FBL-3肿瘤抗原的实验研究:Ⅰ.抗原的预测及合成多肽抗原性的验证

T细胞在肿瘤免疫中发挥着重要作用,它是通过T细胞抗原受体(TCR)识别肿瘤细胞表面由MHC分子呈递的肿瘤源性多肽,其中细胞毒T淋巴细胞(CTL)是通过识别肿瘤细胞MHC I类分子结合并呈递的多肽。研究表明:CTL识别的多肽常由8～9个氨基酸组成,多肽的呈递受MHC分子等位型的限制,不同的MHC分子具有不同的等位型特异的多肽结合基序(peptidebinding motif)。FBL-3是B6小鼠(H-2~b)源由Friend病毒诱导的小鼠白血病细胞,灭活FBL-3免疫B6小鼠可诱导FBL-3特异CTL产生,从而建立FBL-3特异的免疫应答,保护小鼠免受FBL-3的致瘤性。因此,实验中根据H-2D~b多肽结合基序和Friend病毒gag基因序列,预测出7个可能编码FBL-3抗原多肽的片段。根据预测的结果人工合成了7个九肽(gag1-gag7)。实验结果显示:合成多肽免疫B6小鼠可诱导多肽特异的CTL产生,但用体外实验方法不能诱导初次免疫应答;FBL-3特异CTL可以杀伤gag3,gag5致敏的靶细胞EL-4(H-2~b),但体内实验表明gag3,gag5不象FBL-3一样,它们体内免疫后不能建立有效的抗FBL-3肿瘤免疫效应。表明预测的7个多肽可能不是FBL-3的肿瘤抗原。文中讨论了多肽抗原性预测的复杂性和可能的对策。     

PATHOLOGICAL STUDIES ON THE ANTI－INVASIVE CHARACTER BY RECOMBINANT HUMAN INTERLEUKIN－6 GENE－TRANSFECTED MOUSE LEUKEMIA CELLS

ＰＡＴＨＯＬＯＧＩＣＡＬＳＴＵＤＩＥＳＯＮＴＨＥＡＮＴＩ－ＩＮＶＡＳＩＶＥＣＨＡＲＡＣＴＥＲＢＹＲＥＣＯＭＢＩＮＡＮＴＨＵＭＡＮＩＮＴＥＲＬＥＵＫＩＮ－６ＧＥＮＥ－ＴＲＡＮＳＦＥＣＴＥＤＭＯＵＳＥＬＥＵＫＥＭＩＡＣＥＬＬＳＧｅＬｉｎｆｕ葛林阜，Ｃａｏ...     

小鼠红白血病细胞FBL-3的生物学特性

 目的　探讨小鼠红白血病细胞FBL-3的生物学特性。方法　观察小鼠红白血病FBL-3细胞光镜下的形态、生长曲线、体外克隆形成率及细胞免疫组织化学染色情况;流式细胞术分析细胞周期分布以及MHC分子的表达情况;常规细胞遗传学方法分析染色体核型,PCR检测性别基因Sry;MTT法分析化疗药物敏感性;经尾静脉接种后观察C57BL／6小鼠肝、脾、肺、肾的病理变化。结果　FBL-3细胞呈梭形或多角形贴壁生长;细胞群体倍增时间为24.12 h。14 d和21 d克隆形成率分别为（35.23±1.44）%和（60.27±5.56）%。糖原染色阳性、氯醋酸染色部分阳性,过氧化物酶、碱性磷酸酶、丁酸染色均阴性。细胞周期：G0／G1细胞占（50.9±2.5）%,S期细胞占（36.3±1.4）％、G2／M期细胞占（13.8±0.8）％。对化疗药物阿糖胞苷、长春地辛、顺铂、丝裂霉素、甲氨蝶呤的半数抑制浓度分别为（0.49±0.04）、（0.87±0.09）、（3.77±0.32）、（1.66±0.16）μmol／L和（2.77±0.24）nmol／L。FBL-3细胞染色体数目在34～41条之间,表达H-2b分子,Sry基因阳性。静脉接种小鼠100 %发病;接种的细胞数量与存活时间呈线性关系;白血病细胞可以浸润肝、肺、脾和肾。结论　FBL-3细胞具有白血病细胞的特性,体外易于培养,容易建立小鼠模型,可作为研究白血病的理想动物模型。     

