负载食管癌抗原的DC诱导特异性CTL对食管癌细胞体外杀伤作用观察

目的观察负载食管癌抗原的树突状细胞（DC）活化的特异性细胞毒性T淋巴细胞（CTLs）对食管癌细胞的体外杀伤作用。方法冻融法获取食管癌细胞抗原,联合应用粒细胞—巨噬细胞集落刺激因子（rhGM-CSF）、白细胞介素4（IL-4）和肿瘤坏死因子α（TNF-α）诱导培养外周血DC并负载肿瘤抗原,激活自体T淋巴细胞,制备特异性CTLs。将其加入食管癌细胞中培养48 h,用MTT法检测食管癌细胞裂解率,ELISA法检测γ干扰素（γ-IFN）水平。结果负载食管癌抗原的DC激活的CTLs表现出对食管癌Eca109细胞的特异性杀伤作用,γ-IFN水平为（1 625±37.55）pg/ml;而对A549细胞仅有微弱的杀伤作用,γ-IFN水平为（169.04±13.81）pg/ml。未负载食管癌抗原DC刺激的CTLs对食管癌细胞几无杀伤作用。结论负载食管癌抗原的DC激活的CTLs在体外对食管癌细胞能产生高效而特异性的杀伤作用。     

两种不同抗原致敏方式负载DC疫苗对Lovo细胞的体外杀伤作用

目的比较重组痘苗病毒负载人癌胚抗原（CEA）基因转染方式与Lovo细胞裂解物诱导方式对产生DC疫苗的影响。方法分别采用重组痘苗病毒负载CEA基因转染方式及Lovo肿瘤细胞裂解物转染方式转染未成熟DC,诱导特异性细胞毒性T淋巴细胞（CTL）。体外培养CTL并检测其活性;MTT法检测两组CTL对kovo细胞的杀伤作用。结果两种方式致敏DC均可诱导激活CTL并分泌INF-γ,而重组痘苗病毒转染Dc组CTL诱导率高于细胞裂解物组;重组痘苗病毒转染组诱导的CTL对Lovo细胞的特异性杀伤活性显著高于细胞裂解物组。结论两种不同抗原致敏方式负载DC疫苗均能够诱导高效而特异的CTL杀瘤活性,而使用重组痘苗病毒转染CEA至DCs的方式明显优于肿瘤细胞裂解物的抗原负载方式,为DC疫苗用于结肠癌的免疫治疗奠定基础。     

负载胰腺癌抗原的树突状细胞疫苗诱导T淋巴细胞抗肿瘤效应的研究

培养树突状细胞(DC),反复冻融法裂解培养的负载胰腺癌细胞(PC-3),提取细胞抗原,致敏DC,获得负载胰腺癌抗原的DC疫苗后诱导特异性细胞毒性T淋巴细胞(CTL)的生成,MTT法检测CTL对不同肿瘤细胞的杀伤作用.发现负载PC-3细胞抗原的DC疫苗能诱导产生肿瘤特异性的CTL,其对PC-3细胞具有明显地杀伤效应,而对人乳腺癌细胞MCF-7、人肝癌细胞7721细胞杀伤作用弱.认为负载胰腺癌抗原的DC疫苗能够诱导高效而特异地CTT杀瘤活性,为将来DC疫苗在胰腺癌的免疫治疗中提供了实验依据.     

体外负载冻融抗原的脐血树突状细胞诱导的抗肝癌效应

目的评价体外应用肝癌细胞冻融抗原负载的脐血树突状细胞（DC）所诱导的抗肝癌效应。方法采集健康足月剖宫产孕妇胎盘端脐血,分离脐血单个核细胞（CBMNC）及T淋巴细胞,用GM-CSF、IL-4及TNF-α联合诱导CBMNC分化为DC,观察形态学变化并以流式细胞术鉴定,选培养的第3天以肝癌细胞冻融抗原负载的CBMNC-DC,以负载抗原的DC刺激自体淋巴细胞活化为自体细胞毒性T淋巴细胞（CTL）,并用CTL对肝癌细胞进行杀伤,MTT法测定活化的自体淋巴细胞的相对数量和CTL对肝癌细胞的杀伤率。结果体外负载肿瘤冻融抗原的脐血DC可诱导显著的自体效应淋巴细胞增殖及抗肝癌效应。结论体外负载抗原的脐血DC可诱导显著的抗肝癌效应,是具有临床应用前景的肝癌疫苗。     

