WO2018072105A1 - 一种干细胞样记忆性t细胞体外诱导剂及方法 - Google Patents
一种干细胞样记忆性t细胞体外诱导剂及方法 Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
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- the invention relates to a cell culture inducer and a method for inducing cells, in particular to a stem cell-like memory T cell in vitro inducer and method.
- Mature T cells are immunologically active and can be recirculated through lymphatic vessels, peripheral blood, and tissue fluids to perform functions such as cellular immunity and immune regulation.
- the recycling of T cells facilitates extensive exposure to antigenic substances entering the body, enhances the immune response, and maintains immune memory over a longer period of time.
- TSCM Stem Memory Cell
- TCM central memory T cells
- Effector memory T cells (TEM) and effector T cells (TEF) which in turn produce a large number of effector molecules such as IFN- ⁇ , produce more powerful lethality, and have strong self-renewal ability while having differentiation potential.
- TEM Effector memory T cells
- TEZ effector T cells
- TSCM cells show stronger anti-tumor ability, their self-renewal ability in vivo, the intensity of the second immune response, and the retention time in the body are stronger than the memory cells in the traditional sense.
- the in vitro transplantation experiment shows that it has Very strong survivability.
- TSCM cells have a strong viability to prolong the survival time of cells implanted in vivo, and their own anti-tumor function cytokines make TSCM cells the most effective cell treatment cell type. Therefore, treatment with in vitro expanded TSCM will become a new approach and means of cell therapy.
- T cells with stem cell characteristics for treatment will change the traditional drug and immunotherapy mode, opening up new technologies, new methods, and new ideas for cell therapy.
- the key to truly utilizing TSCM cells for cell therapy is to fully understand the characteristics of TSCM cells and find a way to maximize the expansion of TSCM cells in vitro. Therefore, a purposeful and targeted method for advancing the expansion of TSCM cells in vitro is found to be economical and convenient. Cytokines or drugs with small side effects are of paramount importance.
- the object of the present invention is to provide an in vitro inducer and method for stem cell-like memory T cells.
- a stem cell-like memory T cell in vitro expansion induction medium the main body of which is a T cell culture medium, and a histamine H1 receptor antagonist is added to the medium.
- the histamine H1 receptor antagonist is selected from the group consisting of diphenhydramine, promethazine, loratadine, cetirizine.
- the histamine H1 receptor antagonist is diphenhydramine, and its final concentration in the medium is 10 to 50. ⁇ M, more preferably 25 ⁇ M.
- a method for inducing expansion of stem cell-like memory T cells in vitro comprises the following steps:
- the inducer Anti-CD3 antibody, Anti-CD28 antibody, and histamine H1 receptor antagonist are added to the culture medium to continue the induction culture, and the histamine H1 receptor antagonist is timely supplemented during the induction culture;
- the histamine H1 receptor antagonist is selected from the group consisting of diphenhydramine, promethazine, loratadine, cetirizine.
- the histamine H1 receptor antagonist is diphenhydramine, and its final concentration in the medium is 10 to 50. ⁇ M, more preferably 25 ⁇ M.
- Histamine H1 receptor antagonists have long been tested in clinical practice for human safety, avoiding the long cycle of new drug development. At the same time, histamine H1 receptor antagonist has the advantages of low cost and good safety as a mature drug.
- TSCM cells which are induced to differentiate and expand by histamine H1 receptor antagonist can be used for returning without special treatment. It provides a strong practical basis for the treatment of tumor cells, with important development value and promotion significance.
- Figure 1 shows the effect of different histamine H1 receptor antagonists on the induction of TSCM cells in vitro
- Figure 2 is a statistical analysis of the effect of different histamine H1 receptor antagonists on the in vitro induction of TSCM cells
- Figure 4 is a statistical analysis of the in vitro induced expansion of TSCM cells by different concentrations of histamine H1 receptor antagonist Diphenhydramine;
- Figure 5 shows the cytotoxicity of diphenhydramine at different concentrations on CD8 + T cells.
- a stem cell-like memory T cell in vitro expansion induction medium the main body of which is a T cell culture medium, and a histamine H1 receptor antagonist is added to the medium.
- the histamine H1 receptor antagonist is selected from the group consisting of a first generation histamine H1 receptor antagonist, a second generation histamine H1 receptor antagonist, and a third generation histamine H1 receptor antagonist.
- the histamine H1 receptor antagonist is selected from the group consisting of diphenhydramine, promethazine, chlorpheniramine, cyproheptadine, dechlorpromazine, hydroxyzine, loratadine, and citrate.
- the histamine H1 receptor antagonist is selected from the group consisting of diphenhydramine, promethazine, loratadine, cetirizine.
- the H1 receptor antagonist may be used singly or in combination. One skilled in the art can experiment to determine the appropriate mode of use.
