Chimeric antigen receptor T (CAR-T) cell has achieved excellent efficacy in hematological tumors, especially for lymphoma. Many products have been approved to market all over the world, and 2 products targeting CD19 have been approved to treat relapsed and refractory large B-cell lymphoma in China. The current experiences of using CAR-T cells come from previous clinical studies. How to use CAR-T cells in a standardized and rationalized way is still a challenge faced by our clinicians. Based on the CAR-T cell treatment experiences from Peking University Cancer Hospital and the latest research progresses in CAR-T in China and abroad, this article will elaborate on patient screening, peripheral blood mononuclear cell collection, bridging treatment, lymphocyte depletion chemotherapy, CAR-T cell infusion, the monitoring and treatment of adverse events after infusion, and long-term follow-up after infusion, in order to guide clinicians to better use CAR-T cell and to bring maximum benefits to patients.

Objective:To assess the efficacy and toxicity of chemoradiotherapy in the treatment of early stage extranodal nasal-type NK/T-cell lymphoma (ENKTCL).Methods:Retrospective review was conducted for 174 patients with pathological proved early stage ENKTCL who were treated in the Department of Radiation Oncology, Peking University Cancer Hospital & Institute. The Kaplan-Meier survival analysis was adopted to calculate the local-regional control (LRC), overall survival (OS), and progression free survival (PFS), and the Log-rank test COX regression model were applied to univariate and multivariate analyses.Results:The patients in this study included 102 and 72 patients diagnosed with Ann Arbor stage-Ⅰ and stage-Ⅱ, respectively. Among them, two patients received radiotherapy alone and 172 patients were treated with combined chemoradiotherapy. The overall response rate of all the patients was 94.2%, with a complete response (CR) rate of 87.9% (153). Furthermore, the rates of 5-year OS, PFS, and LRC were 87.3%, 83.1%, and 91.9%, respectively. The most common toxicities during the chemotherapy and radiotherapy included myelosuppression and oral mucositis, with grade ≥ 3 myelosuppression and grade ≥ 3 oral mucositis accounting for 62.1% and 10.9% of all patients, respectively. As shown by multivariate analysis, the adverse prognostic factors for OS included age > 60, B symptoms, and stage Ⅱ, while the adverse prognostic factors for PFS included age > 60 and stage Ⅱ. Meanwhile, the PFS rate was significantly improved by increasing the radiation dose (≥ 50 Gy vs.<50 Gy), and the 5-year PFS rates of the two groups were 83.5% and 76.5%, respectively [hazard ratio ( HR) 0.374; 95% CI, 0.169-0.826; P=0.015]. Conclusions:A good therapeutic effect can be achieved for early stage NK/T-cell lymphoma and the toxicities after combined chemoradiotherapy can be tolerated.

Abstract Background The treatment outcomes and prognosis of lymphoma are affected by various factors such as hospital types. This study was to describe the temporal trend in the survival of lymphoma in an academic center in China. Methods A total of 3840 consecutive patients with lymphoma diagnosed between 1996 and 2015 were reviewed. Eighty patients were excluded, and finally, 3760 patients were analyzed in this study. The cohort was divided into four groups according to calendar periods at diagnosis: 1996‐2000, 2001‐2005, 2006‐2010, and 2010‐2015. The overall survival (OS) rates among the four groups were compared. Results The 5‐ and 10‐year OS for the whole cohort were 62% and 52%, respectively. The 5‐year OS of patient with classic Hodgkin lymphoma (cHL), mature B‐cell lymphoma (BCL), and peripheral T‐cell lymphoma (PTCL) were 79%, 63%, and 50%, respectively. Among mature BCL, the 5‐year OS was highest in follicular lymphoma (77.8%), followed by Burkitt lymphoma (76.5%), marginal zone lymphoma (74.1%), diffuse large B‐cell lymphoma (61.5%), small lymphocytic lymphoma/chronic lymphocytic leukemia (55.1%), and mantle cell lymphoma (44.3%). Among PTCL, the 5‐year OS was highest in ALK+anaplastic large cell lymphoma (79.0%), followed by ALK−anaplastic large cell lymphoma (63.1%), natural killer/T‐cell lymphoma (57.7%), angioimmunoblastic T‐cell lymphoma (34.9%, and peripheral T‐cell lymphoma not otherwise specified (27.6%). Significant improvement in the survival of lymphoma was observed, with the 5‐year OS increasing from 48% in 1996‐2000 to 65% in 2011‐2015 (P < .001). The 5‐year OS of patients with cHL, mature BCL, and PTCL changed from 55%, 49%, and 41% in 1996‐2000 to 79%, 65%, and 51% in 2011‐2015, respectively (P values were .014, .002, and .592, respectively). Conclusion The survival of most types of lymphoma such as cHL and mature BCL, rather than PTCL, was improved significantly during the past two decades.

