免疫學(xué)技術(shù)在科研中的應(yīng)用

.,免疫新技術(shù)在科研中的應(yīng)用,廈門(mén)大學(xué)醫(yī)學(xué)院高豐光,.,抗原或抗體的檢測(cè),抗原抗體反應(yīng)的特點(diǎn)抗原抗體反應(yīng)的影響因素常用的抗原抗體反應(yīng),.,抗原抗體反應(yīng)的特點(diǎn),高度的特異性可逆性抗原抗體結(jié)合除了空間構(gòu)象互補(bǔ)外，主要以氫鍵、靜電引力、范德華力和疏水鍵等分子表面的化學(xué)基團(tuán)之間的非共價(jià)方式結(jié)合。這種非共價(jià)鍵不如共價(jià)鍵結(jié)合穩(wěn)定，極易受溫度、酸堿度和離子強(qiáng)度的影響而解離?？乖贵w比例影響免疫復(fù)合物的大小抗原抗體反應(yīng)的兩個(gè)階段,.,抗原抗體反應(yīng)的影響因素,抗原抗體濃度與比例電解質(zhì)抗原抗體特異性結(jié)合后，親水性降低，易受電解質(zhì)的影響而使表面失去較多的負(fù)電荷。溫度適當(dāng)?shù)臏囟瓤稍黾涌乖c抗體分子的碰撞機(jī)會(huì)，加速抗原抗體復(fù)合物的形成。酸堿度抗原抗體反應(yīng)的最適pH在68之間，pH過(guò)高或過(guò)低，即過(guò)堿或過(guò)酸，均可影響抗原、抗體的理化性質(zhì)。,.,常用的抗原抗體反應(yīng),凝集反應(yīng)沉淀反應(yīng)免疫標(biāo)記技術(shù),.,凝集反應(yīng),直接凝集反應(yīng)血型鑒定肥大反應(yīng)間接凝集反應(yīng)間接凝集抑制試驗(yàn)微粒捕獲酶免疫分析技術(shù),.,間接凝集反應(yīng),將可溶性抗原或抗體吸附在與免疫無(wú)關(guān)的顆粒載體上，形成致敏顆粒，再與相應(yīng)抗體或抗原進(jìn)行反應(yīng)產(chǎn)生的凝集反應(yīng)，稱(chēng)為間接凝集反應(yīng)。顆粒載體有紅細(xì)胞、聚苯乙烯乳膠顆粒、活性炭顆粒，而相應(yīng)的凝集現(xiàn)象分別稱(chēng)為間接血球凝集、間接乳膠凝集、間接炭粒凝集反應(yīng)。,.,.,微粒捕獲酶免疫分析技術(shù),將已知特異性抗體致敏的免疫微粒與生物素親和素酶放大系統(tǒng)相結(jié)合，最后酶作用于熒光底物，使之發(fā)熒光，通過(guò)檢測(cè)熒光強(qiáng)度判斷待測(cè)抗原的含量。檢測(cè)腫瘤標(biāo)記物CAl99、CEA、CAl25、AFP、性激素、甲狀腺素T-T+、葉酸、HIV抗體、HCG等微量可溶性抗原。,.,.,沉淀反應(yīng),速率散射比濁法免疫比濁法瓊脂擴(kuò)散法單向瓊脂擴(kuò)散雙向瓊脂擴(kuò)散火箭電泳對(duì)流免疫電泳免疫印跡技術(shù),.,速率散射比濁法免疫比濁法,將已知抗體與相應(yīng)抗原在液相中按一定比例混合形成可溶性免疫復(fù)合物，這些復(fù)合物微小粒子對(duì)一定波長(zhǎng)的光照射發(fā)生散射，可通過(guò)三個(gè)光路系統(tǒng)測(cè)定光散射(OD值)?？贵w含量固定并處于抗體過(guò)剩時(shí)，免疫復(fù)合物的多少直接取決于抗原的濃度，抗原的終濃度通過(guò)標(biāo)準(zhǔn)品繪出的標(biāo)準(zhǔn)曲線查出。提高了靈敏度，通過(guò)自動(dòng)化，可同時(shí)檢測(cè)多個(gè)樣品并進(jìn)行精確的定量分析。檢測(cè)前白蛋白、酸性蛋白酶、巨球蛋白、轉(zhuǎn)鐵蛋白、尿微量蛋白及IgG、IgM、IgA及補(bǔ)體，藥物含量分析。,.,.,.,免疫印跡技術(shù),將用SDS聚丙烯酰胺凝膠電泳PAGE分離得到的按分子量大小排列的非標(biāo)記蛋白轉(zhuǎn)移到固相載體膜上，再用標(biāo)記的特異性的抗血清或單克隆抗體對(duì)蛋白質(zhì)進(jìn)行定性及定量分析的技術(shù)，其鑒定蛋白質(zhì)的敏感性為15ng。檢測(cè)可溶性抗原、細(xì)胞成分的鑒定與分析，檢測(cè)與自身變性細(xì)胞核成分結(jié)合的抗體(抗核抗體)，HIV的明確診斷。,.,免疫印跡法的基本步驟,電泳分離蛋白抗原，SDS是一種陰離子去污劑，與蛋白質(zhì)牢固結(jié)合，十二烷基磺酸根帶負(fù)電荷，使樣品中各種蛋白質(zhì)與SDS形成SDS-蛋白質(zhì)復(fù)合物，由于蛋白質(zhì)表面均帶有相同密度的負(fù)電荷，使分子構(gòu)型幾乎相同(線性)。