WO2018032619A1 - 可溶性蛋白baff在b细胞体外培养及扩增的应用 - Google Patents

可溶性蛋白baff在b细胞体外培养及扩增的应用 Download PDF

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WO2018032619A1
WO2018032619A1 PCT/CN2016/104807 CN2016104807W WO2018032619A1 WO 2018032619 A1 WO2018032619 A1 WO 2018032619A1 CN 2016104807 W CN2016104807 W CN 2016104807W WO 2018032619 A1 WO2018032619 A1 WO 2018032619A1
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cells
cell
baff
cd40l
peptide
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张辉
张译文
高士麟
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Sun Yat Sen University
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Definitions

  • the invention relates to a method for constructing cells and an application thereof, in particular to a method for constructing a secretory BAFF cell line and an application thereof for improving in vitro expansion and antigen presentation of human peripheral blood B cells.
  • T cells play an important role in the immune system
  • more and more research groups are trying to use the treatment of T cells to treat tumors or viral infections.
  • many research groups at home and abroad are studying how to maintain long-term in vitro culture of T cells, increase the survival time of T cells, and improve the immunological function of T cells for adoptive immunotherapy.
  • the main strategy is to first clone and amplify, and obtain a sufficient amount of T cells with killing function to be co-cultured with antigen presenting cells.
  • B cells have a large number of cells, easy cell acquisition, and strong cell expansion potential, so that a large number of B cells having antigen presenting function are easily obtained. These B cells re-present antigens to T cells, and the role of promoting T cell culture time and amplification efficiency as well as enhancing T cell function has been confirmed. Large-scale expansion of antigen-specific human peripheral blood B cells in vitro and enhancement of antigen presentation function play an important role in tumor and infected T cell adoptive therapy.
  • BAFF B-cell activating factor
  • BAFF-R three receptors, TACI, BCMA and BAFF-R.
  • BAFF-R the binding of BAFF to BAFF-R is the main reason for promoting the survival of B cells.
  • BAFF can also synergize with CD40L to increase the efficiency of B cell expansion (Do et al., 2000; Moore et al., 1999).
  • BAFF is a transmembrane protein that requires the action of proteolytic enzymes to release active water soluble fragments. Due to this special property of BAFF protein, BAFF has not been introduced into the system of in vitro expansion and long-term culture of human B cells.
  • the object of the present invention is to provide an application of soluble protein BAFF in vitro culture and expansion of B cells.
  • the secreted BAFF peptide has an amino acid sequence consisting of 133-285 of BAFF and its N-terminal IL-2 peptide.
  • the amino acid sequence of 133-285 of BAFF is: AVQGPEETVTQDCLQLIADSETPTIQKGSYTFVPWLLSFKRGSALEEKENKILVKETGYFFIYGQVLYTDKTYAMGHLIQRKKVHVFGDELSLVTLFRCIQNMPETLPNNSCYSAGIAKLEEGDELQLAIPRENAQISLDGDVTFFGALKLL (SEQ ID NO: 1), IL-2 peptide
  • the amino acid sequence is: MYRMQLLSCIALSLALVTNS (SEQ ID NO: 2).
  • An expression vector for a secreted BAFF peptide having a nucleotide sequence expressing the above-described secreted BAFF peptide having a nucleotide sequence expressing the above-described secreted BAFF peptide.
  • the vector is further inserted with a nucleotide sequence which expresses membrane type CD40L.
  • a cell expressing a secreted BAFF peptide transfected with the above expression vector A cell expressing a secreted BAFF peptide transfected with the above expression vector.
  • the cells were also transfected with a vector expressing the membrane type CD40L.
  • the cells are 293T cells.
  • a method for amplifying a human peripheral B cell body comprising co-culturing human peripheral B cells with cells simultaneously expressing a secreted BAFF peptide and a membrane type CD40L.
  • human peripheral B cells are cultured in an IMDM medium supported by insulin and transferrin, and CpG2006/2219 and cyclosporine A are added to stimulate B cell expansion.
  • Human IL-4, IL-2, IL-10 were added to maintain cell growth.
  • the secreted BAFF peptide of the present invention the expressed peptide can be efficiently secreted outside the cell to produce a corresponding biological activity.
  • the cell line of the invention can continuously secrete the biologically active water-soluble BAFF protein during the culture process, and can be co-cultured with the B cell as a tool cell, thereby effectively promoting the in vitro expansion and antigen presentation effect of human B cells.
  • Figure 1 is a schematic representation of CD40L and soluble BAFF expression plasmids
  • Figure 2 shows the expansion of B cells with 293T, 293T-CD40L or 293T-CD40L-sBAFF cell lines as feeder cells; A) B cell amplification curve; B) 35 days post-sampling statistics B cell expansion Double the number;
  • Figure 3 shows the results of amplification flow CFSE detection of B cells and 293T, 293T-CD40L or 293T-CD40L-sBAFF cell lines as feeder cells for 7 days, and A) flow cytometry analysis of expanded B cell ratio And B) ratio statistics;
  • Figure 4 shows B cells co-cultured with 293T, 293T-CD40L or 293T-CD40L-sBAFF cell lines as feeder cells, and BCR cells were co-cultured to CD80, CD86, CD70 and CD275 on day 5 or day 30.
