Mouse Homologue of the Human SART3 Gene Encoding Tumor-rejection Antigen

We recently isolated a human SART3 ( hSART3 ) gene encoding a tumor-rejection antigen recognized by HLA-A2402-restricted cytotoxic T lymphocytes (CTLs). The hSART3 was also found to exist as an RNA-binding nuclear protein of unknown biological function. In this study, we cloned and analyzed the homologous mouse SART3 ( mSART3 ) gene in order to understand better the function of hSART3, and to aid in establishing animal models of specific immunotherapy. The cloned 3586-bp cDNA encoded a 962-amino acid polypeptide with high homology to hSART3 (80% or 86% identity at the nucleotide or protein level, respectively). Nonapeptides recognized by the HLA-A2402-restricted CTLs and all of the RNA-binding motifs were conserved between hSART3 and mSART3. The mSART3 mRNA was ubiquitously expressed in normal tissues, with low level expression in the liver, heart, and skeletal muscle. It was widely expressed in various organs from as early as day 7 of gestation. mSART3 was mapped to chromosome 5, a syntenic region for human chromosome 12q23–24, and its genomic DNA extended over 28-kb and consisted of 19 exons. This information should be important for studies of the biological functions of the SART3 protein and for the establishment of animal models of specific cancer immunotherapy.     

Expression of Tumor-rejection Antigens in Gynecologic Cancers

We recently reported the four tumor-rejection antigens (SART1₂₅₉ SART2, SART3, and ART4) that possess tumor epitopes capable of inducing HLA-A2402-restricted cytotoxic T lymphocytes (CTLs) in cancer patients. This study investigated the expression of these tumor antigens in gynecologic cancers, including 33 ovarian cancers, 38 cervical cancers, and 40 endometrial cancers. SART1₂₅₉ antigen was detected in 56%, 35%, and 30% of ovarian, cervical and endometrial cancers, while SART2 antigen was detected in 46%, 66%, and 30% of these cancers, respectively. Both SART3 and ART4 antigens were detectable in the majority of these gynecologic cancers tested. In contrast, none of these antigens was detectable in any of the normal ovarian and uterine tissues tested. Peripheral blood mononuclear cells (PBMCs) of HLA-A24⁺ patients with gynecologic cancers were found to produce significant levels of interferon-γ in response to HLA-A24⁺ SART3⁺ gynecologic cancer cells after having been stimulated three times in vitro with either SART3_109–118 or SART3_315–323 peptide. These PBMCs lysed HLA-A24⁺ SART3⁺ gynecologic cancer cells, but not HLA-A24^- SART3⁺ gynecologic cancer cells or HLA-A24⁺ normal cells. Therefore, these four antigens and their peptides, including SART3 peptides, would be appropriate molecules for use in specific immunotherapy of HLA-A24⁺ gynecologic cancer patients.     

Expression of the SART-1 Antigens in Uterine Cancers

We recently reported that the SART-1 gene, encoding the SART-1₂₅₉ tumor antigen which is recognized by HLA-A26-restricted cytotoxic T lymphocytes (CTLs), is expressed in the cytosol of squamous cell carcinomas and adenocarcinomas. The present study deals with the expression of SART-1₂₅₉ and SART-1₈₀₀ antigens in uterine cancers. The SART-1₂₅₉ antigen was detected in the cytosol fraction of 4 of 8 uterine cancer cell lines, 24 of 74 (32%) uterine cancer tissues, 0 of 7 uterine myomas, and 0 of 5 non-tumorous uterine tissues. The SART-1₈₀₀ antigen was expressed in the nuclear fraction of all the uterine cancer cell lines, 41 of 74 (55%) uterine cancer tissues, 0 of 7 myomas, and 3 of 5 non-tumorous uterine tissues. The SART-1₂₅₉⁺ uterine cancer cells were recognized by HLA-A24 restricted and SART-1 specific CTLs. Therefore, SART-1₂₅₉ antigen could be an appropriate vaccine candidate for a relatively large number of uterine cancer patients.     