因此SDS-PAGE中，SDS多肽復(fù)合物的電泳遷移率只與其分子質(zhì)量有關(guān)，而不受所帶電荷及分子形態(tài)的影響。將SDS-PAGE分離到的蛋白條帶轉(zhuǎn)移至固相的硝酸纖維素膜上。蛋白條帶可用酶、同位素標(biāo)記的一抗或二抗進(jìn)行特異性反應(yīng)，加入顯色底物或放射自顯影以顯示結(jié)果。,.,.,免疫標(biāo)記技術(shù),免疫酶測(cè)定法免疫熒光技術(shù)放射免疫測(cè)定法免疫膠體金技術(shù),.,免疫酶測(cè)定法,是一種用酶標(biāo)記一抗或二抗檢測(cè)特異性抗原或抗體的方法。將抗原抗體反應(yīng)的高度特異性與酶對(duì)底物的高效催化作用有效地結(jié)合起來(lái)，通過(guò)酶分解底物產(chǎn)生有色物質(zhì)(也可作用于熒光底物，產(chǎn)生熒光)，肉眼觀察顏色深淺或酶標(biāo)儀測(cè)定光密度值(OD)，以反映抗原或抗體的含量。本法靈敏度高，檢測(cè)可溶性抗原或抗體、組織或細(xì)胞表面特異性抗原。,.,.,ELISA,雙抗體夾心法(sandwichassay)檢測(cè)血清、腦脊液、胸、腹水等各種液相中的可溶性抗原間接法測(cè)定細(xì)胞及組織表面抗原,.,.,酶聯(lián)免疫斑點(diǎn)試驗(yàn)(enzymelinkedimmunospotassay，ELISPOT),用已知細(xì)胞因子的抗體包被固相載體，加入待檢效應(yīng)細(xì)胞，溫育一定時(shí)間后洗去細(xì)胞，如待檢效應(yīng)細(xì)胞產(chǎn)生相應(yīng)細(xì)胞因子，則與已包被的抗體結(jié)合，再加入酶標(biāo)記抗該細(xì)胞因子抗體，加底物顯色。一般選擇硝酸纖維素膜(NC)或聚偏二氟乙烯(PVDF)膜覆蓋微量反應(yīng)板作為固相，在分泌相應(yīng)細(xì)胞因子的細(xì)胞所在局部呈現(xiàn)有色斑點(diǎn)，一個(gè)斑點(diǎn)表示一個(gè)分泌相應(yīng)細(xì)胞因子的細(xì)胞，通過(guò)計(jì)數(shù)可推算出分泌某種細(xì)胞因子細(xì)胞的頻率。,.,.,.,免疫熒光技術(shù),用熒光素標(biāo)記一抗或二抗，檢測(cè)特異性抗原或抗體的方法。常用的熒光素有異硫氰酸熒光素(nuoresceinisothiocyanate，F(xiàn)ITC)、藻紅蛋白(phycoerythrin，PE)等。在激發(fā)光的作用下，可直接發(fā)射熒光，前者發(fā)黃綠色熒光，后者發(fā)紅色熒光。,.,ImmunofluorescentstainofimmunoglobulinG(IgG)showinglinearpatterninGoodpasturessyndrome,.,天皰瘡免疫熒光染色,.,放射免疫測(cè)定法,用放射性同位素標(biāo)記抗原或抗體進(jìn)行的免疫測(cè)定。既有同位素的敏感性又有抗原抗體結(jié)合的特異性，同時(shí)具有重復(fù)性好、準(zhǔn)確性高、標(biāo)本用量少等優(yōu)點(diǎn)。廣泛應(yīng)用于激素、藥物等微量物質(zhì)的檢測(cè)。,.,免疫膠體金技術(shù),氯金酸(HAuCl+)在還原劑作用下，產(chǎn)生分散狀態(tài)的膠體金顆粒。堿性條件下，金顆粒表面帶負(fù)電荷，與蛋白質(zhì)正電荷基團(tuán)結(jié)合。膠體金可標(biāo)記白蛋白、免疫球蛋白、糖蛋白、激素、脂蛋白、植物血凝素、卵白素等。大分子以單層形式吸附在金顆粒表面。不同還原劑作用于氯金酸，產(chǎn)生的膠體金粒徑大小不相同(550nm)，小粒徑的膠體金由于穿透性好，電子密度高，常被用于免疫電鏡技術(shù)。這些小粒徑的金顆粒，經(jīng)銀顯影液處理后，金粒子還原銀離子生成銀顆粒而吸附在金顆粒周?chē)屎诤稚?，從而放大了金顆粒的顯色效果，又稱(chēng)免疫金銀法。膠體金顏色隨顆粒大小而變化，大于20nm的金顆粒在光鏡下呈現(xiàn)磚紅色，可在光鏡水平行免疫分析，也可用銀顯影劑增強(qiáng)，進(jìn)一步提高靈敏度。當(dāng)膠體金的粒徑較大、濃度密集時(shí)肉眼水平即可觀察，即膠體金斑點(diǎn)滲濾試驗(yàn)和膠體金斑點(diǎn)免疫層析試驗(yàn)。,.,.,免疫細(xì)胞的檢測(cè),免疫細(xì)胞的分離磁珠分離法fluorescence-activatedcellsorter，F(xiàn)ACS免疫細(xì)胞功能的測(cè)定T細(xì)胞B細(xì)胞,.,磁珠分離法,特異性分離所需淋巴細(xì)胞的方法。