  • Figure 5 is a B cell cultured using 293T-CD40L-sBAFF cell line as an antigen-presenting cell to educate CD8 cells; a, b) B cell cultured CTL IFN- ⁇ secretion under different culture conditions, c) CTL killing ability Detection; d) Schematic diagram of CTL in vivo model; e) In vivo experimental tumor volume detection.
  • the amino acid sequence of the secreted BAFF peptide consists of 133-285 of BAFF and IL-2 peptide coupled to its N-terminus.
  • the amino acid sequence of 133-285 of BAFF is: AVQGPEETVTQDCLQLIADSETPTIQKGSYTFVPWLLSFKRGSALEEKENKILVKETGYFFIYGQVLYTDKTYAMGHLIQRKKVHVFGDELSLVTLFRCIQNMPETLPNNSCYSAGIAKLEEGDELQLAIPRENAQISLDGDVTFFGALKLL
  • the amino acid sequence of IL-2 peptide is: MYRMQLLSCIALSLALVTNS.
  • nucleotide sequence corresponding to BAFF 133-285 bits gccgttcagggtccagaagaaacagtcactcaagactgcttgcaactgattgcagacagtgaaacaccaactatacaaaaggatcttacacatttgttccatggcttctcagcttttaaaaggggaagtgccctagaagaaaaagagaataaaatattggtcaaagaaactggttacttttttatatactgataagacctacgccatgggacatctaattcagaggaagaaggtccatgtctttggggatgaattgaattgagtctggtgaattgagtctggtgaattgagtctggtgaattgagtctggtgaattgagtctggtgaattga
  • nucleotide sequence corresponding to the IL-2 peptide is: atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacaaacagt (SEQ ID NO: 4).
  • An expression vector for a secreted BAFF peptide having a nucleotide sequence expressing the above-described secreted BAFF peptide having a nucleotide sequence expressing the above-described secreted BAFF peptide.
  • the vector is further inserted with a nucleotide sequence which expresses membrane type CD40L.
  • a cell expressing a secreted BAFF peptide transfected with the above expression vector A cell expressing a secreted BAFF peptide transfected with the above expression vector.
  • the cells were also transfected with a vector expressing the membrane type CD40L.
  • the cells are 293T cells.
  • human peripheral B cells are cultured in an IMDM medium supported by insulin and transferrin, and CpG2006/2219 and cyclosporine A are added to stimulate B cell expansion. add a person IL-4, IL-2, IL-10 maintain cell growth.
  • the templates are all cDNAs of PBMC cells.
  • the PCR enzyme used is 2 ⁇ Premix HS.
  • the primers used are shown in the table:
  • the PCR system is shown in the following table:
  • the PCR conditions were 94 ° C for 30 s, 62 ° C for 30 s, 72 ° C for 1 min, 72 ° C for 7 min, and finally 4 ° C to stand.
  • the PCR conditions were 94 ° C for 30 s, 58 ° C for 30 s, 72 ° C for 30 s, 72 ° C for 7 min, and finally 4 ° C to stand.
  • the PCR product was electrophoresed, and after the target band was observed under blue light, the target gene band was excised and then recovered by a gel recovery kit.
  • the target gene fragment and the corresponding vector are subjected to double digestion reaction, respectively.
  • the obtained target gene fragment and the obtained target vector fragment were subjected to an overnight ligation reaction at 16 ° C using Takara T4 DNA ligase.
  • the CD40L fragment is linked to the cPPT-mini-MMIG vector
  • the BAFF fragment is linked to the cPPT-IRES-RFP vector.
  • a 10 cm cell culture dish was treated with a 1 ⁇ polylysine solution; the cell density was 40% transfected with the virus packaging plasmid; after transfection for 36 hours, the virus concentrate was prepared, and the supernatant was filtered with 0.45 ⁇ m. The head was filtered and added to the concentrate at 4 ° C overnight; after 24 h, the virus supernatant was centrifuged at 4000 g for 5 min, the supernatant was discarded, and resuspended in 1 ml of DMEM medium;
  • PBMC peripheral blood cells
  • Human peripheral blood was taken from the Guangzhou Blood Center.
  • the peripheral blood was added to 1 ⁇ PBS buffer (containing 2% BSA and 0.5% EDTA) and mixed uniformly. Lymphocyte separation solution was slowly added thereto, and the ratio of the two was 1:1, and centrifuged at 1500 rpm for 40 minutes. Carefully pipette the peripheral blood mononuclear cell layer (white floc, ie, the white membrane layer), place it in another centrifuge tube, add 5 volumes of 1 ⁇ PBS (containing 0.5% BSA and 2% EDTA), and mix well. Centrifuge at 380g for 8 min. Heavy Repeat the above step once.
  • 1 ⁇ PBS buffer containing 2% BSA and 0.5% EDTA
  • the feeder cells were prepared. After the 12-well plate was previously treated with a 1 ⁇ polylysine solution, the 293T cell line, the 293T-CD40L-sBAFF cell line and the 293T-CD40L cell line were plated into a 12-well plate. When the cell density was as long as about 30%, the cells were irradiated with 95 Gy of irradiation cells or mitomycin C for 3 hours to stop the growth of the cells. After 12 hours of observation, it was confirmed that the cells stopped growing.