Production of Murine Leukemia RL♂1 Rejection Antigen Peptide pRL1a by Proteolysis of Natural Precursor pRL1b

In this study, we demonstrated that NH₂-terminal Ser and Ile residues of pRL1b (SI-pRL1a) (SIIPGLPLSL) are not involved in the recognition by RL♂1-specific cytotoxic T lymphocyte. The sensitization activity observed with pRL1b (SI-pRL1a) was not greater than that of peptides substituted with irrelevant amino acids at these positions. In serum-free medium, pRL1a retained sensitization activity, but pRL1b (SI-pRL1a) did not. Furthermore, addition of bestatin to serumcontaining medium blocked sensitization by pRL1b (SI-pRL1a). On the other hand, the addition of captopril enhanced it, probably by inhibiting the degradation of pRL1a by ACE. pRL1a-D peptide with D-Ile in place of the L-Ile residue of pRL1a (IPGLPLSL) showed sensitization, but SI-pRL1a-2,3D peptide, which has D-Iles in place of the L-Ile residues of pRL1b (SI-pRL1a), and which was not cleaved between the two D-Iles, did not. The findings suggest that pRL1a is the antigenic peptide bound to L^d molecules and pRL1b (SI-pRL1a) peptide is its natural precursor, which generates pRL1a via proteolysis.     

Separation of the Tumor Rejection Antigen of Rous Sarcoma Virus-induced Murine Fibrosarcoma

The tumor antigen capable of inducing tumor resistance (tumor rejection antigen; TRA) was separated and some of its physicochemical properties were characterized. Cytosol and plasma membrane fractions were separated from Rous sarcoma virus (RSV)-induced CSA1M tumor cells. Immunization with membrane but not cytosol fraction of these tumor cells together with complete Freund's adjuvant resulted in complete protection against subsequent challenge with viable CSA1M cells. The TRA activity contained in the membrane fraction was recovered in the sodium dodecyl sulfate (SDS)-solubilized fraction after the SDS-extraction of CSA1M membranes. This CSA1M SDS-solubilized preparation gave protection against syngeneic RSV-induced CSA9F tumor cells as well as the homologous tumor cell type, but failed to induce resistance to RSV-unrelated tumor cells. The membrane or SDS-solubilized fraction from RSV-unrelated tumor cells was unable to generate anti-CSA1M protective immunity. Physicochemical analyses have demonstrated that TRA activity in the SDS-solubilized fraction was completely abolished by treatment with proteinase K but was only marginally affected after treatment with glycosidase mixture. When the SDS-solubilized preparation was applied to a Sephacryl S-300 superfine column, TRA activity was recovered in the range of molecular weight of 50-90 kD. Further fractionation of this TRA-positive fraction by SDS-polyacrylamide gel electrophoresis revealed that the molecular size of TRA is 56–68 kD. These results indicate that membrane proteins which were isolated from CSA1M tumor cells and have a molecular size of about 60 kD are capable of inducing RSV-induced tumor-specific in vivo protective immunity.     

Inhibition of B6RV2 Leukemia Growth by Immunization with Purified Unique Antigen

Monoclonal antibody (mAb) NU7-99 reacted with only B6RV2 cells, not with 28 other leukemia cell lines, fibroblasts or normal tissues. Biochemical analyses of the unique antigen on B6RV2 cells that reacted with NU7–99 mAb indicated its relationship to xenotropic murine leukemia virus gp70. The antigen that reacted with NU7-99 mAb was extracted from the surface of B6RV2 cells with n-butanol and purified by ion-exchange chromatography and affinity chromatography. Growth of B6RV2 tumors in semi-syngeneic mice was inhibited by immunization of the mice with a purified preparation of this unique antigen.     

白血病细胞可溶性蛋白抗原诱导抗白血病作用

目的探讨白血病细胞可溶性蛋白抗原诱导的抗急性白血病的特异性CTL作用。方法采用急性粒细胞白血病细胞可溶性蛋白抗原致敏体外培养的DC,并通过MTT法测定CTL活性。结果利用反复冻融法可获取可溶性蛋白抗原;在效应细胞:靶细胞为20∶1时,可溶性蛋白抗原组CTL活性达(38.83%±5.42%),不同效靶比对CTL活性无明显影响。结论可溶性蛋白抗原可诱导产生一定程度的抗白血病特异性CTL,为急性白血病微小残留病免疫治疗提供了1种有临床应用前景的方法。     