將特異性抗體(如抗CD3、抗CD4、抗CD8等)吸附在鐵顆粒(磁珠)上，加至細(xì)胞懸液中，具有相應(yīng)抗原的細(xì)胞與磁珠上的特異性抗體結(jié)合。反應(yīng)管置于磁場(chǎng)中，鐵顆粒受磁場(chǎng)的吸引，攜帶有相應(yīng)細(xì)胞的磁球吸附于靠近磁鐵的管壁上。棄細(xì)胞懸液，重新解離細(xì)胞與磁珠。,.,.,fluorescence-activatedcellsorter，F(xiàn)ACS,.,.,.,T細(xì)胞鑒定及功能測(cè)定,使用酶、免疫熒光標(biāo)記單抗進(jìn)行鑒定淋巴細(xì)胞轉(zhuǎn)化試驗(yàn)E花環(huán)形成試驗(yàn)混合淋巴細(xì)胞培養(yǎng)CTL介導(dǎo)的細(xì)胞毒試驗(yàn),.,抗原肽MHC分子四聚體技術(shù)tetramer,用生物素化的抗原肽MHC分子復(fù)合物與熒光標(biāo)記的親合素結(jié)合，由于1個(gè)熒光素標(biāo)記的親合素可結(jié)合4個(gè)生物素分子，能使4個(gè)MHC抗原肽復(fù)合物形成一個(gè)復(fù)合體，將該復(fù)合體標(biāo)記熒光素后，即成抗原特異性四聚體?？乖禺愋运木垠w能與樣品中的特異性T細(xì)胞的TCR結(jié)合，由于四聚體能同時(shí)結(jié)合一個(gè)T細(xì)胞表面的4個(gè)TCR，親和力大大提高。用流式細(xì)胞術(shù)即可確定待檢標(biāo)本中抗原特異性CTL細(xì)胞的頻率。MHC分子可為工類(lèi)或類(lèi)分子，與抗原肽形成的四聚體復(fù)合物，可分別鑒定表達(dá)特異性TCR的CD8+T細(xì)胞及CD4+T細(xì)胞的頻率。,.,.,B細(xì)胞的鑒定及功能測(cè)定,檢測(cè)B細(xì)胞分化抗原測(cè)定B細(xì)胞產(chǎn)生抗體的能力溶血空斑試驗(yàn)ELISPOT,.,細(xì)胞因子的檢測(cè),生物活性檢測(cè)細(xì)胞增生或增生抑制法細(xì)胞病變抑制法趨化作用測(cè)定法免疫學(xué)檢測(cè)法分子生物學(xué)技術(shù),.,基因敲除技術(shù)和轉(zhuǎn)基因動(dòng)物,應(yīng)用基因同源重組，將外源有功能基因(基因組中原先不存在、或已失活的基因)，轉(zhuǎn)入細(xì)胞與基因組中的同源序列進(jìn)行同源重組，插人到基因組中，在細(xì)胞內(nèi)獲得表達(dá)。通過(guò)同源重組產(chǎn)生目標(biāo)基因缺失或失活的轉(zhuǎn)基因動(dòng)物可以確定被敲除的基因在體內(nèi)代謝過(guò)程中的作用，還可確定被敲除基因在分化、發(fā)育、生存等過(guò)程中的作用和必要性。轉(zhuǎn)基因動(dòng)物可以作為疾病模型。可以用于藥物篩選的動(dòng)物模型。轉(zhuǎn)基因動(dòng)物可作為“生物反應(yīng)器”生產(chǎn)藥物。,.,Th細(xì)胞在mCTL介導(dǎo)的腫瘤保護(hù)中作用的研究,.,相關(guān)說(shuō)明,OT-1mice:表達(dá)SIINFEKL特異性TCROVA：雞卵白蛋白OVACTLepitope:SIINFEKLOVAThepitope:ISQAVHAAHAEI-NEAGROVA（OVT）RAG-1KOmice:C57BL/6mice:IFA/CFA/KLHEG7celllineEL4cellline,.,相關(guān)步驟,mCTLandeCTLgenerationOT-1細(xì)胞轉(zhuǎn)輸RAG-1-基因敲除小鼠，2天后以O(shè)T-1TCR特異性表位多肽SIINFEKL免疫；specificThgenerationOVA特異性和非特異性Th表位多肽免疫C57BL/6小鼠以產(chǎn)生特異性和非特異性Th細(xì)胞；mCTLtransfer記憶性CTL轉(zhuǎn)輸已產(chǎn)生特異性和非特異性Th的C57BL/6小鼠；tumorchallenge接種表達(dá)OVA抗原的腫瘤細(xì)胞EG7；,.,GenerationofOvalbumin-specificMemoryCD8TCells.,OT-1lymphnodescellsweretransferredtoRAG-/-micethroughtailveininjection,andmicewereimmunizedwith50ugofSIINFEKLpeptideinCFA1daylater.