  • B cells were then plated into 12-well plates and co-cultured with feeder cells. After a series of conditions, the inventors finally located the density of B cells at 5 ⁇ 10 5 /well.
  • B cells were suspended in IMDM medium at 5 ⁇ 10 5 /ml, and a final concentration of 2 ⁇ g/ml CpG-ODN2006/2219, 0.625 ⁇ g/ml CsA (Cyclosporine A), 10 ng/mL IL-2 was added to the medium. , 10 ng/ml IL-10 and 20 ng/mL IL-4. Subsequently, the cell suspension was added to a 12-well plate in an amount of 1 ml per well, and co-cultured with the feeder cells. B cells were separated from feeder cells by differential centrifugation or density gradient centrifugation every 4 days, counted by trypan blue staining, and transferred to a new feeder cell at a density of 5 ⁇ 10 5 /well. Orifice plate.
  • the ELISPOT assay for measuring cellular IFN- ⁇ was performed using Daktronics' human IFN- ⁇ precoated ELISPOT Kit.
  • the adjusted cell suspension was added to the experimental well, and the experimental group was added with CD8+ T cells co-cultured with CD40L-B cells or CD40L-sBAFF-B cells, and the positive control group was added with PHA-stimulated CD8+ T cells, negative.
  • the control group was added with unstimulated CD8+ T cells.
  • the density of CD8+ T cells was set at 105/well. Background Negative control group was added to an equal volume of RMPI 1640. After the sample was added, incubate at 37 ° C for 16-20 h.
  • the medium in the well was decanted, and the cells were lysed by adding deionized water at 4 ° C for 10 min at 4 °C. After washing 5-7 times with 1 ⁇ ELISPOT eluate, the antibody working solution was added and incubated at 37 ° C for 1 h. The antibody was then decanted, washed 5-7 times with 1 x ELISPOT eluate, and then HRP working solution was added for 1 h at 37 °C.
  • the cells were washed 5-7 times with 1 ⁇ ELISPOT eluent, and the prepared AEC color developing solution was added to the room temperature for 25 min. After the reaction was completed, the cells were washed 5 to 7 times with deionized water, and the plate was placed in a cool place. After drying, the number of positive spots was measured with a CTL Immunospot S5 core analyzer, and the spot count and statistical analysis were performed by CTL ImmunoSpot software.
  • the inventors used 293T cells to construct feeder cell lines.
  • the packaged lentivirus carrying the membrane type CD40L sequence was infected with human 293T cells, and a stable cell line (293T-CD40L cell line) was obtained after multiple rounds of screening by flow cytometry.
  • the amino acid of 1-46 of human BAFF is the intracellular segment, the 47-73 position is the transmembrane region, and the 74-285 is the extracellular segment.
  • amino acids 133-285 form soluble BAFF, which is the main form of function of BAFF. Therefore, the inventors cloned amino acids 133-285 of BAFF.
  • the inventors In order to make BAFF better secreted extracellularly, the inventors carefully designed to insert a secretory peptide of IL-2 at the N-terminus of BAFF. Meanwhile, in order to facilitate the later detection of the amount of BAFF secreted into the supernatant, the inventors expressed the sequence of the Flag tag fusion at the C-terminus of the soluble BAFF.
  • the fusion protein was cloned into the lentiviral vector cPPT-IRES-RFP, and the 293T-CD40L cell line was co-transfected with the packaging plasmid psPAX and the envelope protein VSVG to package the virus.
  • a stable GFP-RFP double positive cell line (293T-CD40L-sBAFF) was obtained after multiple rounds of selection of RFP positive cells by flow cytometry.
  • the plasmid constructed in this experiment is shown in Figure 1.
  • the inventors cultured human peripheral blood primary B cells in vitro using the newly constructed 293T-CD40L-sBAFF cell line, as described above.
  • the inventors amplified the effect of 293T-CD40L-sBAFF cell line on B cells and the 293T-CD40L reported in the previous article.
  • activated B cells can express MHC class molecules (MHC-I, MHC-II), costimulatory factors (CD80, CD86) and adhesion molecules (CD54), they have the ability to present antigens and activate T cells, while costimulatory molecules
  • MHC-I, MHC-II MHC class molecules
  • CD80, CD86 costimulatory factors
  • CD54 adhesion molecules
  • the expression levels of CD80 and CD86 are closely related to the antigen presentation function of B cells.
  • B cells were harvested after co-culture and surface CD80 and CD86 levels were detected using flow cytometry. The results showed that B cells co-cultured with the 293T-CD40L-sBAFF cell line had higher expression levels on the surface of CD80 and CD86 molecules after co-culture.
  • other co-stimulatory molecules involved in B cell antigen presentation such as CD275 (ICOSLG) and CD70 (CD27L), were up-regulated, as shown in Figure 4.