Cytokines Required for Induction of Histocompatibility Leukocyte Antigen-class I-restricted and Tumor-specific Cytotoxic T Lymphocytes by a SART1-derived Peptide

Although there have been several reports on peptides of human tumor-rejection antigens capable of inducing histocompatibility leukocyte antigen (HLA)-class I-restricted and tumor-specific cytotoxic T lymphocytes (CTLs), it is not yet clear which cytokines are required for CTL induction. This study has investigated the cytokine combinations required for optimal induction of CTLs by SARTI^690–698 peptide, which is capable of inducing HLA-A24-restricted and tumor-specific CTLs in peripheral blood mononuclear cells (PBMCs). Pretreatment of PBMCs as a source of antigen-presenting cells (APCs) with interferon (IFN)-γ, or to some extent with IFN-α, but not with any of the other cytokines tested, augmented the peptide-induced CTL activity in HLA-A24 heterozygotes, but not in HLA-A24 homozygotes. This IFN-γ -mediated augmentation was inhibited by either interleukin (IL)-4 or IL-10. IL-2 alone in culture, along with weekly stimulation by peptide-pulsed APCs, was sufficient for the differentiation and proliferation of CTLs for the initial several weeks of culture. This IL-2-mediated activation of CTLs was inhibited by the addition of IFN-γ, IL-4, or IL-10 to the IL-2 culture. For further expansion of the CTLs, dendritic cells (DCs) induced from PBMCs with IL-4 and granulocyte macrophage colony-stimulating factor (GM-CSF) were required as APCs. These results indicate that IFN-γ and IL-2 are important in the activation of APCs and CTLs, respectively, while GM-CSF and IL-4 are needed for the induction of DCs, which in turn are required for further expansion of mature CTLs. These results are important in allowing for a better understanding of the cellular and molecular basis of tumor-specific immunity, and also for the development of peptide-based specific immunotherapy.     

The Expression of Tumor Rejection Antigen on Rat Fetus Fibroblasts Transformed by the ras Oncogene

The expression of tumor rejection antigens (TRA) was analyzed on clones of rat fetus-derived fibroblasts, WFB, transformed or transfected by oncogenes. It was shown that a tumorigenic W14 clone, which is an activated H- ras transformant of parental WFB, expressed TRA in transplantation experiments using syngeneic WKA rats. The data also showed that W14 TRA was acquired in the event of cell transformation, since it was not detected on parental non-transformed WFB cells or Wmyc-4 clone which is a transfectant of WFB by mouse plasmacytoma-derived c- myc DNA. However, TRA was not expressed or at least was not detected on highly tumorigenic W31, another clone of H- ras transformants of parental WFB, in the transplantation experiments. We also assessed the level of expression of major histocompatibility antigens (MHC) class I molecules on these cells by using R4-8B1 Mab that reacts specifically with rat class I antigen. The data indicated that it was decreased on W14, W31, and Wmyc-4, but not on parental WFB. Although this molecule was weakly positive on W14 cells, W31 and Wmyc-4 showed even greater decreases. These data may indicate that the TRA expression and its recognition by syngeneic hosts are dependent upon the transformed clones, although their parental cell is the same. We discuss in detail this difference of expression and recognition of TRA in the context of the cell transforming process.     

卵巢癌细胞系SKOV3冻融抗原诱导CTL特异性杀瘤效应的研究

目的探讨卵巢癌冻融抗原对T淋巴细胞的增殖作用及其诱导的细胞毒性T淋巴细胞(CTL)在体外杀伤卵巢癌细胞的抗原特异性细胞毒性效应。方法采用免疫磁珠分离法(magnetic activated cell sorting，MACS)分离纯化正常人外周血CD3^ 细胞，体外以SKOV3卵巢癌细胞中提取的可溶性肿瘤抗原加以刺激。溴标法检测肿瘤抗原对T细胞增殖的影响；MTT法测定肿瘤抗原诱导的CTL体外杀伤卵巢癌细胞的能力；利用绒毛膜癌细胞抗原对比观察肿瘤抗原诱导CTL杀伤肿瘤细胞的抗原特异性。结果卵巢癌细胞抗原有很强的促进T淋巴细胞增殖的作用，且存在时间依赖性，在24h时促进作用最强。SKOV3抗原诱导的CTL在体外对SKOV3细胞的杀伤率为68．38％，显著高于它对JAR绒毛膜癌细胞的杀伤率(18．31％)；而JAR细胞抗原诱导的CTL对JAR和SKOV3细胞的杀伤率分别为61．02％和14．79％，有显著的抗原特异性(P=0．0001)。结论卵巢癌细胞冻融抗原诱导的CTL在体外具有很强的增殖能力和抗原特异性杀伤卵巢癌细胞的作用。     

