Immunomodulating property of Ocimum sanctum by regulating the IL-2 production and its mRNA expression using rat's splenocytes

Objective:To investigate the regulatory effect of aqueous extract of leaves of Ocimum sanctum on IL-2 cytokine production in vivo and in vitro,and the effect of leaves extract on general blood picture including T& B lymphocytes.Methods:For in vivo studies albino Wistar rats were treated with aqueous crude leaves extract of Ocimum sanctum for 20 consecutive days.Spleen cells were harvested and assayed for IL-2 production by using sandwich enzyme-linked immunosorbent assay(ELISA) and mRNA expression methods.For in vitro study aqueous Ocimum sanctum leaves extract= in different concentrations(25-500μ/mL) was added into culture plates containing ConA stimulated splenocytes.To study the overall effect on blood picture,density gradient purified lymphocytes analysis and conventional methodology for total and differential leukocyte count and hemoglobin level were also done.Results:It indicated that the rats treated with Ocimum sanctum leaves extract had significantly enhanced(P【0.001) ability of spleen cells to secrete IL-2.Investigation in vitro also showed regulation of IL-2 production.Blood study exhibited leucocytosis and augmentation of T& B lymphocytes by 25%approximately.4-5%increase in Hemoglobin value was also noticed.Conclusion:Aqueous Ocimum sanctum leaves extract may have stimulatory effect on T & B lymphocytes particularly on Th 1 subset of lymphocytes as shown by enhancement in IL-2 production.

Donor-derived CD 19 CAR-T Cells versus Chemotherapy Plus Donor Lymphocyte Infusion for Treatment of Recurrent CD 19-positive B-ALL after Allogeneic Hematopoietic Stem Cell Transplantation

Objective:This study aimed to compare the efficacy of anti-CD19 chimeric antigen receptor T cells(CAR-T cells)versus chemotherapy plus donor lymphocyte infusion(chemo-DLI)for treating relapsed CD 19-positive B-cell acute lymphoblastic leukemia(B-ALL)after allogeneic hematopoietic stem cell transplantation(allo-HSCT).Methods:Clinical data of 43 patients with B-ALL who relapsed after allo-HSCT were retrospectively analyzed.Twenty-two patients were treated with CAR-T cells(CAR-T group),and 21 with chemotherapy plus DLI(chemo-DLI group).The complete remission(CR)and minimal residual disease(MRD)-negative CR rates,leukemia-free survival(LFS)rate,overall survival(OS)rate,and incidence of acute graft-versus-host disease(aGVHD),cytokine release syndrome(CRS)and immune effector cell-associated neurotoxicity syndrome(ICANS)were compared between the two groups.Results:The CR and MRD-negative CR rates in the CAR-T group(77.3%and 61.5%)were significantly higher than those in the chemo-DLI group(38.1%and 23.8%)(P=0.008 and P=0.003).The 1-and 2-year LFS rates in the CAR-T group were superior to those in the chemo-DLI group:54.5%and 50.0%vs.9.5%and 4.8%(P=0.0001 and P=0.00004).The 1-and 2-year OS rates in the CAR-T versus chemo-DLI group were 59.1%and 54.5%vs.19%and 9.5%(P=0.011 and P=0.003).Six patients(28.6%)with grade 2-4 aGVHD were identified in the chemo-DLI group.Two patients(9.1%)in the CAR-T group developed grade 1-2 aGVHD.Nineteen patients(86.4%)developed CRS in the CAR-T group,comprising grade 1-2 CRS in 13 patients(59.1%)and grade 3 CRS in 6 patients(27.3%).Two patients(9.1%)developed grade 1-2 ICANS.Conclusion:Donor-derived anti-CD19 CAR-T-cell therapy may be better,safer,and more effective than chemo-DLI for B-ALL patients who relapse after allo-HSCT.

Generation of cytotoxic T lymphocytes specific for B-cell acute lymphoblastic leukemia family-shared peptides derived from immunoglobulin heavy chain framework region