In vitro andin vivo chemotactic effect of mip-lα gene transfected tumor vaccine on immune effector cells

Vaccination with chemokine gene-transfected tumor cells may be a new apporach to cancer treatment. Macrohage inflammatory protein-la (MIP-lα ) is a new type of chemokines which has chemotactic activity on effector cells. In the present study, the B16 melanoma cells were transfected with recombinant adenovirus harboring murine MIP-1α gene. The biological characteristics of the MIP-lα gene transfected B16 melanoma cells were investigated. The level of MlP-lα in the supernatant of gene-transfected melanoma cells was 368±24 ng/ml/10⁶/24hr. This supernatant showed strong chematactic activity for NK cells, CD4⁺ T cells, CD8⁺ T cells or the freshly isolated peritoneal macrophages. Though thein vitro growth were not altered, the tumorigenicity of the gene-transfected B16 melanoma cells decreased signicantly. The infiltration of inflammatory cells into the tumor mass formed by MIP-lα gene-transfected B16 cells were much more obvious than that by wild-type B16 cells or B16 cells transfected with the control gene. However, the survival time of the mice bearing B16 melanoma cells transfected with MIP-1 a gene was not prolonged and the NK, CTL activity remianed unchanged. We suggested that thein vivo phenomenon may be related to the high toxicity of the MIP-1 α as a strong non-specific inflam-matory mediator.     

Characterization of the conversion between CD133+ and CD133- cells in colon cancer SW620 cell line

The state of cancer stem cells (CSC) under reversible fluctuations, which has been revealed in breast cancer cells most recently, suggests that subpopulations with distinct phenotypes and functions within cancer cells can undergo inter-conversion. To investigate the possibility in colon cancer cells, we employed CD133 as the CSC marker, and characterized CD133 expression pattern and the biological features of the CD133⁺ and CD133^- subsets. Flow cytometry revealed that CD133 was bimodally expressed in SW620 cells among eight colon cancer cell lines. The CD133⁺ clonal SW620 cells displayed a differential gene expression profile, higher cellular reactive oxygen species (ROS), enhanced tumorigenesis and resistance to 5-fluorouracil. The conversion in term of the CD133 phenotype of the sorted cells was observed in vitro and in vivo. The fraction of the CD133⁺ cells decreased from 99% to 80% in the sorted CD133⁺ population while rising from 5 to 10% in the sorted CD133^- population during the first 20-day cultivation and then stayed almost unchanged. A fraction (about 20%) of the CD133⁺ clonal cells lost their CD133 marker while about 10% of the CD133^- clonal cells acquired the CD133 marker. 5-Azacytidine enhanced the fraction of the CD133⁺ cells in both of the CD133⁺ and CD133^- clonal cells. Our data demonstrate that CD133 expression is dynamic and reversible, and reveal the inter-conversion between the CD133⁺ and the CD133^- SW620 cells, suggesting that the CD133 phenotype of SW620 cell population is retained by the conversion between the two cell subsets.     

FBL-3肿瘤抗原的研究:Ⅱ.从多肽池中分析肿瘤抗原

在抗肿瘤免疫效应中细胞毒T淋巴细胞(CTL)是通过识别肿瘤细胞MHC I类分子结合的肿瘤源性多肽.为分析肿瘤细胞的抗原性及肿瘤抗原的多肽性质,本文选用FBL-3肿瘤细胞,用pH3.3的枸橼酸-磷酸盐缓冲液间隔24～36小时多次酸洗肿瘤细胞,以获得MHC I类分子结合的多肽,并分析酸洗液中多肽混合物的肿瘤抗原性,以及RP-HPLC分离的部分收集液多肽的肿瘤抗原性,为FBL-3肿瘤细胞抗原的确定提供基本数据.结果表明:酸洗法能有效获得肿瘤细胞MHC I类分子相关的多肽抗原,肿瘤细胞多肽抗原的分离、纯化、HPLC分析以及抗原性鉴定是肿瘤抗原性确定的一个重要方法.     