  • the inventors obtained an increase in costimulatory molecules involved in cell antigen presentation, and the inventors further verified whether B cells cultured in the 293T-CD40L-sBAFF cell line can enhance the ability of tumor-specific antigen presentation.
  • Melanoma is the best tumor treatment model.
  • the inventors chose the melanoma-specific epitope NY-ESO-1, and the B cells cultured with the 293T-CD40L-sBAFF cell line were incubated with the antigen peptide for antigen delivery. Presented.
  • CD8 cells derived from B cells presented by 293T-CD40L-sBAFF cell line can secrete more IFN- ⁇ in the ELISPOT assay, and in cell killing experiments, The killing efficiency is almost 100%.
  • CD8 cells derived from B cell-presenting antigens cultured using the 293T-CD40L-sBAFF cell line also had better antitumor activity in vivo, and the inventors constructed an in vivo experimental model of melanoma. As shown in Fig. 5d, e, the inventors successively returned the educated CTL cells to the mice and measured the tumor size. The results showed that the B cells presented by the 293T-CD40L-sBAFF cell line were presented. The ability of the antigen is enhanced, so the CTL obtained by education also has better antitumor activity in vivo.
  • BLyS member of the tumor necrosis factor family and B lymphocyte stimulator. Science, 285 (5425), 260-263.
  • CD40-activated human B cells an alternative source of highly Efficient antigen presenting cells to generate autologous antigen-specific T cells for adoptive immunotherapy. Journal of Clinical Investigation, 100(11), 2757.
  • B cell maturation antigen the receptor for a proliferation-inducing ligand and B cell -activating factor of the TNF family, induces antigen presentation in B cells.

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Abstract

本发明公开了可溶性蛋白BAFF在B细胞体外培养及扩增的应用，本发明的分泌型BAFF肽，表达出的肽可以有效地分泌到细胞外，产生相应的生物活性。本发明的细胞系，在培养过程中可以持续分泌出具有生物活性的水溶性BAFF蛋白，作为工具细胞与B细胞共培养，可以有效促进人B细胞体外扩增及抗原递呈效果。

Description

可溶性蛋白BAFF在B细胞体外培养及扩增的应用 技术领域
本发明涉及一种细胞的构建方法及其应用，特别涉及分泌型BAFF细胞系的构建方法及其在提高人外周血B细胞体外扩增和抗原递呈的应用。
背景技术
近年来由于T细胞在免疫系统中起着重要的作用，越来越多的研究小组尝试运用T细胞的过继治疗的方式来进行肿瘤或病毒感染的治疗。目前，国内外的多个研究小组正在研究如何维持T细胞的体外长期培养，提高T细胞的存活时间，提高T细胞的免疫学功能以用于过继免疫治疗。在进行T细胞治疗时，主要策略是先通过克隆扩增，得到足够量的具有杀伤功能的T细胞可以与抗原提呈细胞共培养。相对于通过构建人工抗原提呈细胞，利用人体自身的抗原递呈细胞大量扩增用于免疫治疗的T细胞，拥有不易引入外源细胞，不易受异体抗原干扰，呈递过程更接近生理状态，扩增成功率高的优点。
在人体主要的三种专职抗原呈递细胞中，B细胞拥有细胞数量多，细胞获得容易，细胞扩增潜力强，从而易得到大量具有抗原呈递功能的B细胞。这些B细胞再呈递抗原给T细胞，促进T细胞的体外培养时间与扩增效率以及增强T细胞功能方面的作用已被证实。体外大量扩增抗原特异性的人外周血B细胞，并提高其抗原呈递功能对于肿瘤和感染的T细胞过继治疗具有非常重要的作用。
