel: , Fax: drjejung@chonnam.ac.kr

2 박명숙외 : 다발성골수종세포주로부가된수지상세포 187 Conclusion: These results suggest that DCs pulsed with the myeloma cell line lysates can generate potent myeloma cell line-specific CTLs for the myeloma cell-based immunotherapeutic approach in MM. (Korean J Hematol 2006;41: ) Key Words: Dendritic cells, Myeloma, Lysate, Cell line 서 다발성골수종은 B세포클론에서유래된악성형질세포질환으로, 고식적인항암화학요법또는고용량항암화학요법및자가조혈모세포이식등의도입에도불구하고여전히완치가어려운질환으로남아있다. 1,2) 근래에동종골수이식후재발한다발성골수종에서공여자림프구주입술로임상적인반응을유도할수있음이잘알려져있다. 3-5) 이러한일련의보고는임상적으로매우중요한면역-매개성 (immune-mediated) 동종이식편대골수종 (graft-versus-myeloma) 효과가유도될수있음을시사하고있으며, 이는다발성골수종에서면역세포를이용한세포면역치료가새로운치료방법중의하나로도입될수있는이론적근거를제시하고있다. 3) 그동안다발성골수종에서수지상세포를이용한세포면역치료에대한기초연구및임상연구가다양하게시도되었는데, 대표적으로는종양항원으로유전형 (idiotype, ID) 단백질을이용하는것이다. ID는 B세포림프종이나다발성골수종에서생산되는면역글로블린에발현되어있는 paraprotein의결정체로서, 종양세포에만국한되어있어서종양-특이표지자로간주되며, 면역치료에있어서종양항원으로이용될수있다. 5-10) 종양항원으로 ID가부가된수지상세포를이용하여세포면역치료를시행하였을때, 다발성골수종환자의 25~100% 에서특이적인면역반응이유도되었지만, 임상적인반응으로는소수의환자에서만이 M-단백이감소되는실망스러운결과를보였는데, 이는종양항원으로이용된 ID의항원력이매우약해서이러한임상결과를보인것으로생각되고있다 ) 따라서다발성골수종에서수지상세포를이용한세포면역치료의효과를증진시키기위해서는종양항원으로이용되는 ID 단백질을대체할수있는새로운종양항원이요구되겠으며, 다른종양에서와유사하게환자골수의다발성골수종세포를직접이용하는방법이있겠다. 근래에골수종세포용해질 (lysates) 을부가한수지상세포, 13) 수지상세포에골수종세포 RNA를이입 (transfection) 시키 론 는법, 14) 골수종-유래 Heat shock protein (Hsp) 을부가한수지상세포, 15) 또는골수종세포와수지상세포를융합 (fusion) 시키는방법 16-19) 등이보고되고있다. 저자들은다발성골수종세포주의용해질로부가된단구-유래수지상세포로자극된골수종-특이 T 림프구를이용한세포면역치료의가능성을알아보고자본연구를수행하였다. 대상및방법 1. 세포주준비인간다발성골수종세포주로 HLA-A2 양성인 IM-9 (HLA-A0201 +, HLA-A ) 과 ARH-77 (HLA-A ) 세포주, HLA-A2 음성인 RPMI-8226 (HLA-A40 +, HLA- A68 + ) 세포주를순천향의대원종호선생님으로부터받았으며, 수지상세포에서 IL-12 생산을자극하기위하여사용된 CD40L-transfected J558 세포주는 Pawel Kalinski (University of Pittsburgh, PA, USA) 에게서받았다. 다발성골수종세포주는 RPMI-1640 (Gibco-BRL, Grand Island, NY, USA) 에 10% FBS (Gibco-BRL) 와 1% penicillin-streptomycin (GIBCO-BRL) 을첨가하여배양하였으며, CD40L-transfected J558 세포주는 IMDM (Gibco-BRL) 에 10% FBS (GIBCO-BRL) 와 1% penicillin-streptomycin (GIBCO-BRL) 을첨가하여배양하였다. 2. 단구-유래미성숙수지상세포의제조 HLA-A 정상성인으로부터헤파린처리된튜브에말초혈액을 100mL 채혈한후, Ficoll-Hypaque (Lymphoprep TM, Nycomed, Oslo, Norway) 을이용한비중원침법으로단핵구를분리하였다. Mini-Magnetic-activated cell sorting (Mini-MACS) (Miltenyi Biotec, Auburn, CA, USA) 을이용하여단핵구로부터 CD14 + 세포를분리한후, 배양액 1mL당 세포의농도로 GM-CSF (LG biochemicals, Daejeon, Korea) 50ng/mL, IL-4 (R&D systems, Minneapolis, MN, USA) 50ng/mL을첨가하여배양하였다. 실험에사용된배양액은 RPMI (Gibco-BRL) 에 10% FBS (Gibco-BRL) 와 1% peni-