化疗药物诱导的肺癌死亡细胞致敏树突状细胞的抗肿瘤免疫研究

目的探讨化疗药物诱导的肺癌死亡细胞致敏树突状细胞（DC）的免疫应答及对肺癌细胞的免疫杀伤效果。方法用粒-巨噬细胞集落刺激因子（GM—CSF）和白介素4（IL-4）从胎儿脐血中分化、诱导DC。用足叶乙甙与顺铂在体外诱导培养的人肺癌系A549细胞死亡。DC与死亡的肿瘤细胞培育后，对DC表型变化及抗肿瘤效果进行检测。结果负载死亡的A549细胞之后并不能促使未成熟DC（iDC）分化为成熟DC（mDC），而在TNF—α的作用下能够成为mDC。死亡的A549细胞负载后的mDC由其所激发和扩增的T细胞细胞数以及对肺癌细胞的杀伤效果显著。结论化疗药物诱导的肺癌死亡细胞在体外能有效地致敏DC产生抗肿瘤免疫反应。     

负载MB49肿瘤细胞抗原的DC疫苗对膀胱癌的抑制效应及机制

目的探讨负载MB49肿瘤细胞抗原的树突状细胞疫苗（DC疫苗）治疗膀胱癌的可行性及机制。方法采用反复冻融法提取MB49抗原并用其致敏DC2.4,制备DC疫苗。将24只膀胱癌荷瘤小鼠随机分为两组各12只。DC组皮下注射DC疫苗,对照组注射PBS。注射3周观察两组肿瘤体积变化,计算肿瘤抑制率;分离两组脾脏T淋巴细胞进行培养,MTT法检测细胞毒性T淋巴细胞（CTL）对MB49的杀伤率;ELISA法检测培养上清中IFN-γ水平。结果 DC组肿瘤抑制率及细胞杀伤率均明显高于对照组,P〈0.01;IFN-γ水平亦明显高于对照组,P〈0.01。结论以DC2.4为基础的DC疫苗体内具有抑制肿瘤细胞MB49增长的作用;其机制可能为激活T淋巴细胞。     

负载肿瘤抗原的DC体外诱导抗肿瘤免疫反应的实验研究

目的 研究自体及同种异体树突状细胞(DC)体外负载肿瘤抗原后刺激T淋巴细胞增殖及诱导抗肿瘤免疫反应的能力.方法 利用细胞因子诱导人骨髓单个核细胞生成DC,尼龙毛柱法分离T淋巴细胞,3H-TdR掺入法检测负载肺癌细胞凋亡小体的自体及同种异体DC体外刺激T细胞增殖反应,乳酸脱氢酶(LDH)释放法检测负载肺癌细胞凋亡小体的自体及同种异体DC刺激的T细胞对肺癌细胞和乳腺癌细胞系MCF-7的杀伤作用.结果 骨髓细胞诱生的自体及同种异体DC负载肿瘤抗原后,均具有刺激T淋巴细胞增殖的能力,负载肿瘤抗原的自体及异体DC激活的T淋巴细胞后均可杀伤两种靶细胞,T细胞对MCF-7的杀伤力明显低于对患者肺癌细胞的杀伤力. 结论人自体或异体DC体外负载细胞性肿瘤抗原后,可有效地刺激T淋巴细胞的增殖,产生特异性肿瘤杀伤作用.     

NDV联合负载胃癌细胞抗原DC-CIK对胃癌细胞的杀伤效果及机制

目的探讨新城疫病毒（NDV）联合负载胃癌细胞抗原（Ag）的树突状细胞（DC）及细胞因子诱导的杀伤细胞（CIK）对胃癌细胞的杀伤效果及机制。方法用人外周血单个核细胞诱导培养DC及CIK,体外培养人胃癌细胞SGC-7901,反复冻融法制备胃癌细胞Ag悬液冲击致敏DC,CIK与负载胃癌细胞Ag的DC共培养。建立胃癌裸鼠模型,将其随机分为四组各6只。PBS组瘤内注射PBS100μl／只;NDV组瘤内注射NDV0．25ml／只;Ag-DC-CIK组瘤内注射负载抗原的DC诱导的CIK细胞0．2ml／只;NDV＋Ag-DC-CIK组先瘤内注射NDV0．25ml／只,1d后再注射负载抗原的DC诱导的CIK细胞0．2ml／只;各组均隔日注射1次,共3次。14d后测量肿瘤体积,计算抑瘤率。观察肿瘤坏死情况并评分。结果NDV＋AS—DC-CIK组肿瘤平均体积小于NDV组和Ag—DC-CIK组,抑瘤率、肿瘤坏死面积评分高于NDV组和Ag-DC—CIK组;P均〈0．05。结论NDV和负载胃癌细胞Ag的DC-CIK能协同杀伤胃癌细胞,杀伤效果强于二者单用效果;其机制为NDV可增加胃癌细胞的抗原性,为CIK细胞提供更多靶点。     