目的:在临床前及临床研究中探讨抗CD19嵌合抗原受体T细胞IM19的安全性.方法:通过静脉血管刺激(新西兰兔)、过敏(豚鼠)和溶血实验检测IM19试剂的安全性;通过急性毒性和成瘤性检查(小鼠)分析IM19对机体的毒性;通过基因测序检测方法分析IM19潜在的致癌性.入组复发难治B细胞淋巴瘤患者接受IM19 CAR-T细胞治疗,观察其安全性数据.结果:IM19对新西兰兔局部注射没有刺激;未使豚鼠产生过敏反应;溶血结果为阴性.IM19对实验动物安全,未见明显毒性,未见潜在的致癌性和成瘤性.3例复发难治B细胞淋巴瘤患者接受IM19 CAR T细胞治疗,无患者发生严重的细胞因子释放综合征或神经毒性,患者仅接受对症支持治疗.结论:临床前及临床研究结果显示,IM19 CAR-T细胞具有较好的安全性,值得进一步扩大临床研究.

目的:评价抗CD19嵌合抗原受体T(chimeric antigen receptor T,CAR-T)细胞IM19在复发难治B细胞血液系统恶性肿瘤中的疗效及安全性.方法:12例复发难治B细胞血液系统恶性肿瘤患者接受IM19治疗,包括6例B细胞非霍奇金淋巴瘤和6例急性B淋巴细胞白血病患者.患者接受3×105～10×105cells·kg-1 CAR-T细胞输注.细胞回输后1和3个月评估疗效,并监测不良反应事件的发生情况,同时检测细胞扩增以及细胞因子释放.结果:12例患者中有1 1例达到完全缓解,患者体内可以检测到IM19扩增,以及白介素-6和白介素-10水平升高.没有患者发生≥3级的细胞因子释放综合征和CAR-T细胞相关的神经系统毒性.结论:IM19治疗复发难治B细胞血液系统恶性肿瘤安全有效.

Introduction: Patients with relapsed or refractory (R/R) B-cell non-Hodgkin lymphoma (NHL) have poor outcomes with currently available strategies. Based on the promising results seen in patients treated with anti-CD19 CAR T-cell therapy in R/R B-NHL, we initiated the phase I study evaluating the safety and efficacy in patients with R/R B-NHL (NCT02842138). Autologous T cells were genetically modified to express a chimeric antigen receptor consisting of an anti-CD19-scFv domain with CD3ζ and 4-1BB signaling domains. Methods: Patients received CAR T-cell infusion after a conditioning regimen of cyclophosphamide (250 mg/m2) and fludarabine (25 mg/m2) daily for 3 days. Three CAR T cell doses were tested: 5 × 104, 1 × 105, and 1 × 106 CAR T cells/kg, respectively. Primary endpoints include safety and pharmacokinetics (PK) of CAR T cells. Secondary endpoints include complete and overall response (CR, OR) rates and duration of response (DOR) per International Working Group Criteria (Cheson, J ClinOncol2007). Results: As of March 2017, 14 patients were enrolled, 10 patients underwent response evaluation. Median age of the 10 evaluable patients was 37 years (range 24–68), 4 females and 6 males, 3 follicular lymphoma (FL) and 7 diffuse large B-cell lymphoma (DLBCL). Median number of prior therapies was 2.5 (range 2–7). The first group of three patients (2 FL and 1 DLBCL) received 5 × 104 CAR T cells/kg. Two FL patients achieved partial remission (PR) on day 28, and one of the patients achieved complete remission (CR) 3 months later. The second group of three patients (1 FL and 2 DLBCL) received 1 × 105 CAR T cells/kg. The FL patient achieved PR on day 28. The last group of four patients (all were diagnosed with DLBCL) received 1 × 106 CAR T cells/kg. Three patients achieved PR on day 28 (75%). One patient attained PD on day 28 and died 10 days later due to disease progression. A significant CAR T-cell expansion was detected in this case. On day 26, 26.8% of her PBMC were CAR T cells. Interestingly, PD1 expression was significant in her expanded CAR T cells. Approximately 78.3% of CD8+ CAR T cells and 71.4% of CD4+ CAR T cells expressed PD1 on day 26, as compared to 22% and 42% on day 13, respectively. Increased PD1-expressing CAR T cells were also found in other patients' peripheral blood. No serious adverse event (sAE) was observed in all patients. Two patients experienced mild fever (grade 1–2). In vivo expansion of CAR T cells was detected in all patients between day14 and 28. All responding patients are still in remission at the last follow-up (Range: 2.5–9 months). Conclusions: This study demonstrated early promising activity of anti-CD19 CAR T cells in patients with R/R B-NHL. The toxicities of CRS were mild and manageable. Our study also provided the first clinical evidence that expanded CAR T cells could express PD1. Blocking PD1 pathway may enhance the effector function of CAR T cells and improve the clinical outcomes of CAR T-cell therapy. Keywords: B-cell lymphoma; CD19; immune system.