The prognostic benefit of tumour-infiltrating Natural Killer cells in endometrial cancer is dependent on concurrent overexpression of Human Leucocyte Antigen-E in the tumour microenvironment

BackgroundHuman Leucocyte Antigen- E (HLA-E) has been reported as both a positive and negative prognostic marker in cancer. This apparent discrepancy may be due to opposing actions of HLA-E on tumour-infiltrating immune cells. Therefore, we evaluated HLA-E expression and survival in relation to the presence of intratumoural natural killer (NK) cells and cytotoxic T cells (CTLs).MethodsTissue microarrays (TMAs) of endometrial tumours were used for immunohistochemical staining of parameters of interest. The combined impact of clinical, pathological and immune parameters on survival was analysed using log rank testing and Cox regression analyses.ResultsUpregulation of HLA-E was associated with an improved disease-free and disease-specific survival in univariate analysis (HR 0.58 95% CI 0.37–0.89; HR 0.42 95% CI 0.25–0.73, respectively). In multivariate analysis, the presence of NK cells predicts survival with a hazard ratio (HR) 0.28 (95% confidence interval (CI) 0.09–0.91) when HLA-E expression is upregulated; but it is associated with a worse prognosis when HLA-E expression is normal (HR 13.43, 95% CI 1.70–106.14). By contrast, the prognostic benefit of T cells was not modulated by HLA-E expression.ConclusionsTaken together, we demonstrate that the prognostic benefit of NK cells, but not T-cells, is influenced by HLA-E expression in endometrial cancer (EC) and propose a model to explain our observations.     

T-cell receptor Vbeta gene usage by T cells reactive with the tumor-rejection antigen SART-1 in oral squamous cell carcinoma

We recently described that the SART-1(690-698) peptide could induce HLA-A24-restricted cytotoxic T lymphocytes (CTLs), which recognize the SART-1(259) (+) tumor cells from peripheral blood mononuclear cells (PBMCs) of HLA-A24(+) cancer patients. In our study, in 5 of 14 HLA-A24(+) patients with oral squamous cell carcinomas (SCCs), CTLs could be induced with the SART-1(690-698) peptide from the PBMCs. In 2 of the patients from whom the highest CTL activities were induced, the T-cell receptor (TCR) Vbeta repertoire expressed by the SART-1(690-698)-specific CTLs was found to be restricted and multiple Vbeta families were predominantly expressed in each patient. Although the predominant Vbeta families were different between the 2 patients, Vbeta7 was highly and commonly predominant. The same predominant Vbeta families were also detected in the tumor-infiltrating lymphocytes (TILs) from each patient, and each Vbeta family contained one or more unique T-cell clonotypes. The unique T-cell clonotypes were found to be common between the TILs and SART-1(690-698)-specific CTLs from each patient, and especially 2 T-cell clonotypes with Vbeta7 were identical even in the 2 patients. One of the 2 T-cell clonotypes with Vbeta7 was detected in the TILs from 11 of 14 HLA-A24(+) patients and another was found in those from 8 of HLA-A24(+) patients, while none of 10 HLA-A24(-) patients demonstrated both T-cell clonotypes. These results strongly suggest that the T-cell clonotypes with Vbeta7 are major TCR Vbeta genes expressed by SART-1(690-698)-specific CTLs. Furthermore, autologous tumor cells from one of the HLA-A24(+) patients stimulated the PBMCs and regional lymph node cells (LNCs) to expand the same T-cell clonotypes as those in the SART-1(690-698)-specific CTLs. These results strongly suggest that the SART-1(690-698)-specific CTLs clearly accumulate in vivo, especially in the TILs, as a consequence of in situ antigenic stimulation by autologous tumor cells. The identification of the unique TCR Vbeta genes used by SART-1(259)-specific CTLs should help to improve the diagnosis of the specific immune response in patients with SART-1(259) (+) cancers, especially during anticancer immunotherapy.     