3 188 Korean J Hematol Vol. 41, No. 3, September, 2006 cillin-streptomycin (GIBCO-BRL) 을첨가하여사용하였다. 배양 2일과 4일째에동일한농도의사이토카인을추가하였으며, 배양 6일째에미성숙수지상세포를수확하였다. 3. 골수종세포주용해질 (myeloma cell line lysates) 의제조골수종세포주용해질은 HLA-A 세포주인 IM- 9과 ARH-77을이용하였으며, 세포를 PBS (Gibco-BRL) 로 1,000rpm에서 10분동안원심분리기를이용하여 2 회세척한후, protease 억제제가첨가된 PBS 1mL에재부유하여동결 ( 질소액 ) 과해빙 (37 o C) 을 5차례반복하여골수종세포주용해질을제조하였으며, 용해질은 SDS-PAGE로확인하였고, Bradford assay를사용하여단백정량을분석하였다. 4. 골수종세포주용해질의부가및성숙수지상세포의유도미성숙수지상세포 당골수종세포주용해질 10μg 을부가한후 2시간동안 37 o C, 5% CO 2 배양기에서반응시켰다. 이후새로운배양액에 TNF-α (R&D systems) 50ng/mL를첨가하여 2일동안배양하여성숙수지상세포를유도하였다. 5. 배양된세포의형태학적관찰배양후수확한세포 ( 세포 ) 를 400rpm에서 5 분동안세포원심분리기를이용하여슬라이드에부착시킨후, May-Grünwald-Giemsa 염색을실시하였고, 광학현미경으로관찰하였다. 6. 유세포분석기를이용한면역표현형분석수확한수지상세포를마우스단클론항체를이용하여유세포분석기로면역표현형을알아보았다. 사용된항체는 CD14-FITC, HLA-DR-FITC, CD86-FITC, CD40- FITC (fluorescein isothiocyanate), CD54-PE (phycoerythrin), CD1a-PE, CD80-PE, CD83-PE, CD54-PE (Phar- Mingen, San Diego, CA, USA) 이었다. 또한 isotype control을각각반응시켰으며, 각튜브당최소한 5,000 개의세포를 FACS Caliber (Becton Dickinson, San Jose, CA, USA) 로얻었으며, 데이터의분석은 CELLQUEST software (Becton Dickinson) 을이용하였다. 7. Interleukin-12p70 분비능 well당 세포로 96-well plate에넣은후에 CD40Ltransfected J558 cells을 well당 세포를추가한후에배양하였다. 배양 24시간째얻은부유액에서 IL-12p70 ELISA (Endogen, Woburn, MA) 를시행하였다. 8. 세포독성 T 림프구 (cytotoxic T lymphocytes, CTLs) 의제조및세포독성능 (cytotoxicity) 수지상세포의유도에이용된동일한정상성인의말초혈액단핵구로부터 Mini-MACS (Miltenyi Biotec) 를이용하여 CD3 + 세포를분리하였다. 세포독성 T 림프구의제조는이전의보고와같이수행하였다 ) 골수종세포주용해질이부가된성숙수지상세포를수확하여방사선을조사 (30Gy) 한후, CD3 + 세포와 1:5의비율로 RPMI1640 배양액에서 2일동안배양하였다. 배양 2일후세포를 IL-2 (R&D systems) 10ng/mL와 IL-7 (R&D systems) 5ng/mL이함유된배양액 (RPMI1640: AIM-V=1:1, containing 10% FBS) 에서 5일동안배양하였다. 배양 7일째세포를수확하여동일한수지상세포로다시한번자극을시행한후 IL-2 (10ng/mL) 와 IL-7 (5ng/mL) 이함유된배양액에서다시 7일동안배양하여세포독성 T 림프구를제조하였다. 배양기간동안배양액의절반을 3~4일간격으로교환해주었다. 제조된세포독성 T 림프구 ( 작동세포, effector cell: E) 의세포독성시험은표적세포 (target cell: T) 로다발성골수종세포주 (IM-9, ARH-77, RPMI-8226) 를이용하여 IFN-γ release Elispot 측정기로분석하였다. 작동세포 (E) 와표적세포 (T) 의비율을 40:1, 20:1, 10:1 로일차 IFN-γ 단클론항체가코팅된 nitrocellulosebottomed 96-well multitestplates (MTP) 에 37 o C, 5% CO 2 배양기에서공조배양하였다. 