Identification and characterisation of malignant cells using PT-PCR on single flow-sorted cells

In an attempt to optimise stem cell graft evaluation we have developed a method of quantifying the number of cells in a phenotypically defined population of cells, expressing a gene of interest by combining an RT-PCR method working on whole single cells with flow cytometry. The clinical potential is illustrated by two examples. First, the phenotypes of clonal cells in the bone marrow (BM) of a patient with multiple myeloma (MM), were determined by sorting cells phenotypically defined by their expression of surface antigens and then performing RT-PCR on the individual sorted cells using the rearranged immunogiobulin heavy chain (IgH) gene as clonai marker. All plasma cells with the phenotype CD38⁺⁺/CD45RA^-expressed the clonai marker, whereas it could not be detected in plasma cells with the phenotype CD38⁺⁺/CD45RA⁺ A minor population of clonai cells with the CD38⁺CD45RA^- phenotype was found. Second, the number of committed (CD34⁺/CD38⁺) and non-committed (CD34⁺/CD38^-) stem cells, expressing the chimeric fusion gene p210 BCR/ABL in the autografi from a patient with chronic myeloid leukemia (CML), was determined. The number of cells expressing BCR/ABL mRNA was nearly equal in the CD34VCD38⁺ and CD34⁺CD38^- compartment (8.1 and 8.5%). The method presented can easily be applied to determine the phenotype of malignant cells, where a unique mRNA species exist. Furthermore, the method allows one to predict the outcome of antibody mediated purging experiment.     

Immunization with type 5 adenovirus recombinant for a tumor antigen in combination with recombinant canarypox virus (alvac) cytokine gene delivery induces destruction of established prostate tumors

Prostate‐specific antigen (PSA) is expressed by prostate epithelial cells and has a highly restricted tissue distribution. Prostatic malignancies in 95% of patients continue to express PSA, making this antigen a good candidate for targeted immunotherapy. The goals of our studies are to generate a recombinant PSA adenovirus type 5 (Ad5‐PSA) that is safe and effectively activates a PSA‐specific T‐cell response capable of eliminating prostate cancer cells, and to characterize the immunologic basis for this rejection. Here we show that immunization of mice with Ad5‐PSA induced PSA‐specific cellular and humoral immunity that was protective against a subcutaneous challenge with RM11 prostate cancer cells expressing PSA (RM11psa), but not mock‐transfected RM11 tumor cells (RM11neo). Mice immunized with recombinant adenovirus type 5 encoding β‐galactosidase (Ad5‐lacZ) did not generate protective immunity. Antitumor activity was predominantly mediated by CD8⁺ T lymphocytes. Although Ad5‐PSA immunization prior to RM11psa challenge was protective, Ad5‐PSA immunization alone was not able to control the growth of existing RM11psa tumors. In contrast, established RM11psa tumors ranging in size from 500 to 1,000 mm³ were efficiently eliminated if Ad5‐PSA priming was followed 7 days later by intratumoral injection of recombinant canarypox viruses (ALVAC) encoding interleukin‐12 (IL‐12), IL‐2, and tumor necrosis factor‐α. In this case, antitumor immunity was still dominated by CD8⁺ T lymphocytes, but natural killer cells became necessary for a maximal response. These data provide information on the effector cell populations in a protective immune response to prostate cancer and demonstrate the utility of an Ad5‐PSA vaccine combined with cytokine gene delivery to eliminate large established tumors that are refractory to other interventional methods. © 2001 Wiley‐Liss, Inc.     