因此，如何大量扩增抗原特异性的人外周血B细胞，并提高其抗原呈递功能，便成为了近年研究的热点。Shultze于1997年首先报道了由稳定表达CD40L的NIH-3T3细胞刺激并活化从而大量扩增体外B细胞的方法(Schultze et al.,1997)，并证明经过体外培养的B细胞可以高表达CD80，CD86等抗原呈递的必要分子。随后von Bergwelt-Baildon等又对该系统进行了多次的改进。尽管经过改进后，B细胞的体外扩增效率大大提高，但由于活化的B细胞高表达Fas并极易发生凋亡(Garrone et al.,1995；Zhang et al.,1996)，而CD40L的持续刺激并不能抵消凋亡造成的影响，故若要进一步提高B细胞扩增效率，需要减少激活引起的B细胞凋亡。
BAFF(B-cell activating factor)属于TNF超家族，拥有三种受体，分别为TACI,BCMA和BAFF-R。其中，BAFF对BAFF-R的结合是促进B细胞存活的主要原因。除了能够促进B细胞存活，BAFF还可以协同CD40L增加B细胞的扩增效率(Do et al.,2000；Moore et al.,1999)。BAFF是一种跨膜蛋白，需要在蛋白水解酶的作用下才可以释放出具有活性的水溶性片断。 由于BAFF蛋白的这一特殊性质，导致BAFF尚未被引入人B细胞体外扩增与长期培养的体系中。
发明内容
本发明的目的在于提供可溶性蛋白BAFF在B细胞体外培养及扩增的应用。
本发明所采取的技术方案是：
分泌型BAFF肽，其氨基酸序列由BAFF的133-285位和其N端的IL-2肽组成，BAFF的133-285位的氨基酸序列为：AVQGPEETVTQDCLQLIADSETPTIQKGSYTFVPWLLSFKRGSALEEKENKILVKETGYFFIYGQVLYTDKTYAMGHLIQRKKVHVFGDELSLVTLFRCIQNMPETLPNNSCYSAGIAKLEEGDELQLAIPRENAQISLDGDVTFFGALKLL(SEQ ID NO：1)，IL-2肽的氨基酸序列为：MYRMQLLSCIALSLALVTNS(SEQ ID NO：2)。
一种分泌型BAFF肽的表达载体，其插入有表达上述分泌型BAFF肽的核苷酸序列。
作为上述表达载体的进一步改进，载体还插入有表达膜型CD40L的核苷酸序列。
一种表达分泌型BAFF肽的细胞，其转染有上述的表达载体。
作为上述细胞的进一步改进，细胞还转染有表达膜型CD40L的载体。
作为上述细胞的进一步改进，细胞为293T细胞。
一种人外周B细胞体扩增方法，包括将人外周B细胞与同时表达分泌型BAFF肽和膜型CD40L的细胞共培养。
作为上述人外周B细胞体扩增方法的进一步改进，将人外周B细胞培养在有胰岛素和转铁蛋白支持的IMDM培养基中，并添加CpG2006/2219和环孢素A刺激B细胞扩增，添加人IL-4，IL-2，IL-10维持细胞生长。
本发明的有益效果是：
本发明的分泌型BAFF肽，表达出的肽可以有效地分泌到细胞外，产生相应的生物活性。
本发明的细胞系，在培养过程中可以持续分泌出具有生物活性的水溶性BAFF蛋白，作为工具细胞与B细胞共培养，可以有效促进人B细胞体外扩增及抗原递呈效果。
附图说明
图1是CD40L和可溶性BAFF表达性质粒的示意图；
图2是B细胞与293T，293T-CD40L或293T-CD40L-sBAFF细胞系作为饲养层细胞共培养的扩增情况；A)B细胞的扩增曲线；B)35天后收样统计B细胞的扩增倍数；
图3是B细胞与293T，293T-CD40L或293T-CD40L-sBAFF细胞系作为饲养层细胞共培养7天后收样的扩增流式CFSE检测结果，A)流式细胞术分析扩增B细胞比例和B)比例 统计；
图4是B细胞与293T，293T-CD40L或293T-CD40L-sBAFF细胞系作为饲养层细胞共培养后，流式检测B细胞共培养至第5天或第30天时CD80,CD86,CD70和CD275的表达情况；
图5是利用293T-CD40L-sBAFF细胞系培养得到的B细胞作为抗原递呈细胞，教育CD8细胞；a、b)不同培养条件的B细胞教育CTL的IFN-γ分泌情况，c)CTL杀伤能力检测；d)CTL体内模型示意图；e)体内实验肿瘤体积检测。
具体实施方式
分泌型BAFF肽，其氨基酸序列由BAFF的133-285位和偶联在其N端的IL-2肽组成，BAFF的133-285位的氨基酸序列为：AVQGPEETVTQDCLQLIADSETPTIQKGSYTFVPWLLSFKRGSALEEKENKILVKETGYFFIYGQVLYTDKTYAMGHLIQRKKVHVFGDELSLVTLFRCIQNMPETLPNNSCYSAGIAKLEEGDELQLAIPRENAQISLDGDVTFFGALKLL，IL-2肽的氨基酸序列为：MYRMQLLSCIALSLALVTNS。
作为示例，BAFF的133-285位对应的核苷酸序列为：gccgttcagggtccagaagaaacagtcactcaagactgcttgcaactgattgcagacagtgaaacaccaactatacaaaaaggatcttacacatttgttccatggcttctcagctttaaaaggggaagtgccctagaagaaaaagagaataaaatattggtcaaagaaactggttacttttttatatatggtcaggttttatatactgataagacctacgccatgggacatctaattcagaggaagaaggtccatgtctttggggatgaattgagtctggtgactttgtttcgatgtattcaaaatatgcctgaaacactacccaataattcctgctattcagctggcattgcaaaactggaagaaggagatgaactccaacttgcaataccaagagaaaatgcacaaatatcactggatggagatgtcacattttttggtgcattgaaactgctgtga(SEQ ID NO：3)。IL-2肽对应的核苷酸序列为：atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacaaacagt(SEQ ID NO：4)。