肝癌细胞裂解物致敏的树突状细胞瘤苗体外诱导特异性抗肝癌免疫

目的 探讨肝癌患者肿瘤细胞裂解物致敏的树突状细胞(DC)瘤苗体外诱导自体T淋巴细胞特异性抗肝癌免疫效应。 方法 从肝癌患者外周血单个核细胞中诱导D C,用重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)和白细胞介素-4(rhIL-4)刺激活化,经自体肝癌细胞裂解物致敏。用流式细胞仪检测D C细胞表面分子的表达,酶联免疫吸附法检测T淋巴细胞培养上清液中干扰素(I F N)γ和白细胞介索-12(IL-12)的含量,液体闪烁计数仪测定肝癌细胞裂解物致敏的D C刺激自体T淋巴细胞增殖效应,四甲基偶氮唑盐法检测肝癌细胞裂解物致敏D C诱导的细胞毒T淋巴细胞对自体肝癌细胞的特异性杀伤作用。 结果 肝癌细胞裂解物致敏的DC瘤苗可上调DC表面CD1 a、CD40、CD86和人类白细胞抗原-DR分子表达水平,其与T淋巴细胞共培养产生的IFN γ、IL-12的浓度明显高于未致敏的D C组(t值分别为2.30、2.14,P<0.05),肝癌细胞裂解物组(t值分别为14.01、15.40,P<0.01)和对照组(t值分别为14.85、16.87,P<0.01)。同时肝癌细胞裂解物致敏的瘤苗可明显诱导T淋巴细胞的增殖,其诱导的细胞毒性T淋巴细胞对自体肝癌细胞的杀伤率(81.72％±9.49％)显著高于对HepG2的杀伤率(49.37％±11.21％)和人鼻咽癌肿瘤细胞的杀伤率(17.14％±5.65％),P<0.01。 结论 肝癌细胞     

树突细胞与NCI-H460肺癌细胞融合体诱导抗肿瘤作用的实验研究

目的研究树突细胞(DC)与NC I-H460细胞融合体诱导的抗肿瘤作用。方法(1)从人外周血单核细胞诱导DC并与NC I-H460细胞融合。设融合细胞(FC)组、冲击细胞(PC)组及T细胞(TC)组,分别以融合细胞活化的T细胞、抗原冲击DC活化的T细胞及未活化T细胞为效应细胞,用乳酸脱氢酶法测定3种效应细胞对NC I-H460细胞的杀伤作用。(2)皮下注射NC I-H460细胞制备荷瘤裸鼠,将18只荷瘤裸鼠随机分为FC组、PC组及TC组,每组6只,接种上述3种T细胞,比较3组裸鼠肿瘤体积和重量。结果(1)对NC I-H460细胞的杀伤率,FC组为43.54%,PC组26.57%,TC组3.25%,3组杀伤作用比较,差异有统计学意义(F=5.47,P<0.05)。(2)FC组肿瘤体积明显小于PC组和TC组。FC组肿瘤重量为(1 129±123)mg,PC组为(1 709±160)mg,TC组为(3 344±288)mg,3组差异有统计学意义(F=37.05,P<0.01)。结论DC与NC I-H460细胞融合体可有效诱导抗肿瘤免疫,作用优于细胞抗原冲击的DC。     