大肠杆菌CD自杀基因转染诱导肿细胞凋亡及旁观者效应的研究

以重组腺病毒AdCD为载体将大肠杆菌胞嘧啶脱氨酶(CD)基因体外传染小鼠红白血病细胞FBL3,结果显示,转染了CD基因的FBL3细胞对5-氟胞嘧啶(5-FC)的敏感性显著提高,进一步研究发现,AdCD/5-FC系统可以诱导肿瘤细胞发生凋亡;将经AdCD/5-FC处理过的FBL3细胞上清倍比稀释后,加入到野生型FBL3细胞中,发现当上清仅占6.25％时,即对野生型FBL3细胞发挥明显的杀伤作用,提示旁观者效应在AdCD介导的细胞毒作用中起着重要的作用。本实验还观察了CD基因体内转染后的杀伤效果,荷瘤小鼠局部注射AdCD并连续10天给予5-FC(300mg/kg)治疗后,小鼠皮下肿瘤结节的生长受到明显抑制。     

Apoptosis of bladder transitional cell carcinoma T24 cells induced by adenovirus-mediated inducible nitric oxide synthase gene transfection

Objectives：To investigate the effects of adenovirus-mediated inducible nitric oxide synthase gene transfection on bladder transitional cell carcinoma T24 cells,and to provide novel insights and approaches to clinical therapies against bladder transitional cell carcinoma.Methods：Firstly,construct recombinant adenovirus vector pAd-iNOS of iNOS,followed by transfection of pAd-iNOS into HECK293 packaging cells.Thirdly,harvest recombinant adenovirus rAd-iNOS after amplification and purification procedures.Finally,transfect the recombinant adenovirus rAd-iNOS into human bladder carcinoma T24 cells and examine the effect of rAd-iNOS transfection on apoptosis of T24 and possible mechanism.Results：As shown by this study,the recombinant adenovirus rAd-iNOS was constructed successfully.The virus titer was 5.8×108 PFU/mL and recombinant was verified by PCR analysis.Transfection of adenovirus rAd-iNOS into T24 cells could induce secretion of high NO concentration,P53 protein expression upregulation,as well as promotion ofT24 cell apoptosis.Conclusions：The transfection of human bladder carcinoma T24 cells from recombinant adenovirus rAdiNOS was confirmed to induce intracellular iNOS over-expression,high production of NO,up-regulation of intracellular P53 expression and promotion of cell apoptosis.     

Human effector T cells derived from central memory cells rather than CD8T cells modified by tumor-specific TCR gene transfer possess superior traits for adoptive immunotherapy

Adoptive cell therapy provides an attractive treatment of cancer, and our expanding capacity to target tumor antigens is driven by genetically engineered human T lymphocytes that express genes encoding tumor-specific T cell receptors (TCRs). The intrinsic properties of cultured T cells used for therapy were reported to have tremendous influences on their persistence and antitumor efficacy in vivo. In this study, we isolated CD8⁺ central memory T cells from peripheral blood lymphocytes of healthy donors, and then transferred with the gene encoding TCR specific for tumor antigen using recombinant adenovirus vector Ad5F35-TRAV-TRBV. We found effector T cells derived from central memory T cells improved cell viability, maintained certain level of CD62L expression, and reacquired the CD62L⁺CD44^high phenotype of central memory T cells after effector T cells differentiation. We then compared the antitumor reactivity of central memory T cells and CD8⁺T cells after TCR gene transferred. The results indicated that tumor-specific TCR gene being transferred to central memory T cells effectively increased the specific killing of antigen positive tumor cells and the expression of cytolytic granule protein. Furthermore, TCR gene transferred central memory T cells were more effective than TCR gene transferred CD8⁺T cells in CTL activity and effector cytokine secretion. These results implicated that isolating central memory T cells rather than CD8⁺T cells for insertion of gene encoding tumor-specific TCR may provide a superior tumor-reactive T cell population for adoptive transfer.     