Objective:To study the clinical features, therapeutic effects, survival time, and prognosis of patents with mantle cell lymphoma (MCL). Methods:Clinical data of 98 MCL patients admitted from January 2005 to December 2013 were retrospectively an-alyzed. Results:The median age was 61 years old, and the male-to-female ratio was 2.9∶1. Among these cases, 85 (86.8%) were in Ann Arbor stageⅢ-Ⅳ, 46 (46.9%) had bone marrow involvement, 25 (25.5%) had digestive tract involvement, and 53 chose R-CHOP as first-line treatment. The expected 3-year overall survival (OS) of these patients was only 61.4%. A total of 14 cases were treated with R-CHOP followed by ASCT. The expected 5-year OS was 92.3%, and the OS of the ASCT group was significantly higher than that of the R-CHOP group (75.5 months vs. 43.6 months, P=0.039). Elevated ESR,>60 years old, increased LDH level, B symptoms, and Ki-67≥25% were poor prognostic factors. Conclusion: Most patients with MCL were elder adults with bone marrow involvement. R-CHOP followed by ASCT had better clinical efficacy than conventional chemotherapy in the treatment of MCL.

Objective: To explore the value of high-dose therapy/autologous hematopoietic stem cell transplantation (HDT/AHSCT) in the treatment of patients with peripheral T-cell lymphoma (PTCL). Methods: The medical records of 50 patients with PTCL who received HDT/AHSCT were retrospectively analyzed. The followed-up was performed. Results: No HDT/AHSCT-related death occurred. The median follow-up time was 13 months (range: 1-136). The 2-year progression-free survival (PFS) and 2-year overall survival (OS) were 59.0% and 65.0%, respectively. Univariate analysis showed that the patients achieving complete remission (CR) before HDT/AHSCT had superior 2-year PFS and 2-year OS as compared with those of the patients not achieving CR (2-year PFS: 72.8% vs 41.9%, P = 0.003; 2-year OS: 88.2% vs 41.9%, P = 0.002). The 2-year PFS was 76.8% for the patients who were sensitive to the first-line treatment (CR1/PR1) as compared with 30.8% for the patients who were sensitive to the second-line treatment (CR2/PR2) (P = 0.001). The 2-year OS for patients achieving CR1/PR1 was also much better than that for patients achieving CR2/PR2 (81.1% vs 46.2%, P = 0.015). Furthermore, Erythrocyte sedimentation rate (ESR) before transplantation was an important factor for 2-year PFS and 2-year OS (P = 0.004, P = 0.018). Serum lactate dehydrogenase (LDH) level before transplantation was another important factor for 2-year PFS (P = 0.044). Multivariate analysis showed that the therapeutic response (achieving CR) before transplantation was an independent factor for 2-year OS [risk ratio: 4.879 (95% confidence interval: 1.583-15.034), P = 0.006]. No independent factors for 2-year PFS were observed. Subgroup analysis revealed that the patients with angioimmunoblastic T-cell lymphoma and advanced natural killer (NK)/T-cell lymphoma who received HDT/AHSCT during first CR may have benefit in survival from HDT/AHSCT. Conclusion: HDT/AHSCT can be used as safe and effective first-line consolidation therapy or salvage therapy in patints with PTCL and partially improve the prognosis. Prospective randomized controlled trials are necessary to confirm the suitable pathologic subtype and the best pre-transplantation status for HDT/AHSCT. DOI:10.3781/j.issn.1000-7431.2014.33.454

