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目的用免疫球蛋白(Ig)重链框架区九肽体外诱导抗急性B淋巴细胞白血病(BALL)的细胞毒T细胞应答。方法合成Ig重链可变区第1家族框架区3～11位置上的九肽QLVQSGAEV(IgHV13～11),进行T2细胞结合实验。IgHV13～11负荷抗原呈递细胞(APC),体外刺激HLAA0201+正常外周血单个核细胞(PBMNC),每周1次,共3次。HLAA0201/IgHV13～11四聚体监测培养细胞中IgHV13～11特异性CD8+T细胞的增殖。对BALL初治患儿的7个IgHV基因家族进行PCR扩增及PCR产物的测序,选出IgHV1和IgHV3家族单等位基因功能性重排克隆,并鉴定其HLAA0201位点。MTT比色法分析IgHV13～11特异性CD8+T细胞对HLAA0201+IgHV1和IgHV3家族BALL细胞克隆的杀伤活性。结果IgHV13～11可上调HLAA0201分子在T2细胞表面的表达1.63倍。HLAA0201+正常PBMNC中IgHV13～11特异性CD8+T细胞的频率由2轮刺激后1.64%增至3轮刺激后82.57%。IgHV13～11特异性CD8+T细胞在效靶细胞比(E∶T)20∶1时对IgHV1家族B-ALL细胞的杀伤率为18.24%,是IgHV3家族B-ALL细胞的1.8倍(P=0.01)。结论体外诱导的Ig重链框架区九肽特异性CD8+T细胞可杀伤表达该肽的B-ALL细胞。
OBJECTIVE: To induce cytotoxic T cell responses against acute B lymphoblastic leukemia (BALL) in vitro using nonapeptides derived from immunoglobulin (Ig) heavy chain framework regions. Methods The nonapeptide QLVQSGAEV (IgHV13 ~ 11) at position 3 to 11 of the first heavy chain of Ig heavy chain variable region was synthesized and subjected to T2 cell binding assay. IgHV 13-11-loaded antigen-presenting cells (APCs) were stimulated in vitro with HLAA0201 + normal peripheral blood mononuclear cells (PBMNC) once a week for 3 times. Proliferation of IgHV13 ~ 11-specific CD8 + T cells in cultured cells was monitored by HLAA0201 / IgHV13 ~ 11 tetramers. The 7 IgHV gene families of children with BALL were sequenced and PCR products were sequenced. Functional rearrangement clones of single allele of IgHV1 and IgHV3 were selected and HLAA0201 locus was identified. MTT colorimetric analysis of IgHV13 ~ 11-specific CD8 + T cell killing activity of HLAA0201 + IgHV1 and IgHV3 family BALL cell clones. Results IgHV13 ~ 11 up-regulated the expression of HLAA0201 on the surface of T2 cells by 1.63-fold. The frequency of IgHV13 ~ 11 specific CD8 + T cells in HLAA0201 + normal PBMNC increased from 1.64% after 2 rounds of stimulation to 82.57% after three rounds of stimulation. The killing rate of IgHV13 ~ 11-specific CD8 + T cells to IgHV1 family B-ALL cells was 18.24% at 1.8: 1 of target cell ratio (E: T), 1.8 times that of IgHV3 family B-ALL cells 0.01). Conclusions Nine-peptide-specific CD8 + T cells induced in vitro can kill B-ALL cells expressing the peptide.