Background Immunoglobulin heavy chain variable region （IgHV） is a well-characterized tumor antigen for B-cell malignancies. It can function as a target for T cell-mediated immune response. Clinical trials of IgHV protein vaccines against lymphoma have demonstrated induction of tumor-specific cytotoxic T lymphocyte （CTL） responses. However, complementary determining regions-based individual vaccines have disadvantages for wide clinical application. Although a recent study demonstrated that immunogenic peptides are derived from framework regions （FR） shared among patients with B-cell lymphoma, how to choose the appropriate peptides for each patient is still unsolved. The aim of this study was to investigate whether immunoglobulin heavy chain FR-derived peptides shared in each IgHV family are potential CTL epitopes presented by B-cell acute lymphoblastic leukemia （B-ALL）. Such CTL epitopes might be beneficial to shifting vaccination strategies against B-ALL from individual specificity to family specificity.Methods Seven IgHV gene families were amplified respectively by PCR and sequenced directly from 71 childhood B-ALL cases. Bioinformatics was applied in analyzing characteristics of sequences available and predicting HLA-A^＊0201-restricted CTL epitopes for each IgHV family. An antigen-specific T cell expansion system was used to generate peptide-specific CTLs. The cytotoxicity of CTLs against B-ALL cells was assessed in the lactate dehydrogenase release assay. Results Complete IgHV rearrangements were identified in all of the 71 B-ALL cases. All of 40 sequences available showed ≥98% homology with the nearest germline IgHV genes, indicating IgHV genes in B-ALL of germline nature. Twelve nonapeptides of high HLA-A^＊0201-binding scores were obtained from 26 productive IgHV protein sequences. Ten （83%） of the peptides were located in FR1 and FR3 shared among the corresponding IgHV family. CTLs specific for the peptide QLVQSGAEV located in FR1 （3-11） shared among the IgHV1 family could be successfully generated from peripheral blood mononuclear cells of two HLA-A^＊0201＋ healthy donors in vitro and were capable of killing HLA-matched B-ALL cell clones belonging to the IgHV1 family. Conclusion Anti-B-ALL CTLs against immunoglobulin heavy chain FR-derived peptides have family-specific cytotoxicity.

供体淋巴细胞在GVHD模型小鼠组织器官中迁移和分布的动态观察

本研究应用小鼠GVHD模型探讨异基因T淋巴细胞在移植受体体内的迁移和分布。将C57BL/6的骨髓细胞和转基因荧光C57BL/6小鼠的脾淋巴细胞输入经8Gy全身照射的BALB/c小鼠,建立eGFP标记供体淋巴细胞的GVHD小鼠模型;应用荧光显微镜和流式细胞术观测GVHD模型中eGFP+细胞的分布;ELISA法检测GVHD靶组织中趋化因子MIP-1α水平的改变。结果表明:①输入脾细胞和骨髓细胞第8天后出现GVHD临床及病理表现;②GVHD模型中,受体鼠肝、皮肤、肠、脾、肺、舌有eGFP+细胞浸润;③GVHD小鼠肝、脾eGFP+细胞中CD4+、CD8+细胞比例均逐渐升高;④GVHD鼠脾、肝组织中MIP-1α水平升高,脾中MIP-1α水平高峰出现于移植后第3天,肝中MIP-1α水平高峰出现于移植后第7天。结论:除肝、肠、皮肤外,肺、舌可能也是GVHD靶器官。肝、脾组织中供体淋巴细胞浸润伴随MIP-1α水平的升高。

抑制性T淋巴细胞在大鼠同种心脏移植免疫耐受中的作用

目的 探讨抑制性T淋巴细胞在同种心脏移植免疫耐受中的作用。方法 将纯系DA(供体 )、纯系Lewis大鼠 (受体 )各 5 0只随机分为未处理组、脾细胞组、环磷酰胺组、脾细胞 +环磷酰胺组、转移组 ,每组受、供体鼠各 10只 ,各组受体鼠分别采用不处理、门静脉注入供体脾细胞 ( 3× 10 8个 )、腹腔注入环磷酰胺 ( 80mg/kg)、供体脾细胞 +环磷酰胺、脾细胞 +环磷酰胺组心脏移植术后30d受体大鼠脾细胞进行免疫耐受诱导后行腹部心脏移植 ;观察各组供心存活时间、病理改变及供受体间的混合淋巴细胞反应 (MLR)。结果 脾细胞 +环磷酰胺组产生了长期的免疫耐受 ,供心平均存活时间为 ( 71 5± 2 9 1)d ,较未处理组、脾细胞组、环磷酰胺组均显著延长 (t=- 14 0 6 3,- 13 915 ,- 13 777,均P <0 0 1) ,供心仅有少量炎性细胞浸润 ,供受体间MLR特异性降低 (t =2 9 90 2 ,P <0 0 1)。转移组供心平均存活时间为 ( 5 2 3± 7 5 )d ,供心仅有少量炎性细胞浸润 ,供受体间MLR特异性降低 (t=2 3 0 4 7,P <0 0 1)。结论 脾细胞 +环磷酰胺可成功诱导同种大鼠心脏移植的免疫耐受 ,其免疫耐受状态可过继转移给正常的同系受体 。