- the histamine H1 receptor antagonist is diphenhydramine, and its final concentration in the medium is 10 to 50. ⁇ M, more preferably 25 ⁇ M.
- a method for inducing expansion of stem cell-like memory T cells in vitro comprises the following steps:
- the inducer Anti-CD3 antibody, Anti-CD28 antibody, and histamine H1 receptor antagonist are added to the culture medium to continue the induction culture, and the histamine H1 receptor antagonist is timely supplemented during the induction culture;
- the histamine H1 receptor antagonist is selected from the group consisting of diphenhydramine, promethazine, chlorpheniramine, cyproheptadine, dechlorohydroxyzine, and hydroxy Oxazine, loratadine, cetirizine, terfenadine, astemizole, albastatin, fifenadine, aristine, mequitazine, mizolastine, ebasidine, Fexofenadine, demethylastemizole, decarboxylated loratadine. Further, the histamine H1 receptor antagonist is selected from the group consisting of diphenhydramine, promethazine, loratadine, cetirizine.
- the H1 receptor antagonist may be used singly or in combination. One skilled in the art can experiment to determine the appropriate mode of use.
- the histamine H1 receptor antagonist is diphenhydramine, and its final concentration in the medium is 10 to 50. ⁇ M, more preferably 25 ⁇ M.
- the initial CD8 + T cells (CD8 + , CD45RO - , CD62L + ) were sorted by flow cytometry.
- the sorted CD8 + T cells were plated in 96-well plates. According to the experimental design, the expected number of cells per well was 1 ⁇ 10 5 ;
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Abstract
提供一种干细胞样记忆性T细胞（TSCM）体外诱导剂及体外扩增诱导TSCM的方法，该方法包括在T细胞诱导培养基中额外添加组胺H1受体拮抗剂，以使CD8初始T细胞在体外快速诱导分化扩增为TSCM细胞。
Description
技术领域
本发明涉及一种细胞培养诱导剂及细胞的诱导方法，特别涉及一种干细胞样记忆性T细胞体外诱导剂及方法。
背景技术
成熟的T细胞具有免疫活性，可通过淋巴管、外周血和组织液等进行再循环，发挥细胞免疫及免疫调节等功能。T细胞的再循环有利于广泛接触进入体内的抗原物质，加强免疫应答，较长期保持免疫记忆。通过体外诱导扩增记忆性T细胞然后回输至体内，有望治疗某些特定的疾病。
记忆性T细胞的诱导扩增方法中，比较成熟的是使用抗CD3和抗CD28抗体联合刺激诱导分化，刺激分化的T细胞明显增殖活化，原态细胞减少，明显转为记忆细胞表型。然而这种T细胞在体内存活的时间较短，疗效难以满足治疗的需要。
干细胞样记忆性T细胞(T Stem Memory Cell, TSCM)
是近年来新发现的一个T细胞亚群，这群细胞处于记忆细胞的分化上游，具有干细胞的特征，具有较强的多向分化潜能，TSCM细胞能分化为中枢性记忆性T细胞（TCM），效应性记忆T细胞（TEM）和效应性T细胞（TEF），进而产生大量的IFN-γ等效应分子，产生更为强大的杀伤力，在具有分化潜能的同时具有较强的自我更新能力。TSCM细胞展现出了更强的抗肿瘤能力，其在体内的自我更新能力，第二次免疫应答的强度，在机体内停留时间均强于传统意义上的记忆性细胞，体外移植实验表明其具有很强的生存能力。