负载不同转移潜能肝癌细胞exosomes的树突状细胞激活T淋巴细胞体外杀伤作用的初步研究

目的分离制备高、低转移潜能肝癌细胞exosomes,并研究负载相应exosomes的树突状细胞（DC）激活的T淋巴细胞在体外杀伤肿瘤细胞的作用。分析不同转移潜能肝癌细胞系分泌的exosomes的成分差异。方法采用四步离心法分离exosomes,电镜观察。分离小鼠DC,并且负载高、低转移潜能细胞株的exosomes,激活T淋巴细胞后,采用^3H-TdR掺入法进行体外混合淋巴细胞反应。应用蛋白质组学弱阳离子交换芯片（SELDI-TOF-MS）检测不同转移潜能肝癌细胞分泌的exosomes蛋白成分的差异。结果电镜观察高转移潜能的肝癌细胞分泌的exoxomes的密度分布、exosomes内容物均不同于低转移组。CD80、CD86、MHC-;Ⅰ、MHC-Ⅱ,在高转移组分别为64.27±5.00、44.89±10.11、84.35±19.89、59.03±19.37,低转移组分别为71.53.±4.85、50.01±9.50、80.68±29.87、58.86±21.11,与对照组相比P〉0.05,负载exosomes后测得cpm值在高转移组为528.40±179.06,低转移组为78.80±24.44,与对照组相比P〈0.01。蛋白质组学弱阳离子交换芯片结果表明高、低转移组细胞系的exosomes蛋白组分有着明显的差异。结论exosomes具有潜在的免疫治疗价值,在肝癌的转移和复发防治以及生物治疗中具有应用前景。     

胃腺癌BGC-823细胞总RNA转染人树突状细胞分泌exosomes的抗肿瘤效应研究

目的研究人外周血树突状细胞（DC）体外经人胃腺癌BGC-823细胞系总RNA转染后,提取培养上清液中DC分泌的外泌体（exosomes）,诱导出特异性抗胃癌效应。方法分离外周血单核细胞,经GM-CSF、IL-4培养5d后,获得未成熟DC（imDC）;体外以脂质体转染BGC-823细胞总RNA。第7天收集上清,利用超速离心法提取exosomes。分别将DC以及exosomes与T淋巴细胞共培养3d,获得细胞毒T淋巴细胞（CTL）,检测CTL对BGC-823细胞的杀伤作用。结果与未转染组相比,转染BGC-823总RNADC来源的exosomes明显促进T细胞对BGC-823的杀伤活性（P〈0．05）。结论应用BGC-823总RNA转染DC分泌exosomes能够诱导出强烈的抗肿瘤免疫反应。     

Preparation of human ovarian cancer ascites-derived exosomes for a clinical trial

Despite initial response to chemotherapy, at least 50% of ovarian cancer patients will relapse within 18 months. Progression-free survival is related to tumour infiltration with cytotoxic T lymphocytes (CTL). We recently demonstrated that CD8+ T cell responses to recall antigens improve following tumour response to chemotherapy. Vaccination designed to expand CTL, specific for tumour-associated antigens, may be a means of improving outcome. We are planning a clinical trial in advanced ovarian cancer patients undergoing chemotherapy using a combination of a Toll-like receptor 3 (TLR3) agonist and tumour-associated ascites-derived exosomes. Tumour-derived exosomes are a potential source of tumour antigens able to induce CD8+ T cell responses when loaded on mature dendritic cells (DC). DC maturation can be achieved with Toll-like receptor (TLR) agonists, such as the GMP-grade synthetic double stranded RNA, poly[I]:poly[C12U] (Ampligen) which is a TLR-3 agonist. Here, we describe the development of a method suitable for the preparation of GMP-grade exosomes from the ascites fluid of ovarian cancer patients, and the methods used for the molecular and immunological characterisation of these exosomes preceding their use in a clinical trial.     