배양 24시간후 plates 를세척하였고, biotin-conjugated secondary anti-human IFN-γ 항체를첨가하여 2.5시간배양한후 PBS-Tween buffer로세척하고 streptavidin-hrp를 2시간동안반응시켰다. 반응된 plates를 PBS-Tween과 PBS buffer로세척한후 3-amino-9-ethylcarbazol substrate를 15~70 분동안반응시켰다. 반응은 tap water 로정지시켰으며 AID Elispot-Reader ELR02 (Ebinger Straβe4 D72479, Straβberg, Germany) 와 computer-assisted video image analysis (CVIA) 를이용하여세포독성 T 림프구에서분비된 IFN-γ spots을측정하였다. IL-12 분비를자극하기위하여수확한수지상세포를

배양후수확한세포독성 T 림프구 (5 10 4 /well) 를방사선이조사된여러용량 (3.2 10 3 ~5 10 4 ) 의수지상세포 ( 골수종세포주용해질이부가된수지상세포와용해질이부가되지않는수지상세포 ) 와 96-well U bottomed culture plate에서배양하였다.

037 MBq) [ 3 H]-methylthymidine을첨가하여 16~18시간동안배양한후, 수확하여 liquid scintillation counter (Beckman, Fullerton, CA, USA) 를이용하여데이터를분석하였고, 그결과는평균 cpm (± 표준편차 ) 을구하여비교하였다.

8일째의세포는미성숙수지상세포보다크기가더컸고돌기모양이돌출된전형적인성숙수지상세포의특징을보였다. 그러나미성숙수지상세포에골수종세포주용해질이부가되어성숙이유도된세포와부가되지않는세포사이에형태학적인차이는없었다.

4 박명숙외 : 다발성골수종세포주로부가된수지상세포 자가세포독성 T 림프구증식반응 (autologous CTL proliferation assay) 골수종세포주용해질이부가된수지상세포로자극되어유도된세포독성 T 림프구의자가수지상세포에대한증식력을측정하기위해서 [ 3 H]-methylthymidine incorporation assay를이용하였다. 배양후수확한세포독성 T 림프구 ( /well) 를방사선이조사된여러용량 ( ~ ) 의수지상세포 ( 골수종세포주용해질이부가된수지상세포와용해질이부가되지않는수지상세포 ) 와 96-well U bottomed culture plate에서배양하였다. 배양 5일째에각 well당 1μCi (0.037 MBq) [ 3 H]-methylthymidine을첨가하여 16~18시간동안배양한후, 수확하여 liquid scintillation counter (Beckman, Fullerton, CA, USA) 를이용하여데이터를분석하였고, 그결과는평균 cpm (± 표준편차 ) 을구하여비교하였다. 결과 1. 수지상세포의형태학적특징및면역표현형발현양상 CD14 + 세포를 6일동안 GM-CSF와 IL-4를이용하여배양하였을때, May-Grünwald-Giemsa 염색에서세포의외부를불규칙한막이둘러싸고있는전형적인미성숙수지상세포의특징을보였으며, TNF-α를첨가하여성숙이유도된배양 8일째의세포는미성숙수지상세포보다크기가더컸고돌기모양이돌출된전형적인성숙수지상세포의특징을보였다. 그러나미성숙수지상세포에골수종세포주용해질이부가되어성숙이유도된세포와부가되지않는세포사이에형태학적인차이는없었다. 유세포분석기를이용한면역표현형검사에서배양 6일째수확한세포는 CD1a, HLA-DR, CD40, CD54의발현이증가되었으며, CD80과 CD86의발현이약간증가되었지만, CD14의발현은감소하여전형적인미성 Fig. 1. Phenotypic analysis of monocytes (top panel), immature dendritic cells (DCs) (middle panel), or mature DCs (bottom panel). CD14 + cells isolated from the MNCs of normal donor by MACS were cultured with a combination of GM-CSF and IL-4. On day 6, the immature DCs (middle panel) were pulsed with tumor antigens, and then maturation was induced by the addition of TNF-α (bottom panel).