Epstein-barr virus (EBV)-related lymphoproliferative disorder with subsequent EBV-negative T-cell lymphoma

A 58-year-old Chinese man presented initially with generalized lymphadenopathy, and lymph-node biopsy showed disturbed architecture with preponderance of large B-blasts mixed with numerous CD8⁺ T lymphocytes, consistent with an acute Epstein-Barr virus(EBV) infection. Immunohistological and gene rearrangement studies confirmed the absence of clonal T or B cells. Polyclonal EBV with lytic infection was detected by Southern blot hybridization (SoBH). Expression of EBV proteins (EBNA2, LMP and ZEBRA) was detected in a proportion of cells by immunostaining. EBV-lytic proteins EA-D, VCA, MA were also detected in rare scattered cells. Double immunostaining showed that the LMP-positive cells were of B and of T phenotype: 73% CD19⁺, 26% CD2⁺ 23% CD3⁺ 8% CD4⁺ 17% CD8⁺. After biopsy, there was spontaneous regression of lymph-node enlargement, but lymphadenopathy recurred 8 months later, and the second lymph-node biopsy showed T-cell lymphoma, confirmed by detection of clonally rearranged T-cell-receptor beta-chain gene. However, EBV genome could not be detected in the second biopsy by SoBH, in situ hybridization for EBV-encoded EBER RNA, and immunostaining for EBNA2, LMP and ZEBRA was also negative. This case is of special interest because an EBV-negative T-cell lymphoma developed shortly after an acute episode of EBV-related lymphoproliferation, even though many EBV-positive T cells were detected during the acute episode. EBV was apparently not a direct cause of the lymphoma, but the close temporal association of the 2 lesions supports the hypothesis that EBV can act as a co-factor in lymphomagenesis. © 1994 Wiley-Liss, Inc.     

腺病毒介导多基因在肺癌细胞中的表达及致凋亡效应

目的:观察重组腺病毒介导入野生型p53,B7-1和GM-CSF基因在肺癌细胞中的表达及致调亡效应.方法:以复制缺陷型重组腺病毒为载体,将入野生型P53,B7-1和GM-CSF基因导入肺癌细胞,分别以免疫组织化学法、流式细胞分析法和ELJSA法检测了外源基因在肺癌细胞中的表达,通过苔盼蓝染色后计数活细胞数绘制细胞生长曲线,末端标记法检出细胞凋亡.结果:观察到重组腺病毒分导的多种外源基因均可在肺癌细胞中高效表达,并可明显抑制肺癌细胞增殖并诱导其凋亡.结论:以上工作为进一步开展肺癌的多基因治疗打下了基础.     

Analysis of T-cell immune response in renal cell carcinoma: Polarization to type 1-like differentiation pattern,clonal T-cell expansion and tumor-specific cytotoxicity

We assessed the naturally occurring T-cell immune response in primary renal cell carcinoma (RCC) tumors from 12 unselected patients. A predominance of CD3⁺ T-cell receptor (TCR)α/β⁺ T cells was observed in tumor-infiltrating lymphocytes (TILs), in contrast with peripheral blood lymphopenia found in some patients. Activation antigen expression on TILs revealed an imbalance in the activation status, with a significant percentage of CD69⁺ and HLA-DR⁺ and a low percentage of CD25⁺ and CD71⁺ TILs. The lymphocyte activation gene-3 (LAG-3) was detected in some TIL subpopulations and especially in one patient in whom TILs were predominantly TCRα/β⁺CD8⁺DR⁺LAG-3⁺. In addition, we found that RCC TILs are polarized to a global type 1-like (Th1/Tc1) differentiation pattern (strong secretion of interferon-γ and interleukin-2 (IL-2) following CD3/TCR cross-linking) but are under the influence of the down-modulatory cytokines IL-6 (secreted by tumor cells) and IL-10, within the tumor microenvironment. In 3 of 5 patients, clonal T-cell expansion at the tumor site was found for several Vβ specificities, suggesting that in situ stimulation of specific clonotypes in response to potential tumor antigens is a frequent event in RCC. Furthermore, in one patient, selective intratumor amplification of a Vβ1 subpopulation (5% of TCR α/β⁺ cells) corresponding to 2 distinct Vβ1-Jβ1.6 and Vβ1-Jβ2.3 tumor-specific MHC class I-restricted cytotoxic T lymphocytes supports the view that discrete T-cell subsets contribute readily to in situ immunosurveillance. Int. J. Cancer 72:431–440, 1997. © 1997 Wiley-Liss, Inc.     