一种分泌型BAFF肽的表达载体，其插入有表达上述分泌型BAFF肽的核苷酸序列。
作为上述表达载体的进一步改进，载体还插入有表达膜型CD40L的核苷酸序列。
一种表达分泌型BAFF肽的细胞，其转染有上述的表达载体。
作为上述细胞的进一步改进，细胞还转染有表达膜型CD40L的载体。
作为上述细胞的进一步改进，细胞为293T细胞。
在现有人外周B细胞体扩增方法的基础上，将人外周B细胞与同时表达分泌型BAFF肽和膜型CD40L的细胞共培养，可以有效促进人B细胞体外扩增及抗原递呈效果。
作为上述人外周B细胞体扩增方法的进一步改进，将人外周B细胞培养在有胰岛素和转铁蛋白支持的IMDM培养基中，并添加CpG2006/2219和环孢素A刺激B细胞扩增，添加人 IL-4，IL-2，IL-10维持细胞生长。
下面，结合实验，进一步说明本发明的技术方案。
载体的构建
本文中所涉及的分子克隆实验，其模板均为PBMC细胞的cDNA。所用到的PCR酶为2×Premix 
Figure PCTCN2016104807-appb-000001
HS。所用到的引物如表所示：
本实验所合成的引物序列表
Figure PCTCN2016104807-appb-000002
PCR体系如下表所示：
膜型CD40L PCR体系
Figure PCTCN2016104807-appb-000003
PCR条件为94℃30s，62℃30s，72℃1min，72℃7min，最后4℃静置。
分泌型BAFF PCR体系
Figure PCTCN2016104807-appb-000004
Figure PCTCN2016104807-appb-000005
PCR条件为94℃30s，58℃30s，72℃30s，72℃7min，最后4℃静置。
PCR反应结束后，对PCR产物进行电泳，在蓝光下看到目的条带后，切下目的基因条带，随后利用胶回收试剂盒回收。
胶回收得到目的片段后，同时目的基因片段和相应载体分别进行双酶切反应。酶切产物回收后，将得到的目的基因片段与得到的目的载体片段利用Takara的T4DNA连接酶进行16℃过夜连接反应。其中，CD40L片段链接cPPT-mini-MMIG载体，BAFF片段连接cPPT-IRES-RFP载体。
293T-CD40L-sBAFF细胞系的构建
1)首先，用1×多聚赖氨酸溶液处理10cm细胞培养皿；待细胞密度达到40％转染病毒包装质粒；转染36h后，配制病毒浓缩液，将上清液用0.45μm的滤头过滤后加入浓缩液中4℃过夜；24h后将病毒上清以4000g的速度离心5min，弃掉上清液后，用1ml DMEM培养基重悬；
2)预先用1×多聚赖氨酸溶液处理12孔板；待感染细胞密度达到75％左右时吸掉上清液并准备感染；
3)向细胞中加入1ml浓缩的病毒液和终浓度为6ug/ml的Polybrene，随后以1500g的速度离心感染30min；感染后8h换液；随后进行扩大培养；
4)将扩大培养的细胞系使用胰酶消化后，用1×PBS缓冲液洗涤细胞两次，运用流式细胞仪分选FITC通道阳性细胞(293T-CD40L)或FITC通道与PE通道双阳性细胞(293T-CD40L-sBAFF)；收集管使用2ml DMEM收集细胞；细胞收集后，继续扩大培养并再次进行分选；
5)经过3-5轮分选后，细胞阳性率保持在90％(293T-CD40L)或80％(293T-CD40L-sBAFF)以上，且阳性细胞比例保持长期稳定后停止分选。
人外周血细胞(PBMC)的分离
人外周血取自广州血液中心。将外周成分血加入到1×PBS缓冲液(含2％BSA和0.5％EDTA)混合均匀后，在上方缓慢加入淋巴细胞分离液，二者的比例为1:1混合，1500rpm离心40min。小心吸取外周血单个核细胞层(白色絮状，即白膜层)，置入另一离心管中，加入5倍体积的1×PBS(含0.5％BSA和2％EDTA)稀释，混合均匀后，380g离心8min。重 复以上一步骤一次。
使用阴选磁珠分离B细胞
按照每107的PBMC细胞加入100μl 1×PBS缓冲液(含0.5％BSA和2％EDTA)及50μl CD19+B细胞阴选一抗，在室温下孵育15min。加入10倍体积的1×PBS缓冲液稀释，混合均匀后，380g离心8min，弃掉上清液。每107细胞加入50μl带有磁珠的二抗，在室温下孵育30min后，过磁力柱进行细胞分选，380g离心8min后得到B细胞，加入含10％胎牛血清和1％双抗的IMDM培养基重悬细胞。使用计数板数细胞总数。其他细胞的阴选磁珠用法与此类似。
使用阳选磁珠分离B细胞
按照每107的PBMC细胞加入100μl 1×PBS缓冲液(含0.5％BSA和2％EDTA)及50μl CD19+B细胞阳选磁珠，在室温下孵育20min。加入10倍体积的1×PBS缓冲液稀释，混合均匀后，380g离心8min，弃掉上清液。重悬后过柱进行细胞分选，380g离心8min后得到B细胞，加入含10％胎牛血清和1％双抗的RPMI1640培养基重悬细胞。使用计数板数细胞总数。其他细胞的阳选磁珠用法与此类似。
B细胞扩增