The immunogenicity of dendritic cell-derived exosomes

Exosome production represents an alternate endocytic pathway for secretion. Multivesicular endosomes (MVE) fuse with the plasma membrane expelling internal vesicles or exosomes from cells. Exosome production has been recently described for immune cells including B cells, dendritic cells (DC), mast cells, macrophages and T cells. Exosomes derived from some DC populations stimulate T lymphocyte proliferation in vitro and have potent capacity to generate anti-tumour immune responses in vivo. These reported studies have involved in vitro grown mature DC expanded from precursors with cytokines. However, immature DC produce higher numbers of exosomes than mature DC and this is thought to be due to a reduction in endocytosis as DC mature, associated with reduced reformation of MVE and reduced exosome formation. This lab pioneered a method to generate immature DC in spleen long-term cultures (LTC). DC produced in cultures represent immature myeloid DC, highly endocytic but with weak capacity to stimulate T cells. LTC-DC produce exosomes and contain many MVE. This prompted a study of immunogenic potential with a view to the potential use of exosomes in vaccination and immunotherapy. DC produced in cultures represent immature myeloid DC, highly endocytic but with weak capacity to stimulate T cells. Exosomes were isolated by differential centrifugation from LTC-DC and shown by marker expression to arise by budding from the LAMP-1+ limiting endosomal membrane of MVE. These LTC-derived exosomes appear however to lack immunostimulatory markers like CD86, CD40, MHC-I and MHC-II. While LTC-DC can stimulate antigen-specific proliferation of CD4+ T cells, exosome preparations derived from antigen-pulsed DC were unable to stimulate purified na?ve T cells in vitro. They were however found to weakly activate allogeneic CD8+ T cells in vitro. Tumour antigen-pulsed LTC-DC or their exosomes could induce a protective response in mice against growth of a transplanted tumour but could not induce a response to clear an existing tumour. Exosomes derived from immature DC can modulate immune responses, but do not function in direct T cell activation in vitro. Modulation of immune responses by exosomes produced by immature DC may be dependent on the presence of other antigen presenting DC subsets in the animal. The possible function of immature DC and their exosomes in maintenance of tolerance and in the induction of immunity is discussed.     

肿瘤相关成纤维细胞外泌体调控PI3K/AKT/mTOR信号通路影响肺癌细胞的生长和转移

目的探究肿瘤相关成纤维细胞外泌体对肺癌细胞生长和转移的影响及机制。方法取肺癌患者的癌组织和癌旁组织分离肿瘤相关成纤维细胞(Cancer-associated fibroblasts,CAFs)和成纤维细胞(Normal Fibroblasts,NFs),免疫荧光和Western blot鉴定CAFs和NFs中α-SMA、FSP-1、FAP、Vimentin表达,差速离心法提取CAFs和NFs细胞外泌体,利用透射电镜和Western blot鉴定外泌体,将外泌体与肺癌细胞A549共孵育后检测细胞增殖、迁移、侵袭、细胞凋亡水平,Western blot检测PI3K/AKT/mTOR信号通路激活水平。结果α-SMA、FSP-1、FAP、Vimentin高表达于CAFs;透射电镜和Western blot鉴定外泌体符合其特征;与PBS组相比,与CAFs外泌体共孵育组A549细胞增殖、迁移、侵袭能力显著增加(P<0.001),细胞凋亡水平显著降低(P<0.001),PI3K、AKT、mTOR磷酸化水平显著增加,而与NFs外泌体共孵育后细胞增殖、迁移、侵袭以及凋亡水平无显著变化,PI3K、AKT、mTOR磷酸化水平无显著变化。结论肿瘤相关成纤维细胞外泌体能够通过激活PI3K/AKT/mTOR信号通路,而促进肺癌细胞的生长和转移。     

不同肺转移潜能的大鼠肝癌细胞外泌体对巨噬细胞转录组的影响

目的 比较不同肺转移潜能的大鼠肝癌细胞来源外泌体对巨噬细胞转录组的影响.方法 通过转录组测序技术,比较经两株不同肺转移潜能的大鼠肝癌细胞(WB1和WN1)来源外泌体处理的巨噬细胞的转录组差异,筛选差异基因并使用KEGG分析、GO分析和蛋白互作网络分析,探索癌细胞来源外泌体影响巨噬细胞功能促进肺转移的潜在机制.结果 对比...     

HBeAg特异性细胞免疫反应体外抗乙肝病毒作用

目的探讨体外HBeAg特异性细胞免疫反应对乙型肝炎病毒(HBV)mRNA表达及蛋白合成的影响。方法自正常人外周血单核细胞中诱导培养树突状细胞(DC),经不同浓度的HBeAg负载后与自身T细胞共同培养,诱导产生HBeAg特异性T细胞,同时设与无HBeAg刺激的DC共同培养的T细胞、单纯T细胞为对照组,作用于HepG2.2.15细胞系,逆转录PCR(RT-PCR)检测HepG2.2.15细胞内乙肝表面抗原(HBsAg)、乙肝核心抗原(HBcAg)的mRNA水平,酶联免疫法(ELISA)检测细胞培养上清中HB-sAg和乙肝e抗原(HBeAg)水平。结果经HBeAg刺激激活的T细胞可以有效地抑制HBsAg、HBcAg mRNA的表达(P0.05)及HBsAg、HBeAg的合成(P0.05),且随着HBeAg刺激浓度的升高,对上述指标的抑制率逐渐上升。结论HBeAg特异性细胞免疫反应可显著抑制HBV mRNA和蛋白的表达,为开发HBeAg相关的治疗性疫苗奠定理论基础。     