5 190 Korean J Hematol Vol. 41, No. 3, September, 2006 숙수지상세포의면역표현형을보였다 (Fig. 1). 8일째수확한세포는 CD80, CD83, CD86과 CD40의발현이현저하게증가되어전형적인성숙수지상세포의면역표현형을보였다 (Fig. 1). 그러나, 골수종세포주용해질이부가되어성숙이유도된세포와부가되지않는세포사이에면역표현형의발현에차이는없었다. 2. 수지상세포에서 IL-12p70의생산수확한수지상세포에서 CD40L-transfected J558 세포주를이용하여 IL-12p70의생산을자극하였을때, 골수종세포주 (IM-9과 ARH-77) 용해질이부가된수지상세포에서 IL-12의분비를확인할수있었다 (Fig. 2). 그러나, 종양항원이부가된수지상세포는종양항원이부가되지않는수지상세포에비하여 IL-12p70의분비 능이저하되었다. 3. 수지상세포로유도된세포독성 T 림프구의강력한세포독성능 HLA-A 정상공여자의말초혈액단구-유래수지상세포에골수종세포주용해질 (IM-9, ARH-77) 을부가한후에자가 CD3 + T 세포를 2차례자극하여골수종세포주-특이세포독성 T 림프구를제조하여서 IFNγ Elispot assay을이용하여세포독성능을평가하였다. 표적세포는자가골수종세포주 (IM-9, ARH-77) 와 HLA-A2 - 인 RPMI-8226 세포주를이용하였다. 골수종세포주 (IM-9, ARH-77) 용해질로부가된수지상세포로자극되어유도된세포독성 T 림프구는 RPMI-8226 표적세포보다자가표적세포 (IM-9, ARH-77) 에대해 Fig. 2. Production of IL-12 by DCs pulsed with or without myeloma cell line lysates (A: ARH-77, B: IM-9) after the stimulation with CD40L-transfected J558 cells for 24 hr. IL-12p70 concentrations in 24 hr supernatants were determined by ELISA. Results, expressed as mean±sd of duplicate culture, are from one representative experiment of two. Fig. 3. ELISPOT data showing the mean number of spots and standard deviation per 1~ T cells. Comparison of IFN-γ secreting cells of CTL generated from stimulation with DCs pulsed with ARH-77 lysates (A) or IM-9 lysates (B) against autologous myeloma cell line lysates or RPMI The data are shown as the mean IFN-γ secreting cell (±SD) of duplicate culture from one representative experiment of two.