首先准备饲养细胞。预先用1×多聚赖氨酸溶液处理12孔板后，将293T细胞系，293T-CD40L-sBAFF细胞系与293T-CD40L细胞系铺入12孔板。待细胞密度长到30％左右时，使用射线以95Gy辐照细胞或丝裂霉素C处理3h，使细胞停止生长。观察12h，确认细胞停止生长。
随后将B细胞铺入12孔板与饲养细胞共培养。经过一系列条件摸索试验后，发明人最终将B细胞的密度定位5×105个/孔。将B细胞按照5×105/ml悬于IMDM培养基中，在培养基中加入终浓度为2μg/ml CpG-ODN2006/2219，0.625μg/ml CsA(Cyclosporine A)，10ng/mL IL-2，10ng/ml IL-10和20ng/mL IL-4。随后将细胞悬液按照每孔1ml的量加入12孔板中与饲养细胞共培养。每隔4天，将B细胞使用差速离心或密度梯度离心的办法与饲养细胞分离，台盼蓝染色计数，并按照5×105个/孔的密度转入铺有新的饲养细胞的12孔板。
ELISPOT实验
测量细胞IFN-γ的ELISPOT实验采用了达科为公司的human IFN-γprecoated ELISPOT  kit。将调整好浓度的细胞悬液加入实验孔，其中实验组加入与CD40L-B细胞或CD40L-sBAFF-B细胞共培养的CD8+T细胞，阳性对照组加入被PHA刺激过CD8+T细胞，阴性对照组加入未刺激过的CD8+T细胞。CD8+T细胞的密度定为105/孔。背景负对照组加入等体积RMPI 1640。加完样品后，37℃孵育16-20h。
第二天，倒去孔内培养基，加入4℃的去离子水4℃下裂解细胞10min。使用1×ELISPOT洗脱液洗涤5-7次后，加入抗体工作液在37℃孵育1h。随后倒去抗体，使用1×ELISPOT洗脱液洗涤5-7次后，再加入HRP工作液在37℃孵育1h。
孵育结束后，再使用1×ELISPOT洗脱液洗涤5-7次，加入配置好的AEC显色液室温避光反应25min。反应结束后，用去离子水洗涤5-7次，将板放于阴凉处，待其晾干后用CTL Immunospot S5core analyzer检测阳性斑点数，CTL ImmunoSpot software进行斑点计数和统计学分析。
实验结果
发明人使用293T细胞来构建饲养细胞系。将包装好的带有膜型CD40L序列的慢病毒感染人293T细胞，经流式细胞术多轮筛选后得到稳定的细胞系(293T-CD40L细胞系)。人BAFF的1-46位氨基酸为胞内段，47-73位为跨膜区域，74-285为胞外段。BAFF在体内被furin蛋白酶切割后，133-285位氨基酸形成可溶性的BAFF，为BAFF发挥功能的主要形式。因此，发明人克隆了BAFF的133-285位氨基酸。为使BAFF能更好的分泌到胞外，发明人通过精心设计，在BAFF的N端插入了一段IL-2的分泌肽。同时，为了方便稍后检测分泌到上清液中的BAFF的量，发明人在可溶性BAFF的C端融合表达了Flag标签的序列。该融合蛋白被克隆到慢病毒载体cPPT-IRES-RFP，与包装质粒psPAX和包膜蛋白VSVG共同转染293T-CD40L细胞系以包装病毒。经流式细胞术多轮筛选RFP阳性细胞后得到稳定的GFP-RFP双阳性细胞系(293T-CD40L-sBAFF)。本实验构建的质粒如图1所示。
随后，发明人使用新构建的293T-CD40L-sBAFF细胞系体外培养人外周血原代B细胞，培养方法如前所述。为了评估293T-CD40L-sBAFF细胞系对人外周血原代B细胞的扩增作用，发明人将293T-CD40L-sBAFF细胞系扩增B细胞的效果与之前文章所报道的293T-CD40L，以及终浓度分别为10ng/ml，20ng/ml，40ng/ml的GMP级重组人可溶性BAFF(rhBAFF)对B细胞的扩增效果做了比较。结果表明，293T-CD40L-sBAFF细胞系对B细胞的扩增能力好于之前报道的293T-CD40L细胞系，以及加入了终浓度为10ng/ml rhBAFF的293T-CD40L细胞系，如图2所示。
除了通过细胞计数观察B细胞的扩增能力外，发明人还通过CFSE染色实验进一步分析 了B细胞的扩增情况。CFSE结果显示，293T-CD40L-sBAFF使B细胞的扩增能力更强，具有扩增活性的细胞比例也更高，提示293T-CD40L-sBAFF细胞系与293T-CD40L细胞系相比具有更强的扩增B细胞的能力，如图3所示。
由于激活的B细胞可以表达MHC类分子(MHC-I,MHC-II)，共刺激因子(CD80，CD86)和粘附分子(CD54)，具有呈递抗原并激活T细胞的能力，而共刺激分子CD80和CD86的表达水平与B细胞的抗原呈递功能密切相关，在共培养后收取B细胞并使用流式细胞术检测表面的CD80和CD86水平。结果显示，在共培养后，与293T-CD40L-sBAFF细胞系共培养的B细胞，其表面CD80，CD86分子有更高的表达水平。除CD80和CD86外，其他参与B细胞抗原呈递功能的协同刺激分子，如CD275(ICOSLG)，CD70(CD27L)表达均有上调，如图4所示。
在体外实验中发明人得到参与细胞抗原递呈的共刺激分子有所抬高，发明人进一步验证293T-CD40L-sBAFF细胞系培养得到的B细胞能否抬高肿瘤特异性抗原递呈的能力，从来在肿瘤过继回输治疗中发挥功能。黑色素瘤作为最好的肿瘤治疗模型，发明人选择了黑色素瘤特异性抗原表位NY-ESO-1，使用293T-CD40L-sBAFF细胞系培养得到的B细胞与抗原肽共孵育，进行抗原的递呈。如图5a，b，c所示，使用293T-CD40L-sBAFF细胞系培养得到的B细胞递呈抗原的CD8细胞，在ELISPOT实验中可以分泌更多的IFN-γ，并且在细胞杀伤实验中，杀伤效率几乎达到了100％。为了进一步验证，使用293T-CD40L-sBAFF细胞系培养得到的B细胞递呈抗原的CD8细胞在体内也有更好的抗肿瘤活性，发明人构建了黑色素瘤的体内实验模型。如图5d，e所示，发明人将教育好的CTL细胞过继回输到小鼠体内，并对肿瘤大小进行测量，实验结果显示，由于293T-CD40L-sBAFF细胞系培养得到的B细胞递呈抗原的能力增强，所以教育得到的CTL在体内也具有更好的抗肿瘤活性。