Objective:To investigate the prognostic value of 18F-fluoro-deoxyglucose positron emission tomography-computed tomography (18F-FDG PET-CT) and some clinicopathological factors for mantle cell lymphoma (MCL).Methods:Clinical records and follow-up information of 19 untreated patients with MCL admitted in Peking University Cancer Hosipital between February 2009 and November 2012 were reviewed.The impacts of 18F-FDG PET-CT and some clinicopathological factors on progression-free survival(PFS) and overall survival (OS) were evaluated.Results:Seventeen patients had an 18F-FDG PET-CT with an abnormal uptake value [the median maximum standardized uptake value (SUVmax) was 7.4 (range:2.5-1 8.3).Bone marrow (BM) biopsies demonstrated disease in 8 patinets,but only 2 of them had 18F-FDG uptakes.There was no statistically significant relationship between SUVmax and Ki-67 (≥30％ vs ＜ 30％).The median follow-up time was 29 months (range:3-58) with an estimated 3-year PFS of 74％ and an estimated 3-year OS of 71％.Univariate analysis showed that Ki-67 and response were associated with PFS (P =0.000; P =0.004) and OS (P =0.002; P =0.004),but International Prognostic Index (IPI),simplified MCL International Prognostic Index (sMIPI) and initial 18F-FDG PET-CT SUVmax were not associated with PFS and OS (P ＞ 0.05).COX proportional-hazards regression model revealed that Ki-67,IPI,sMIPI,initial 18F-FDG PET-CT SUVmax and response were not associated with PFS and OS (P ＞ 0.05).Conclusion:18F-FDG PET-CT at diagnosis is complementary to conventional evaluations,but BM biopsies remain mandatory.More large-sample clinical trials may be needed to further verify the association of Ki-67 and response with the prognosis of MCL.

Background and purpose:Anthracycline has a very important position in the chemotherapy of lymphoma.But its toxicity,particularly cardiac toxicity had limited its application.This study aimed to analyze the response rate and safety of liposomal doxorubicin in lymphoma patients.Methods:Clinical records of 68 lymphoma patients treated with liposomal doxorubicin-based combined chemo-regimen in Peking University Cancer Hospital from Jan.2006 to Oct.2011 were retrospectively analyzed,including 47 primary treated cases and 21 relapsed cases.SPSS 17.0 software was used to evaluate the efficacy and safety of liposomal?doxorubicin.Results:Among the 68 patients,47 cases were newly diagnosed,42 patients were diagnosed as diffuse large B-cell lymphoma.The overall response rate(ORR) was 73.5%,complete remission(CR) rate was 57.4%,and partial remission(PR) rate was 19.1%.In the primary treatment diffuse large B-cell lymphoma(DLBCL) patients,the ORR was 74.3%,complete remission(CR) rate was 60%.The ORR in patients treated with R-CCOP regimen was 81.5%,CR was 66.7%.Myelosuppression was the major toxicity,in which grade 3 and 4 neutropenia accounted for 55.9%.Thirty-seven(54.4%) patients experienced with abnormal electrocardiogram,3 patients suffered from cardiac dysfunction.Only one patient died of severe pulmonary infection.Conclusion:The application of liposomal doxorubicin in lymphoma patients shows a high efficacy and good safety.

Objective: To evaluate the efficacy and safety of fludarabine-based combination chemotherapy in 20 patients undergoing initial treatment for marginal zone B-cell lymphoma (MZL). Methods: Data of 20 patients with initial treatment of MZL in the study hospital between September 2005 and February 2010, including 16 with extranodal MZL, 3 with nodal MZL and 1 with splenic MZL, were reviewed in this study. A fludarabine-based regimen was administered in all patients. Specifically, 14 patients were treated with a FC regimen (Fludarabine and cyclophosphamide) while 6 were treated with R-FC (Rituximab + FC). On average the patients received 4.3 (1-6) therapeutic cycles. Results: Eighteen of the 20 patients (90 %) achieved complete remission (CR) and the other 2 patients (10 %) achieved partial remission (PR). The total effective rate (CR + PR) was 100 % (20/20). In all 20 patients, the 1- and 2-year overall survival rates were 88 %, the progression-free survival (PFS) rate was 88 % and the disease-free survival rate was 85 %. Myelosuppression and mild GI toxicity were the major side effects. The rates of leucopenia, nausea and vomiting and mild liver injury were 51 % (III+IV 9.3 %), 20%, and 8%, respectively. Six patients (30 %) had thrombocytopenia; the incidence of thrombocytopenia was 66.7% (4/6) in patients over 60 years old and 14.3% (2/14) in patients younger than 60 years old (P = 0.037). Pulmonary fungal infection occurred in 1 patient (5 %). Conclusion: The fludarabine-based chemotherapy regimen has shown a satisfactory therapeutic effect on patients undergoing initial treatment for MZL, and the corresponding side effects can be well tolerated.