Atday7,14,21,miceweresacrificed,andsplenocytesorlymphnodescellswereisolatedandanalyzed.ThepercentageofCD44high,SIINFEKL-specificCD8Tcellswasassessedbythree-colorFACSstainingwithSIINFEKLMHCclassItetramerandantibodiestoCD8andCD44.PlotsshownaregatedontheSIINFEKLtetramerpositivelymphocytes;valuesaremeanpercentagesofCD44cellswithintheSIINFEKLtetramer-positivepopulation.,.,.,.,.,.,CD8+MemoryTCellsNeedAntigen-specificCD4+T-helperCellstoAchieveTumorProtection,OvalbuminCTLepitope(SIINFEKL)-specificTcellswereparkedinRag-/-micefor42daystogeneratemCTLandthenadoptivelytransferredtoC57BL/6mice.TwogroupsofrecipientC57BL/6Jmicewereimmunized8dayspriorwith50ugofOVTinincompleteFreundsadjuvantor20ugkeyholelimpethemocyanin(KLH)proteinascontrolinincompleteFreundsadjuvant.Micewerechallengedwithovalbumin-expressingtumorcells(EG7)inthescruffoftheneck1dayafteradoptivetransfer.NormalC57BL/6controlmicewerechallengedwithEG7withoutanytreatment.,.,.,Absenceoftumorprotectioninmicewithoutantigen-specificT-helpercellsisnotbecauseoflowerlevelsoftumorantigen(SIINFEKL)-specificCD8+Tcells.,mCTLsweretransferredtogroupsofC57BL/6micewithorwithoutimmunization8dayspriorwith50ugofOVTorwith20ugofKLHproteincontrol.C57BL/6controlgroupwasgivennotreatment.IFN-Elispotassayswereperformedusingthesplenocytesofthesemice,whichwerechallengedinvitrowith(A)1ug/mlSIINFEKLpeptideor(B)8ug/mlOVAT-helperpeptidetodeterminethepresenceofovalbumin-specificCD8+CTLsandCD4+T-helpercells.,.,.,IncontrasttomCTLs,eCTLsdonotneedThelptokilltumor.,eCTLsgeneratedfromRAG-/-miceweretransferredat(A)day7or(B)day14toC57BL/6micethathadbeenimmunized8daysearlierwith50ugofOVTorascontrol20ugofKLH.C57BL/6micewithoutanytreatmentwereusedascontrols(EG7andEL4controls).Ovalbumin-expressingtumorcells(EG7)wereinjectedunderscruffoftheneckofsomegroupsofmiceat(A)day7or(B)day14.TheparenttumorcelllineEL4thatdoesnotcontainovalbumingenewasinjectedintoothergroupsofmiceatday7(A)asnonspecifictumorcontrol.,.,.,Studyofthelong-livedmemoryCD8+TcellsgeneratedinC57BL/6Jmice.,OvalbuminCTLepitope(SIINFEKL)-specificOT-1cellswereadoptivelytransferredtosyngeneicC57BL/6mice;micewereimmunizedwithSIINFEKL/CFAtoactivateovalbumin-specificCD8+Tcells,andthemicewerekeptfor100daystoensurethatalltransferredOT-1areoflong-livedmemoryphenotypebeforechallengingwithtumor.Eightdaysbeforetumorchallenge,twogroupsofmicewereimmunizedwith50ugofOVTinincompleteFreundsadjuvant.UntreatedC57BL/6micewerealsochallengedwithtumorasnegativecontrol.Micewerechallengedunderscruffoftheneckwithovalbumin-expressingtumorcellline(EG7)orwiththeparenttumorline(EL4)ascontrol.,.,.,.,.,1.RoweHM,LopesL,IkedaY,etal.ImmunizationwithalentiviralvectorstimulatesbothCD4andCD8TcellresponsestoanovalbumintransgeneMOLECULARTHERAPY13(2):310-319FEB2006TimesCited:02.ThompsonJA,DissanayakeSK,KsanderBR,etal.TumorcellstransducedwiththeMHCclassIItransactivatorandCD80activatetumor-specificCD4(+)TcellswhetherornottheyaresilencedforinvariantchainCANCERRESEARCH66(2):1147-1154JAN152006TimesCited:03.SalucciV,MennuniC,CalvarusoF,etal.CD8(+)T-celltolerancecanbebrokenbyanadenoviralvaccinewhileCD4(+)T-celltoleranceisbrokenbyadditionalco-administrationofaToll-likereceptorligandSCANDINAVIANJOURNALOFIMMUNOLOGY63(1):35-41JAN2006TimesCited:04.RadfordKJ,VariF,HartDNJVaccinestrategiestotreatlymphoproliferativedisordersPATHOLOGY37(6):534-550DEC2005TimesCited:15.HuDE,KettunenMI,BrindleKMMonitoringT-lymphocytetraffickingintumorsundergoingimmunerejectionMAGNETICRESONANCEINMEDICINE54(6):1473-1479DEC2005TimesCited:06.VanMeirvenneS,DullaersM,HeirmanC,etal.InvivodepletionofCD4(+)CD25(+)regulatoryTcellsenhancestheantigen-specificprimaryandmemoryCTLresponseelicitedbymaturemRNA-electroporateddendriticcellsMOLECULARTHERAPY12(5):922-932NOV2005TimesCited:27.MiyazakiM,NakatsuraT,YokomineK,etal.DNAvaccinationofHSP105leadstotumorrejectionofcolorectalcancerandmelanomainmicethroughactivationofbothCD4(+)TcellsandCD8(+)TcellsCANCERSCIENCE96(10):695-705OCT2005TimesCited:18.KumaraguruU,BanerjeeK,RouseBTInvivorescueofdefe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