参考文献：
Do,R.K.,Hatada,E.,Lee,H.,Tourigny,M.R.,Hilbert,D.,&Chen-Kiang,S.(2000).Attenuationof apoptosis underlies B lymphocyte stimulator enhancement of humoral immune response.The Journal of experimental medicine,192(7),953-964.
Garrone,P.,Neidhardt,E.-M.,Garcia,E.,Galibert,L.,Van Kooten,C.,&Banchereau,J.(1995).Fas ligation induces apoptosis of CD40-activated human B lymphocytes.The Journal of experimental medicine,182(5),1265-1273.
Mackay,F.,&Schneider,P.(2009).Cracking the BAFF code.Nat Rev Immunol,9(7),491-502.doi:10.1038/nri2572
Moore,P.A.,Belvedere,O.,Orr,A.,Pieri,K.,LaFleur,D.W.,Feng,P.,...Parmelee,D. (1999).BLyS:member of the tumor necrosis factor family and B lymphocyte stimulator. Science, 285(5425),260-263.
Schultze,J.L.,Michalak,S.,Seamon,M.J.,Dranoff,G.,Jung,K.,Daley,J.,...Nadler,L.M.(1997).CD40-activated human B cells:an alternative source of highly efficient antigen presenting cells to generate autologous antigen-specific T cells for adoptive immunotherapy.Journal of Clinical Investigation,100(11),2757.
Yang,M.,Hase,H.,Legarda-Addison,D.,Varughese,L.,Seed,B.,&Ting,A.T.(2005).B cell maturation antigen, the receptor for a proliferation-inducing ligand and B cell-activating factor of the TNF family,induces antigen presentation in B cells.The Journal of Immunology,175(5),2814-2824.
Zhang,X.,Li,L.,Choe,J.,Krajewski,S.,Reed,J.C.,Thompson,C.,&Choi,Y.S.(1996).Up-regulation of Bcl-x L expression protects CD40-activated human B cells from Fas-mediated apoptosis.Cellular immunology,173(1),149-154.

Claims (9)

  1. 分泌型BAFF肽，其氨基酸序列由BAFF的133-285位和其N端的IL-2肽组成，BAFF的133-285位的氨基酸序列为：AVQGPEETVTQDCLQLIADSETPTIQKGSYTFVPWLLSFKRGSALEEKENKILVKETGYFFIYGQVLYTDKTYAMGHLIQRKKVHVFGDELSLVTLFRCIQNMPETLPNNSCYSAGIAKLEEGDELQLAIPRENAQISLDGDVTFFGALKLL，IL-2肽的氨基酸序列为：MYRMQLLSCIALSLALVTNS。
  2. 一种分泌型BAFF肽的表达载体，其插入有表达权利要求1所述分泌型BAFF肽的核苷酸序列。
  3. 根据权利要求2所述的表达载体，其特征在于：载体还插入有表达膜型CD40L的核苷酸序列。
  4. 一种表达分泌型BAFF肽的细胞，其转染有权利要求2所述的表达载体。
  5. 根据权利要求4所述的细胞，其特征在于：细胞还转染有表达膜型CD40L的载体。
  6. 根据权利要求4所述的细胞，其特征在于：细胞转染有权利要求3所述的表达载体。
  7. 根据权利要求4～6任意一项所述的细胞，其特征在于：细胞为293T细胞。
  8. 一种人外周B细胞体扩增方法，其特征在于：将人外周B细胞与同时表达分泌型BAFF肽和膜型CD40L的细胞共培养。
  9. 根据权利要求8所述的人外周B细胞体扩增方法，其特征在于：将人外周B细胞培养在有胰岛素和转铁蛋白支持的IMDM培养基中，并添加CpG2006/2219和环孢素A刺激B细胞扩增，添加人IL-4，IL-2，IL-10维持细胞生长。
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