The objective of this study was to detect the expression levels of VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 in plasma of newly diagnosed lymphoma patients, and analyze their possible relationships with clinicopathological characteristics and prognosis. The expression levels of VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 in plasma from 86 newly diagnosed lymphoma patients were detected by enzyme-linked immunosorbent assay (ELISA). As a results, the multivariate analysis showed that VEGF-C level in non-Hodgkin's lymphoma patients was low, but high in Hodgkin's lymphoma patients; VEGFR-2 level was higher in patients > 60 years, while VEGF-D level was lower in patients with IPI > 2. The univariate analysis showed that VEGF-D level was lower in patients with IPI > 2, while VEGF-D and VEGF-C levels were higher in patients without B symptoms. Relationship analysis between these factors indicated that the relation of VEGF-D expression level with VEGFR-2 and VEGFR-3 was positive. It is concluded that VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 play important roles in the pathogenesis of lymphoma, and may be used as indicators of prognosis evaluation or even guide for the antiangiogenesis treatment of lymphoma.

Objective:To investigate the expression of RIP3(receptor interaction protein-3) in lymphoma patients pathological tis-sues and the sub-cellular localization,and to investigate the relationship of the expression of RIP3 and malignant degree of lymphoma.Methods: The RIP3 expression in 48 cases of the lymphoma and non-neoplastic lymph node pathological tissues was detected by im-munohistochemmistry,and the sub-cellular localization of RIP3 was observed.Results: 1) There were RIP3 expresses in the lymphoma pathological tissues,which mainly located in nucleus but weekly in cytoplasm.2) The expression of RIP3 had a relationship with malig-nant degrees of lymphoma.Conclusion: There was RIP3 expression in lymphoma pathological tissues,which shell new light to the study of mechanism of lymphoma.

目的:分析伯基特淋巴瘤(BL)和伯基特样淋巴瘤(BLL)的临床特点,总结疗效,探讨可能的最佳方案和治疗相关合并症.方法:回顾性分析北京肿瘤医院1996年8月至2008年10月收治的13例经病理确诊为伯基特淋巴瘤和伯基特样淋巴瘤患者的临床资料.所有患者均接受化疗为主的治疗方案,评价疗效和不良反应.结果:13例患者中,男12例,女1例;发病年龄11～62岁,中位年龄15岁;Ⅰ期3例,Ⅱ期2例,Ⅲ期2例,Ⅳ期6例,其中晚期(Ⅲ、Ⅳ期)病例占61.5%,初治时发生骨髓侵犯2例(15.4%),中枢神经系统侵犯4例(30.8%);常见的侵犯部位为浅表淋巴结(61.5%)、腹腔脏器(53.8%)和腹腔及腹膜后淋巴结(38.5%);有B症状7例(53.8%);8/10例(80.0%)血清乳酸脱氢酶(LDH)水平升高,1/10例血清尿酸升高;病理示BL 11例,BLL2例.11例患者获得完全缓解或未经证实的完全缓解,1例部分缓解,总有效率为92.3%.中位随访时间8个月(5～35个月),至随访截止6例患者死亡,1例失访.1年总生存率(OS)、无进展生存率(PFS)和无瘤生存率(DFS)分别为56.98%、32.31%和39.77%.化疗中Ⅲ～Ⅳ度骨髓抑制发生率为69.2%,1例患者出现肿瘤溶解综合征和Ⅳ度全消化道黏膜炎.结论:推荐高强度短疗程化疗方案作为BL和BLL的一线治疗,应积极预防处理化疗不良反应.