Residue substitution enhances the immunogenicity of neoepitopes from gastric cancers

Objective:Neoantigens arising from gene mutations in tumors can induce specific immune responses, and neoantigen-based immunotherapies have been tested in clinical trials. Here, we characterized the efficacy of altered neoepitopes in improving immunogenicity against gastric cancer.Methods:Raw data of whole-exome sequencing derived from a patient with gastric cancer were analyzed using bioinformatics methods to identify neoepitopes. Neoepitopes were modified by P1Y (the first amino acid was replaced by tyrosine) and P2L (the second amino acid was replaced by leucine). T2 binding and stability assays were used to detect the affinities between the neoepitopes and the HLA molecules, as well as the stabilities of complexes. Dendritic cells (DCs) presented with neoepitopes stimulated naïve CD8⁺ T cells to induce specific cytotoxic T lymphocytes. ELISA and carboxyfluorescein succinimidyl ester were used to detect IFN-γ and TNF-α levels, and T cell proliferation. Perforin was detected by flow cytometry. The cytotoxicity of T cells was determined using the lactate dehydrogenase assay.Results:Bioinformatics analysis, T2 binding, and stability assays indicated that residue substitution increased the affinity between neoepitopes and HLA molecules, as well as the stabilities of complexes. DCs presented with altered neoepitopes stimulated CD8⁺T cells to release more IFN-γ and had a greater effect on promoting proliferation than wild-type neoepitopes. CD8⁺T cells stimulated with altered neoepitopes killed more wild-type neoepitope-pulsed T2 cells than those stimulated with wild-type neoepitopes, by secreting more IFN-γ, TNF-α, and perforin.Conclusions:Altered neoepitopes exhibited greater immunogenicity than wild-type neoepitopes. Residue substitution could be used as a new strategy for immunotherapy to target neoantigens.     

腺病毒介导的HBsAg基因修饰树突状细胞瘤苗对HepG2.2.15肝癌细胞的免疫效应

目的研究腺病毒介导的HBsAg基因修饰树突状细胞(DC)体外诱导对HepG2.2.15肝癌细胞特异性的细胞毒效应。方法将HBsAg基因亚克隆到pIND和pShuttle载体中,构建重组载体pShuttle-S。将用PI-SceⅠ和Ⅰ-CeuⅠ双酶切后所获得的HBsAg基因片段与线性化的腺病毒载体pAdeno-X连接,构成腺病毒表达载体AdVHBsAg。经HEK293细胞包装腺病毒后,收获病毒并做滴度测定,再将AdVHBsAg转染人单核细胞来源的DC构建AdVHBsAg-DC肝癌瘤苗。采用Western blot法鉴定转染基因表达;流式细胞仪检测表面分子;3H-胸腺嘧啶核苷(3H-TdR)法检测T细胞增殖反应的能力;乳酸脱氢酶(LDH)法检测CTL活性。结果穿梭质粒插入片段为HBsAg基因;包装的腺病毒载体具有良好的感染性,可在HEK293细胞中形成病毒颗粒;HBsAg基因表达于AdVHBsAg- DC中,表明腺病毒介导的HBsAg基因转染的有效性;AdVHBsAg-DC可高表达CD1a、CD11c、CD80、CD86和HLA-DR;AdVHBsAg-DC刺激自体T细胞增殖功能明显高于AdVLacZ-DC组和未转染的DC组(P<0.05);AdVHBsAg-DC体外诱导CTL能够对表达HBsAg肝癌细胞起到特异性杀伤作用。结论AdVHBsAg可在DC中表达HBsAg肝癌相关抗原;AdVHBsAg-DC瘤苗可诱导T细胞产生高效的针对HepG2.2.15肝癌细胞的细胞毒效应。     

CONSTRUCTION AND EXPRESSION OF THE REPLICATION-DEFI-CIENT ADENONIRUS VECTOR OF HUMAN GM-CSF

ＣＯＮＳＴＲＵＣＴＩＯＮＡＮＤＥＸＰＲＥＳＳＩＯＮＯＦＴＨＥＲＥＰＬＩＣＡＴＩＯＮＤＥＦＩＣＩＥＮＴＡＤＥＮＯＮＩＲＵＳＶＥＣＴＯＲＯＦＨＵＭＡＮＧＭＣＳＦＺｈａｎｇＷｅｉｐｉｎｇ章卫平ＣａｏＸｕｅｔａ曹雪涛ＴａｏＱｕｎ陶群Ｈｉｒｏｆｕｍ...     