Direct stimulation of T cells by membrane vesicles from antigen-presenting cells

Activation of na?ve T cells generally requires T cell receptor-mediated contact with MHC-bound peptides on viable antigen-presenting cells such as dendritic cells (DC). Here evidence is presented that dissociated cell membrane fragments from a DC line can be used as an effective substitute for viable DC. Ultracentrifuged material derived from sonicates of IFN-gamma-matured DC is enriched in small membrane vesicles that closely resemble exosomes. When complexed with MHC class I-restricted specific peptide, vesicles from DC sonicates generate strong responses by purified na?ve CD8(+) cells in vitro in the absence of normal antigen-presenting cells and can also efficiently prime T cells for tumor rejection in vivo. Both in terms of total yields from DC and relative immunogenicity, membrane vesicles from DC sonicates are much more effective than classic exosomes and may be a valuable tool for tumor immunotherapy.     

exosomes在肺癌细胞对顺铂敏感性降低过程中的作用研究

目的研究肺癌细胞分泌的exosomes在同源细胞对顺铂的敏感性调节过程中的可能作用。方法采用超速离心和Exoquick-TC结合的方法从肺癌细胞系A549和H1975的上清液中分离出exosomes,透射电子显微镜观察其形态,western blot检测其CD63蛋白表达;共聚焦显微镜观察细胞胞吞exosomes过程;分别提取顺铂处理肺癌细胞来源exosomes和未处理肺癌细胞来源exosomes,并使用2种exosomes分别处理肺癌细胞,采用CCK-8法检测上述获得的exosomes对肺癌细胞顺铂敏感性的影响。结果透射电子显微镜下观察肺腺癌细胞来源的exosomes具有特征性的盘状结构,直径30-100 nm,western blot结果显示exosomes富含CD63蛋白;显微镜下可观察到exosomes可进入细胞;同时经顺铂处理过肺癌细胞分泌的exosomes可降低同源细胞对顺铂的敏感性。结论减少exosomes的分泌和传递可能会增加顺铂化疗的疗效,这为肺癌的治疗提供了一种新的思路。     

Specificity and efficacy of dendritic cell-based vaccination against tuberculosis with complex mycobacterial antigens in a mouse model

Dendritic cells (DC) likely play important and unique roles in the generation of protective immunity to mycobacteria. In order to clarify their contributions, bone marrow-derived DC loaded with Mycobacterium tuberculosis sonicate antigens were used to stimulate T cell proliferation both in vitro and in vivo and to vaccinate C57BL/6 mice against subsequent challenge with virulent mycobacteria. Antigen-pulsed DC developed in fetal calf serum (FCS-DC), but not DC developed in normal mouse serum (NMS-DC), stimulated significant proliferation of both na?ve and immune T cells in vitro. The difference between cell populations developed in FCS and NMS in the content of CD11c(+) cells and in production of key cytokines indicated that NMS is less supportive for the development of activated DC. However, following adoptive transfer of a single dose of antigen-pulsed DC into naive recipients, NMS-DC induced T cells that proliferated in response to mycobacterial antigen, whereas FCS-DC stimulated strong non-specific proliferation. Vaccination with two doses of antigen-pulsed NMS-DC by the subcutaneous route induced significant protection against intravenous challenge with a moderate dose of virulent M. tuberculosis. DC-vaccinated mice exhibited significant reductions in bacillary loads in the lungs and spleens, and markedly reduced lung pathology. Three doses of antigen-pulsed NMS-DC induced a significant increase in survival time following high dose challenge, which correlated with a significant increase in IFN-gamma-producing cells in both lung and lymphoid tissues, as assessed by the ELISPOT assay. Taken together, these results indicate that DC play a critical role in the induction of protective resistance against virulent mycobacterial challenge accompanied by the development of antigen-reactive, IFN-gamma-producing T cells, and that their antigenic specificity is influenced by the culture conditions under which the DC are developed.