OBJECTIVE: To understand hepatitis B virus(HBV) infection in lymphoma patients.METHODS:Retrospective analysis was conducted in 459 cases from Jan.2008 to Dec.2008,including 276 cases of lymphoma and 183 cases of lung cancer.Electrochemiluminescence assay was used to test serum samples from both groups for HBV-related markers,including HBsAg,HBsAb,HBeAg,HBeAb and HBcAb.RESULTS:Statistical analysis showed that there was a higher prevalence of HBsAg positive rate in lymphoma patients(11.95%,33/276) than that in lung cancer patients(4.92%,9/183,P=0.010 4),diffuse large B-cell lymphoma was the highest among all lymphomas(14.77%,26/176,P=0.005 2).CONCLUSIONS:The current results demonstrated that lymphoma patients have a higher incidence of HBV infection.Compared with lung cancer,lymphoma should not only supervise the detection of HBV,but also pay attention to the prevention and treatment of HBV reactivation.

目的:通过构建单一质粒四环素调控载体pCEP4-tetR-IL-24,将其转染胃癌细胞系后,探讨IL-24对胃癌细胞的初步作用以及此系统能否发挥四环素的调控作用.方法:将hIL-24基因片段从pORF9-hIL-24定向克隆至pcDNA4-TO-Xpress中,再亚克隆到pCEP4-tetR中构建pCEP4-tetR-IL-24;利用脂质体转染法转染细胞;Western blotting检测蛋白表达;MTT法与台盘蓝拒染法检测细胞增殖;annexin V法检测细胞的凋亡.结果:DCEP4-tetR-IL-24转染胃癌细胞系MGC803,BGC823与正常胃黏膜细胞系GES-1后,在强力霉素诱导下均可表达IL-24蛋白;MGC803,BGC823细胞中,强力霉素诱导组的增殖较未诱导组、空载体组与对照组明显受抑制(P＜0.01,0.001-0.006);强力霉素诱导组与未诱导组相比,凋亡细胞比例明显增多(23.5%/25.6%→33.8%/36.7%,P＜0.01);GES-1中,诱导组与未诱导组、空载体组及对照组的差异无统计学意义(P＞0.05).结论:IL-24可以抑制胃癌细胞增殖,诱导胃癌细胞凋亡,对正常胃黏膜上皮细胞无明显毒性;构建的单一质粒四环素调控系统pCEP4-tetR-IL-24可以发挥四环素的调控作用.

原发性乳腺非霍奇金淋巴瘤（PNHLB）临床少见,占所有乳腺恶性肿瘤的0.04%～1.10%,占所有非霍奇金淋巴瘤（NHL）的0.39%。好发于中年女性,无特征性临床表现,诊断依赖病理。弥漫性大B细胞淋巴瘤是最常见的病理类型。以化疗为主的综合治疗疗效较好。手术的主要目的在于取得组织标本以明确诊断。预后可能与分期及年龄有关。