The Tumor Rejection Antigen Separated from Rous Sarcoma Virus-induced Murine Fibrosarcoma Exhibits a Molecular Weight of Approximately 60 kD but Differs from Functional pp60src

The tumor antigen capable of inducing tumor resistance (tumor rejection antigen; TRA) was obtained in a solubilized form by sodium dodecyl sulfate (SDS) extraction of plasma membrane fraction from Rous sarcoma virus (RSV)-induced CSA1M fibrosarcoma cells (BALB/c origin). Analyses by Sephacryl S-300 gel filtration and SDS-polyacrylamide gel electrophoresis revealed that TRA activity was recovered in the fraction with a molecular weight of approximately 60 kD. Unfractionated crude SDS-solubilized preparation contained gp70 as detected by rabbit anti-gp70 antiserum, whereas such reactivity was lost in the fraction exhibiting the molecular weight of about 60 kD. Since this fraction retained pp60 ^src activity, the relation of TRA to pp60 ^src was further investigated. pp60^{v- src} was also obtained from the lysate of v-src-expressing yeast transformant. Immunization of BALB/c mice with such pp60^{v- src} -containing lysate failed to induce any significant tumor protection. The above 60 kD fraction of CSA1M solubilized antigens was allowed to bind to Sepharose beads coupled with anti-pp60 ^src monoclonal antibody and separated into the bead-bound and bead-unbound fractions. The bead-bound fraction that was recovered from pp60 ^src -binding beads (pp60 ^src -positive fraction) did not exhibit the TRA activity. In contrast, immunization with the fraction depleted of pp60 ^src activity (bead-unbound fraction) resulted in potent tumor protection. These results indicate that the solubilized membranous component(s) of CSA1M with a molecular weight of approximately 60 kD, which is distinct from functional pp60 ^src , functions as the TRA against RSV-induced CSA1M tumor cells.     

Mutation Analysis of the WT1 Gene in Myelodysplastic Syndromes

The WT1 tumor suppressor gene was examined for mutations in a panel of 44 patients with myelo-dysplastic syndromes (MDS) including acute myelogenous leukemias (AML) secondary to MDS, using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis and sequencing analysis. A WT1 mutation was detected in one out of 17 cases of AML secondary to MDS. This mutation exists upstream of the zinc finger region and is predicted to produce a truncated WT1 protein lacking the zinc finger region. No mutations were detected in 27 MDS patients who had not progressed to AML. This is the first report of analysis for WT1 mutations in a large number of MDS patients, suggesting that WT1 mutations are uncommon in MDS. Abnormalities in this gene may, however, contribute to a small proportion of cases showing progression from MDS into AML.     

What Is the Role of Cytotoxic T Lymphocyte–Associated Antigen 4 Blockade in Patients with Metastatic Melanoma?

With increasing knowledge of the molecular basis of the immune system and mechanisms of tumor tolerance, novel approaches to treating malignant diseases refractory to standard therapies are being investigated. Monoclonal antibodies (mAbs) that bind cytotoxic T lymphocyte–associated antigen (CTLA)‐4 can block inhibitory signals normally generated through this receptor, thus prolonging and sustaining T‐cell activation and proliferation. These antibodies are being developed and tested in patients with metastatic melanoma. This article reviews data published or presented at scientific congresses describing the clinical safety and antitumor activity of two different anti–CTLA‐4 mAbs: tremelimumab (CP‐675,206) and ipilimumab (MDX‐010). Overall, although the response rate has not been consistently higher than the response rates associated with other treatments, the induction of durable responses and the favorable safety profile observed with anti–CTLA‐4 mAbs are encouraging. However, the true advantage of these new drugs may depend largely on the characterization of predictive biomarkers of activity and subsequent targeting of responsive patients.     