6 박명숙외 : 다발성골수종세포주로부가된수지상세포 191 Fig. 4. T cell-stimulatory capacity of DCs with or without myeloma cell line lysates (ARH-77: A, IM-9: B) for CTLs that were generated from stimulation with DCs with myeloma cell line lysates. The data are shown as the mean cpm (±SD) of triplicate cultures from one representative experiment of two. 서현저한 IFN-γ 분비능을보여서, 골수종세포주-특이세포독성 T 림프구가골수종세포주에대하여강력한세포독성능이있음을보여주었다 (Fig. 3). 4. 세포독성 T 림프구가자가수지상세포에대하여강력한세포증식능력 HLA-A 정상공여자의단구-유래수지상세포에골수종세포주용해질 (IM-9, ARH-77) 을부가한후에제조된골수종세포주-특이세포독성 T 림프구가자가수지상세포에반응해서세포증식력을보이는지를평가하기위해서 [ 3 H]-methylthymidine incorporation assay를이용하였다. 골수종세포주용해질이부가된수지상세포로자극되어유도된세포독성 T 림프구는골수종세포주용해질이부가된자가수지상세포로다시자극하였을때골수종용해질이부가되지않는수지상세포에비하여강력한 T 세포증식력을보였다 (Fig. 4). 고찰본연구에서는수지상세포에부가되는종양항원으로서다발성골수종세포주의용해질을이용하여서골수종세포주-특이세포독성 T 림프구의유도가가능하였고, 제조된세포독성 T 림프구는자가골수종세포주에특이적인세포독성을보였다. Wen 등 13) 도다발성골수종에서종양항원으로종양용해질을이용하여보고하였는데, 이들은환자의골수검체로부터골수종세포용해질을제조하여서수지상세포에부가하였을때 ID 항원을부가한수지상세포와비교하여골수종 세포에보다특이성이높은세포독성 T 림프구가유도되어서, 다발성골수종에서종양의항원성이약한 ID보다골수종세포를직접이용하는방법이더유용함을시사하고있다. 그러나, 근래에 Fiore 등 24) 은골수종세포주를이용하여종양용해질을제조하여수지상세포에부가하였을때와는달리, 환자의골수종세포를이용하였을때는수지상세포내로괴사성종양용해질의유입효율이매우낮고, 환자의골수종세포용해질이부가된수지상세포는 Th1형유도에필수적인사이토카인으로알려진 IL-12의분비능은보이지않고오히려 IL-10을다량분비하였으며, 이러한수지상세포로자극된 T 세포는세포증식력이감소될뿐만아니라 IFN-γ의분비능도감소되어서효과기표현형 (effector phenotype) 보다는오히려조절성표현형 (regulatory phenotype) 을보여서, 다발성골수종에서수지상세포를이용한세포치료에있어서다발성골수종세포로부터괴사성종양용해질을제조하여이용하는데문제점이있음을제시하였는데, 이러한보고에대해서는향후후속연구를통하여검증되어야하겠다. ID의약한항원력을극복하기위한대체종양항원으로골수종세포용해질이외에도여러가지종양항원에대한연구가있었다. 이중가장활발하게연구되고있는분야가미성숙또는성숙수지상세포에다발성골수종세포를직접융합시키는방법이다. Gong 등 16) 은마우스모델에서골수종세포를수지상세포에융합하여서백신치료를하였을때마우스의생존이연장되었고, IL-12에의해서이러한면역반응이더욱증가됨을

7 192 Korean J Hematol Vol. 41, No. 3, September, 2006 보고하였다. Raje 등 17) 은인간다발성골수종세포주를수지상세포에융합하여서다발성골수종세포주-특이세포독성능의유도를보고하였으며, Vasir 등 19) 은다발성골수종환자의골수로부터골수종세포를분리하여수지상세포와융합하였을때 IL-12의분비가증가되었고, 이러한융합세포로자극된자가 T 세포는 IFN-γ 분비능과표적세포인골수종세포에대하여강력한세포독성능을보여서, 종양세포와수지상세포을융합하는방법이향후암백신치료에매력적인방법이될수있음을제시하고있다. 근래보고된또다른종양항원으로는고형암에서이미많이연구가진행되었던 HSP으로, 골수종-유래 Hsp gp96을제조하여서수지상세포에부가하였을때정상세포에는영향을미치지않으면서골수종세포에만강력한살상능을보이는골수종-특이세포독성 T 림프구가제조됨을보고하였는데, 이는이론적으로동종종양세포로부터 Hsp을얻어서다른환자에게백신치료을시행할수있음을시사하고있다. 15) 그러나, 근본적으로다발성골수종환자는정상인에비해서말초혈액에골수성 (myeloid) 및형질세포성 (plasmacytoid) 수지상세포의전구체숫자가매우적고, 또한다발성골수종환자의단구-유래수지상세포는정상대조군에비해서 HLA-DR, CD40 및 CD80의발현이유의하게낮을뿐만아니라동종 T 세포자극력도매우낮은단점을가지고있는데, 이는다발성골수종의병인에매우중요하게관여하는 IL-6가 CD34 + 수지상세포전구체의집락형성을억제하고수지상세포로의분화도억제하고있음이규명되어서, 다발성골수종에서수지상세포를이용한면역치료를시행함에있어서이러한단점을극복하기위한후속연구가필요하겠다. 25) 결론적으로다발성골수종세포주용해질이부가된정상인단구-유래수지상세포로다발성골수종세포주에강력한세포독성능을지닌다발성골수종세포주-특이세포독성 T 림프구의제조가가능하게되었다. 이는다발성골수종에서수지상세포를이용한세포면역치료에있어서기초적인토대를제공하였으며, 향후다발성골수종환자의골수종세포를이용한연구가수반되어야할것으로생각된다. 요 배경 : 다발성골수종에서수지상세포를이용한세포면역치료의종양항원으로유전형단백질이널리이용 약 되어왔으나, 유전형-특이면역반응에대한임상효과는제한적이었다. 따라서이를대체할수있는다른종양항원에대한연구가필수적이다. 