OBJECTIVETo construct the gene co-expression vector of immunoglobulin heavy chain variable region (IgHV) gene and GM-CSF or IL-2 as IgHV family specific nucleic acid vaccine to lymphoma, and study the immune response of the immunized mice against lymphoma.METHODSSix clones with significantly different length in gene fragments were selected from a gene bank constructed from normal fetal umbilical cord blood. These gene fragments in the clones were sequenced. The sequences were translated into IgHV peptides. T cell epitopes in the IgHV were predicted by bioinformatics. Meanwhile 6 clones of IgHV1 constructed before were analyzed. The most typical clone of IgHV1 and IgHV3 containing most of the T cell epitopes were selected. The IgHV gene fragments whose complementary determining region 3 (CDR3) was cut out and composed mainly of framework region were cloned into eukaryotic expression vector. The gene fragments of framework region of IgHV linking with gene of GM-CSF or IL-2 were cloned into pcDNA3.0 to form fusion genes of IgHV (FR)-GM-CSF/IL-2. Then they were transected into COS cells, African green monkey renal cells, by Lipofectin and their transient expression product was detected by ELISA. Twenty male Balb/c mice were randomly divided into 5 groups of 4 mice to be injected with different vectors at the weeks 0, 2, and 4: with IgHV3 (FR)/pcDNA3.0, with IgHV3 (FR)-IL-2/pcDNA 3.0, with blank vector pcDNA3.0 as control, with IgHV1 (FR)/pcDNA3.0, and with IgHV1 (FR)-IL-2/pcDNA3.0. At the weeks 0, 2, 4, 6, and 8 serum was collected from the mice to undergo indirect immunofluorescence examination to detect the antibodies against Namalwa cells, lymphoma cells from Africa green monkey, and normal lymphocytes. ELISA was used to examine the serum interferon (IFN)-gamma.RESULTSAbout 30 of T cell epitopes existed in each IgHV peptides predicted by bioinformatics. Among the bioinformatics prediction, about 90% of the T cell epitopes were in the framework region (FR) of IgHV, and the first 10% with higher prediction score were in FR. The CDR3 of two typical IgHV sequences were cut down and the remaining sequences, mainly composed of FR, were used to construct the IgHV (FR)/pcDNA3.0 expression vectors. The 3' end of IgHV1 (FR) and IgHV3 (FR) were linked to GM-CSF or IL-2 gene successfully. The expression of GM-CSF in the group transfected with IgHV (FR)-GM-CSF/pcDNA3.0 were 200 times higher than in the group transfected with control pcDNA3.0. The expression of IL-2 in the group transfected with IgHV (FR)-IL-2/pcDNA3.0 were 60 times higher than in the group transfected with control pcDNA3.0. The antibody against IgHV1 family lymphoma cell line Namalwa could be detected since the second week in the mice immunized with IgHV1 (FR)-IL-2/pcDNA3.0. The antibody could be detected since the fourth week in the mice immunized with IgHV1 (FR). The antibody against lymphocyte line of IgHV3 family could be detected since the second week in the mice immunized with IgHV3 (FR)-IL-2/pcDNA3.0. The antibody could be detected since the fourth week in the mice immunized with IgHV3 (FR)/pcDNA3.0. The contents of serum IFN-gamma the mice immunized with IgHV1 (FR)-IL-2/pcDNA3.0 and with IgHV3 (FR)-IL-2/pcDNA3.0 was significantly higher than those of the mice immunized with IgHV (FR)/pcDNA3.0 or pcDNA3.0 (all P < 0.01).CONCLUSIONThe gene fragments of IgHV (FR) can be used to construct IgHV family specific nucleic acid vaccine that induces anti-lymphoma immune response in mice by muscle injection. The expressing vectors of IgHV (FR)-GM-CSF/IL-2 induces stronger immunoreaction.

患者男,72岁.因颈部淋巴结肿大、皮肤搔痒、发热,于2001年4月28日住我院治疗.入院查体:血压、体温正常,皮肤未见皮疹、色素沉着;双颈、双腹股沟多发淋巴结肿大,最大2 cm×2 cm,质中,表面光滑,边界不清,活动度差;心肺无异常,肝脾未及,下肢不浮肿.腹部B超示腹腔、腹膜后多发淋巴结肿大,最大3.9 cm×2.4 cm,肝内多发低回声结节,直径0.5 cm左右,双腮腺多发低回声结节,最大1.8 cm×1 cm,脾大12.9 cm×4.7 cm.

OBJECTIVE:To explore the feasibility of construction of the universal nucleic acid vaccine against B-cell lymphoma.METHODS:RT-PCR was employed to obtain the immuncy losulin heavy chain variable region (IgHV1) gene fragments of the Namalwa cell, normal fetal umbilical cord blood and adult peripheral blood. Then these RT-PCR products were cloned into the eukaryotic expression vector pcDNA3.0 and sequenced. Six plasmids that had high identities with the germ line genes were mixed to serve as vaccines. Eighteen Balb/c mice were divided randomly into three groups and were immunized respectively with the six mixed plasmids, the specific IgHV1 fragment of the Namalwa cell and the blank plasmid pcDNA3.0. 100 microg plasmid and 5 microg hIL-6 per mouse were injected into the muscle, 3 times in 4 weeks. Western blotting and indirect immunofluorescence staining were used to assess the humoral immune responses against the Namalwa cell.RESULTS:The specific humoral immune responses against the Namalwa lymphoma cell could be induced in both the IgHV1-mix group and the Na-IgHV1 group, and the antibodies could recognize the nature antigenic determinants in the surface of the Namalwa cell. The antibodies could be detected since the fourth week after the first immunization, and reached the climax at the sixth week. There was no significant difference of the titers of the antibodies between the two groups. The antibody couldn't be found in the negative control group.CONCLUSION:The mixed IgHV plasmids can be used as the universal nucleic acid vaccine against the B-cell lymphoma which expresses the same IgHV1 family genes.