Tumor-specific T Cell Lines: Capacity to Proliferate and Produce Interleukin 2 in Response to Various Forms of Tumor Antigens

Anti-tumor proliferative T cell lines were established from cultures of lymph node cells from BALB/c mice immunized to syngeneic CSAIM fibrosarcoma with the CSAIM tumor cell membrane. The cultures were maintained throughout in the absence of exogenous interleukin 2 (IL2) J. Cell surface phenotypes of all T cell lines established were Thy-1⁺, Ig^-, L3T4⁺ and Lyt-2^-. Their proliferation was induced in a tumor antigen dose-dependent fashion and a tumor antigen-specific way. Such proliferative responses were inhibited by the addition to cultures of anti-class II H-2^d (anti-I-A^d) or anti-L3T4 but not of anti-class I H-2^d or anti-Lyt-2 monoclonal antibody. None of the T cell lines exhibited any cytotoxic T lymphocyte activity but they all produced IL2 upon stimulation with CSAIM tumor antigens, indicating that they represent helper-type T cell (Th) lines. The activation of these tumor-specific Th lines was induced with either CSAIM tumor cells themselves, or their membrane or detergent-solubilized fraction depending on the presence of antigen-presenting cells (APC). Most importantly, activation was also inducible by membranous tumor antigen-pulsed APC, which were capable of producing potent anti-tumor protective immunity when administered in vivo into syngeneic BALB/c mice. These results indicate that the tumor-specific Th lines established here can be activated with various forms of tumor antigens for their expression of helper function. Since Th lines of this type have not been described previously, our Th lines provide an intriguing tool for investigating the cellular and molecular mechanisms by which tumor-specific Th recognize tumor antigens.     

PD-1、CTLA-4、BLTA在食管鳞状细胞癌患者外周血中的表达及临床意义

目的 检测食管鳞癌患者外周血中抑制性协同刺激因子受体PD-1、CTLA-4、BLTA表达情况,并分析其临床意义。方法 选取2016年6月—2017年4月河北医科大学第四医院胸外科90例食管鳞状细胞癌患者(其中50例患者行手术治疗)和40例健康对照者为研究对象,收集其外周血液标本,采用酶联免疫吸附方法检测血清中可溶性PD-1(sPD-1)、可溶性CTLA-4(sCTLA-4)及可溶性BLTA(sBLTA)的表达水平。结果 食管鳞癌组血清中sPD-1、sCTLA-4及sBLTA水平均明显高于对照组(P＜0.05);食管鳞癌组手术前后血清中sPD-1、sCTLA-4及sBLTA水平均无统计学差异(P＞0.05)。sPD-1和sBLTA的表达水平与临床病理特征无相关性,sCTLA-4的表达水平与TNM分期相关(P＜0.05),与T分期、N分期、肿瘤体积大小、肿瘤部位、组织分化程度、性别、年龄无相关性;血清中sPD-1、sCTLA-4及sBLTA两两间无相关性(P＞0.05)。结论 食管鳞癌患者血清中sCTLA-4较正常人表达升高,且sCTLA-4的表达水平与TNM分期相关,说明血清中sCTLA-4表达水平与病情发展变化有一定相关性。     

Analysis of Oncogenes and Tumor Suppressor Genes in Human Breast Cancer

Oncogenes (c- erb B-2, c- myc , and some genes linked to the 11q13 lesion), tumor suppressor genes (retinoblastoma gene, p53) and an antimetastatic gene ( nm 23/nucleoside diphosphate kinase) play important roles in breast cancer progression. Amplification of c- erb B-2, c- myc , and int -2, and expression of RB, p53 (mutant), and NDP kinase were determined in 77 primary breast cancer specimens. nm 23-H1 allelic loss was also studied. c- erb B-2 and c- myc amplification, loss of RB expression, p53(mutant) expression, and nm 23-H1 allelic loss were also found in non-invasive carcinoma, int -2 amplification was significantly correlated with lymph node status ( P =0.02) and a significant association was found between p53(mutant) expression and tumor size ( P =0.04). c- erb B-2 amplification was strongly associated with disease-free and overall survival in multivariate analysis ( P =0.002). All of the c- erb B-2 amplified cases and all but one of the int -2 amplified cases in node-positive patients had relapsed within 2 years post resection. The cancer cells may acquire new proliferative pathways sequentially as a result of multiple genetic alterations which enable them to bypass the estrogendependent proliferation.