저자들은다발성골수종세포주용해질로부가된단구-유래수지상세포로자극된골수종세포주-특이세포독성 T 림프구를이용한세포면역치료의가능성을알아보고자본연구를수행하였다. 방법 : HLA-A 정상공여자의말초혈액으로부터단구 (CD14 + ) 를분리하여 GM-CSF와 IL-4로 6일간배양하여유도된미성숙수지상세포에골수종세포주용해질 (IM-9: HLA0201 +, ARH-77: HLA ) 을부가하였고, TNF-α을첨가하여 2일간배양한후성숙수지상세포를얻었다. 성숙수지상세포로자가 CD3 + T 세포를 2차례자극한후세포독성 T 림프구를제조하였다. 결과 : 골수종세포주용해질이부가된수지상세포는 CD40L-transfected J558 세포주의자극에의해서 IL- 12p70의분비를보였지만, 용해질이부가되지않은수지상세포에비하여 IL-12p70의분비능은낮았다. 골수종세포주용해질로부가된수지상세포로자극되어제조된세포독성 T 림프구는 HLA-A2 - 표적세포 (RPMI- 8226) 와비교하여자가표적세포 (HLA : IM-9 및 ARH-77) 에대하여강력한세포독성능을보였다. 골수종세포주용해질이부가된수지상세포는용해질이부가되지않은수지상세포와비교해서자가세포독성 T 림프구에대하여강력한증식능력을보였다. 결론 : 골수종세포주용해질로부가된수지상세포로강력한골수종세포주-특이세포독성 T 림프구의제조가가능하여서, 향후다발성골수종에서종양용해질로부가된수지상세포를이용한세포면역치료의가능성을제시해주었다. 참고문헌 1) Kyle RA, Rajkumar SV. Multiple myeloma. N Engl J Med 2004;351: ) Sirohi B, Powles R. Multiple myeloma. Lancet 2004; 363: ) Harrison SJ, Cook G. Immunotherapy in multiple myeloma--possibility or probability? Br J Haematol 2005;130: ) Tricot G, Vesole DH, Jagannath S, Hilton J, Munshi N, Barlogie B. Graft-versus-myeloma effect: proof of principle. Blood 1996;87: ) Perez-Simon JA, Martino R, Alegre A, et al. Chronic

8 박명숙외 : 다발성골수종세포주로부가된수지상세포 193 but not acute graft-versus-host disease improves outcome in multiple myeloma patients after non-myeloablative allogeneic transplantation. Br J Haematol 2003;121: ) Bergenbrant S, Yi Q, Osterborg A, et al. Modulation of anti-idiotypic immune response by immunization with the autologous M-component protein in multiple myeloma patients. Br J Haematol 1996;92: ) Osterborg A, Yi Q, Henriksson L, et al. Idiotype immunization combined with granulocyte-macrophage colony-stimulating factor in myeloma patients induced type I, major histocompatibility complexrestricted, CD8- and CD4-specific T-cell responses. Blood 1998;91: ) Wen YJ, Ling M, Bailey-Wood R, Lim SH. Idiotypic protein-pulsed adherent peripheral blood mononuclear cell-derived dendritic cells prime immune system in multiple myeloma. Clin Cancer Res 1998; 4: ) Lim SH, Bailey-Wood R. Idiotypic protein-pulsed dendritic cell vaccination in multiple myeloma. Int J Cancer 1999;83: ) Reichardt VL, Okada CY, Liso A, et al. Idiotype vaccination using dendritic cells after autologous peripheral blood stem cell transplantation for multiple myeloma--a feasibility study. Blood 1999;93: ) Lim SH, Bailey-Wood R. Idiotypic protein-pulsed dendritic cell vaccination in multiple myeloma. Int J Cancer 1999;83: ) Osterborg A, Yi Q, Henriksson L, et al. Idiotype immunization combined with granulocyte-macrophage colony-stimulating factor in myeloma patients induced type I, major histocompatibility complex-restricted, CD8- and CD4-specific T-cell responses. Blood 1998;91: ) Wen YJ, Min R, Tricot G, Barlogie B, Yi Q. Tumor lysate-specific