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本研究分析白血病细胞免疫时自体树突细胞(DC)和多种细胞因子诱导外周血淋巴细胞后T细胞克隆性的变化。采集21例化疗缓解后行细胞免疫治疗的白血病患者的骨髓和外周血,加入细胞因子培养,获得树突细胞(DC)和激活T细胞。用RT-PCR扩增代表T细胞克隆性的T细胞受体β链可变区(T-cell receptor β variable region,TCRBV),用TCRBV基因扫描的方法观察培养前后T细胞克隆的特征性变化;对T细胞克隆的TCRBV进行序列分析。流式细胞术(FCM)检测CD3+、CD4+、CD8+、CD56+和CD4+CD25str+FOXP3+,确定其细胞毒T细胞、辅助T细胞、调节T细胞和NK-T细胞的比例变化。结果表明:临床缓解后的白血病患者的T细胞呈寡克隆分布,分属在24个TCRBV家族中一些TCRBV基因家族的淋巴细胞克隆消失,仅少数保持多克隆分布,其中TCRBV24基因家族均为克隆性的分布(100%)。分析患者的TCRBV24基因序列特征时发现,3例在CDR3区共用motif VAG,2例共用GGG,表明这些TCRBV24有可能识别同一抗原,其中1例(例5)患者除TCRBV24在CDR3区有motif GGG外,TCRBV8也有GGG,也可能会识别同一抗原。外周血淋巴细胞经过13天的体外抗原和细胞因子培养后,培养前出现的一些寡克隆以及克隆完全缺失的状态随即出现了明显变化,培养后都出现完整多克隆分布。将培养前后T细胞进行流式细胞术分析显示,培养后淋巴细胞数目为培养前的3.38±1.20倍,CD3+数培养前为(71.1±11.8)%细胞,培养第13天后为(95.4±3.2)%,明显增加;CD4+数培养前为(26.7±11.4)%,培养第13天后为(27.0±13.1)%,无明显变化(p>0.01);CD8+数培养前为(35.7±12.9)%,培养第13天后为(55.5±13.8)%,明显增加(p<0.01);CD3+CD56+数培养前为(3.1±1.6)%,培养第13天后为(9.8±6.1)%,增加2倍多(p<0.01);CD4+CD25str+FOXP3+数培养前为(0.12±0.1)%,培养第13天后为(0.22±0.18)%(p<0.01)。结论:T淋巴细胞诱导培养方法的主要作用是促进CTL和NK细胞增殖,缓解后白血病患者体内淋巴细胞群往往呈寡克隆增殖,一些克隆会共用同样的TCRBVCDR3模体,识别同样的抗原,细胞因子联合诱导与DC共培养后淋巴细胞恢复多克隆免疫状态,产生以CTL和NK-T为主的效应细胞。
This study analyzed the changes of T cell clonality after peripheral blood lymphocytes were induced by autologous dendritic cells (DCs) and various cytokines in leukemia cells. Twenty-one patients with leukemia underwent chemotherapeutic cytotoxic chemotherapy were collected for bone marrow and peripheral blood. Cytokines were cultured to obtain dendritic cells (DCs) and activated T cells. The T cell receptor β variable region (TCRBV), which represents the clonality of T cells, was amplified by RT-PCR. The characteristic changes of T cell clones before and after culturing were observed by TCRBV gene scanning. TCRBVs from T cell clones were sequenced. Flow cytometry (FCM) detected CD3 +, CD4 +, CD8 +, CD56 + and CD4 + CD25str + FOXP3 + to determine the cytotoxic T cells, helper T cells, regulatory T cells and NK-T cell ratio changes. The results showed that the T cells in leukemia patients after clinical remission presented an oligoclonal distribution. The lymphocyte clones of some TCRBV gene families belonging to the 24 TCRBV families disappeared, only a few kept the polyclonal distribution. The TCRBV24 gene families were all clonal Distribution (100%). Analysis of the patient’s TCRBV24 gene sequence found that 3 patients shared the motif VAG in the CDR3 region and 2 patients shared GGG, indicating that these TCRBV24 may recognize the same antigen, of which 1 patients (Example 5) except TCRBV24 motif GGG in the CDR3 region , TCRBV8 also has GGG, may also recognize the same antigen. Peripheral blood lymphocytes after 13 days of in vitro antigens and cytokines cultured before the emergence of some oligoclonal and cloned complete absence of the state immediately after the significant changes in the culture showed a complete polyclonal distribution. Flow cytometry analysis of T cells before and after culture showed that the number of lymphocytes cultured before culturing was 3.38 ± 1.20 times before culturing, that of (71.1 ± 11.8)% before culturing CD3 +, and (95.4 ± 3.2) days after culturing on day 13, %, And the number of CD4 + cells was (26.7 ± 11.4)% before training and 27.0 ± 13.1% after 13 days of culture, with no significant change (p> 0.01) The percentage of CD3 + CD56 + cells was (3.1 ± 1.6)% before culture and (9.8 ± 6.1)% after culture for 13 days and increased more than 2 folds (0.12 ± 0.1)% before culture and (0.22 ± 0.18)% (p <0.01) after 13 days culture, respectively. CONCLUSION: The main function of T lymphocyte-induced culture is to promote the proliferation of CTL and NK cells. In the leukemia patients, the lymphocyte population often shows oligoclonal proliferation. Some clones share the same TCRBVCDR3 motif and recognize the same antigens, cytokines After co-cultured with DC, polyclonal immune status of lymphocytes was restored and CTL and NK-T-based effector cells were produced.