Melanoma-specific CD4+ T lymphocytes recognize human melanoma antigens processed and presented by epstein-barr virus-transformed B cells

While much emphasis has been placed on the role of MHC class I-restricted CDS⁺ T cells in the recognition of tumor-specific antigens (Ag), evidence has accumulated that CD4⁺ T cells also play a critical role in the anti-tumor immune response. However, little information exists on the nature of MHC class II-restricted human tumor Ag. In an attempt to develop in vitro systems to characterize such Ag, we examined the ability of Epstein-Barr virus(EBV) transformed B cells to present melanoma-associated Ag to melanoma-specific CD4⁺ cells. CD4⁺ T cells cultured from lymphocytes infiltrating a s.c. melanoma metastasis secreted TNF-α and GM-CSF specifically in response to autologous cultured melanoma cells expressing MHC class II molecules. These CD4⁺ cells also recognized MHC class II-compatible EBV-B cells pulsed with extracts of autologous melanoma cells, but failed to recognize EBV-B cells pulsed with autologous non-transformed cells or a variety of allogeneic tumors or normal cells. B cells pre-fixed with paraformaldehyde were incapable of Ag presentation, suggesting that intracellular processing events were occurring. Antibody-blocking studies defined HLA-DR as the dominant if not exclusive restriction locus in this T-B interaction, and HLA-DR genocyping revealed DRBI 0404 to be the probable restriction element. In a second patient, a CD4⁺ T-cell clone cultured from a melanoma lesion recognized autologous tumor Ag presented by autologous EBV-B; no cross-reactivity was observed with the other tumor system investigated, nor with autologous CD4⁺ T cells specific for tetanus toxoid. These findings demonstrate that tumor Ag can be processed and presented by EBV-transformed B cells to MHC class II-restricted tumor-specific CD4⁺ T cells. They also provide a model system for direct identification of these tumor-derived antigens. © 1994 Wiley-Liss, Inc.     

T-cell co-stimulation by the CD28 ligand B7 is involved in the immune response leading to rejection of a spontaneously regressive tumor

Cell variants from experimental tumors may lose their tumorigenicity or give rise to tumors that regress after a short period of progression in immunocompetent syngeneic animals. Rejection of these tumor cells is often T-cell-dependent. It has recently been reported that, besides the specific signal delivered through the clonogenic receptor, T-cell activation requires a co-stimulatory signal, delivered through its CD28 receptor by B7-1 and/or B7-2 molecules expressed at the surface of the antigen-presenting cells. CTLA4Ig, a fusion molecule that specifically inhibits B7-1 and B7-2 binding to their receptor on T cells, was used to investigate the role of B7 in the spontaneous regression of the tumors induced in syngeneic rats by REGb cells, a regressor cell line established from a chemically induced colon carcinoma. When rats received either 1 or 3 CTLA4Ig injections, REGb tumors grew 3 or 7 times larger than in control animals, respectively. However, in most animals, single or repeated CTLA4Ig injections delayed rather than suppressed REGb tumor rejection. Antibodies to CTLA4Ig appeared in treated rats and could explain this transient effect. Neither REGb cells nor freshly isolated MHC class-11⁺ antigen-presenting cells infiltrating REGb tumors expressed B7, establishing that the target of CTLA4Ig was not located inside the tumor. In contrast, MHC class-II⁺ B7⁺ accessory cells were found in the REGb tumor-draining lymph nodes, suggesting that lymphoid tissue, rather than the tumor itself, was the site of tumor-antigen presentation to tumor-specific T cells. These results establish the role of the B7/CD28 co-stimulation pathway in the control of a spontaneously regressive tumor. © 1996 Wiley-Liss, Inc.