cytotoxic T lymphocytes in multiple myeloma: promising effector cells for immunotherapy. Blood 2002;99: ) Milazzo C, Reichardt VL, Muller MR, Grunebach F, Brossart P. Induction of myeloma-specific cytotoxic T cells using dendritic cells transfected with tumorderived RNA. Blood 2003;101: ) Qian J, Wang S, Yang J, et al. Targeting heat shock proteins for immunotherapy in multiple myeloma: generation of myeloma-specific CTLs using dendritic cells pulsed with tumor-derived gp96. Clin Cancer Res 2005;11: ) Gong J, Koido S, Chen D, et al. Immunization against murine multiple myeloma with fusions of dendritic and plasmacytoma cells is potentiated by interleukin 12. Blood 2002;99: ) Raje N, Hideshima T, Davies FE, et al. Tumour cell/ dendritic cell fusions as a vaccination strategy for multiple myeloma. Br J Haematol 2004;125: ) Hao S, Bi X, Xu S, et al. Enhanced antitumor immunity derived from a novel vaccine of fusion hybrid between dendritic and engineered myeloma cells. Exp Oncol 2004;26: ) Vasir B, Borges V, Wu Z, et al. Fusion of dendritic cells with multiple myeloma cells results in maturation and enhanced antigen presentation. Br J Haematol 2005;129: ) Lee JJ, Takei M, Hori S, et al. The role of PGE(2) in the differentiation of dendritic cells: how do dendritic cells influence T-cell polarization and chemokine receptor expression? Stem Cells 2002;20: ) Lee JJ, Nam CE, Nam JH, et al. Generation of cytotoxic donor CD8+ T cells against relapsing leukemic cells following allogeneic transplantation by stimulation with leukemic cell- or leukemic lysate pulsed donor cell-derived dendritic cells. Leuk Res 2004;28: ) Lee JJ, Kook H, Park MS, et al. Immunotherapy using autologous monocyte-derived dendritic cells pulsed with leukemic cell lysates for acute myeloid leukemia relapse after autologous peripheral blood stem cell transplantation. J Clin Apher 2004;19: ) Lee JJ, Choi BH, Nam JH, et al. The generation of leukemic dendritic cells from acute myeloid leukemia cells is potentiated by the addition of CD40L at the terminal maturation stage. J Clin Apher 2004;19: ) Fiore F, Nuschak B, Peola S, et al. Exposure to myeloma cell lysates affects the immune competence of dendritic cells and favors the induction of Tr1-like regulatory T cells. Eur J Immunol 2005;35: ) Ratta M, Fagnoni F, Curti A, et al. Dendritic cells are functionally defective in multiple myeloma: the role of interleukin-6. Blood 2002;100:230-7.

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012임수진 Received : 2012. 11. 27 Reviewed : 2012. 12. 10 Accepted : 2012. 12. 12 A Clinical Study on Effect of Electro-acupuncture Treatment for Low Back Pain and Radicular Pain in Patients Diagnosed with Lumbar