临床上细胞治疗的过继回输面临着处于终末阶段的细胞很难在体外培养的条件下长期生存，并且难以获得足够量的细胞，而TSCM细胞所具备的这些特征解决了临床上移植细胞体外扩增难的问题。并且，TSCM细胞有很强的生存能力使得植入体内细胞的生存时间延长，自身产生的抗肿瘤功能的细胞因子也使得TSCM细胞成为最有效的细胞治疗细胞类型。所以利用体外扩增的TSCM进行治疗将成为细胞治疗新的途径与手段。
利用具有干细胞特性的T细胞进行治疗的方式将改变传统的药物与免疫治疗的模式，开辟细胞治疗的新技术，新方法，新思路。真正利用TSCM细胞进行细胞治疗的关键在于充分认识TSCM细胞特性并且找到在体外使得TSCM细胞大量扩增的方法，因此，有目的、有针对性的推进体外扩增TSCM细胞的技术方法，找到经济方便副作用小的细胞因子或者药物，具有极为重要的意义。然而，现有技术中缺乏较为简便高效的TSCM体外扩增方法。
发明内容
本发明的目的在于提供一种干细胞样记忆性T细胞体外诱导剂及方法。
一种干细胞样记忆性T细胞体外扩增诱导培养基，其主体为T细胞培养基，培养基中添加有组胺H1受体拮抗剂。
作为上述培养基的进一步改进，组胺H1受体拮抗剂选自苯海拉明、异丙嗪、氯雷他定、西替利嗪。
作为上述培养基的进一步改进，组胺H1受体拮抗剂为苯海拉明，其在培养基中的终浓度为10～50
μM，更佳为25μM。
一种干细胞样记忆性T细胞体外扩增诱导方法，包括如下步骤：
1) 分选出CD8+ T 细胞，置于T细胞培养液中培养；
2)
在培养基中加入诱导剂Anti-CD3抗体、Anti-CD28抗体、组胺H1受体拮抗剂，继续诱导培养，在诱导培养的过程中，适时补充组胺H1受体拮抗剂；
3) 培养结束，分选出需要的TSCM。
作为上述干细胞样记忆性T细胞体外扩增诱导方法的进一步改进，组胺H1受体拮抗剂选自苯海拉明、异丙嗪、氯雷他定、西替利嗪。
作为上述干细胞样记忆性T细胞体外扩增诱导方法的进一步改进，组胺H1受体拮抗剂为苯海拉明，其在培养基中的终浓度为10～50
μM，更佳为25μM。
发明人通过实验发现可以在T细胞诱导培养基中额外添加组胺H1受体拮抗，可以在体外快速将CD8初始T细胞诱导分化扩增为TSCM细胞，且TSCM细胞占CD8细胞比例上有明显抬高，TSCM细胞的绝对数有显著抬高。诱导分化扩增的TSCM细胞保持了TSCM细胞的特性和功能。
组胺H1受体拮抗剂，对人体的安全性早已经过临床实践的检验，避免了新药研发漫长的周期。同时，组胺H1受体拮抗剂作为一种成熟的药物，具有成本低廉、安全性好的优点，加入组胺H1受体拮抗剂诱导分化扩增的TSCM细胞无需进行特别的处理即可用回输，为肿瘤细胞治疗提供了强有力的实践基础，具有重要的开发价值和推广意义。
附图说明
图1 为不同组胺H1受体拮抗剂对TSCM细胞体外诱导扩增效果；
图2 为不同组胺H1受体拮抗剂对TSCM细胞体外诱导扩增效果的统计学分析；
图3
为不同浓度的组胺H1受体拮抗剂苯海拉明(Diphenhydramine)对TSCM细胞的体外诱导扩增效果；
图4为不同浓度的组胺H1受体拮抗剂苯海拉明(Diphenhydramine)对TSCM细胞的体外诱导扩增效果统计学分析；
图5为不同浓度苯海拉明对CD8+ T细胞细胞毒性检测。
具体实施方式
一种干细胞样记忆性T细胞体外扩增诱导培养基，其主体为T细胞培养基，培养基中添加有组胺H1受体拮抗剂。
作为上述培养基的进一步改进，组胺H1受体拮抗剂选自第一代组胺H1受体拮抗剂、第二代组胺H1受体拮抗剂和第三代组胺H1受体拮抗剂。
作为上述培养基的进一步改进，组胺H1受体拮抗剂选自苯海拉明、异丙嗪、氯苯那敏、赛庚啶、去氯羟嗪、羟嗪、氯雷他定、西替利嗪、特非那丁、阿斯咪唑、艾巴斯丁、非索那丁、阿化斯丁、甲喹吩嗪、咪唑斯丁、依巴斯丁、非索非那丁、去甲基阿司咪唑、脱羧基氯雷他啶。进一步的，组胺H1受体拮抗剂选自苯海拉明、异丙嗪、氯雷他定、西替利嗪。
H1受体拮抗剂既可以单独使用，也可以组合使用。本领域技术人员可以进行尝试，确定合适的使用方式。
作为上述培养基的进一步改进，组胺H1受体拮抗剂为苯海拉明，其在培养基中的终浓度为10～50
μM，更佳为25μM。
一种干细胞样记忆性T细胞体外扩增诱导方法，包括如下步骤：
1) 分选出CD8+ T 细胞，置于T细胞培养液中培养；
2)
在培养基中加入诱导剂Anti-CD3抗体、Anti-CD28抗体、组胺H1受体拮抗剂，继续诱导培养，在诱导培养的过程中，适时补充组胺H1受体拮抗剂；
3) 培养结束，分选出需要的TSCM。
作为上述干细胞样记忆性T细胞体外扩增诱导方法的进一步改进，组胺H1受体拮抗剂选自苯海拉明、异丙嗪、氯苯那敏、赛庚啶、去氯羟嗪、羟嗪、氯雷他定、西替利嗪、特非那丁、阿斯咪唑、艾巴斯丁、非索那丁、阿化斯丁、甲喹吩嗪、咪唑斯丁、依巴斯丁、非索非那丁、去甲基阿司咪唑、脱羧基氯雷他啶。进一步的，组胺H1受体拮抗剂选自苯海拉明、异丙嗪、氯雷他定、西替利嗪。
H1受体拮抗剂既可以单独使用，也可以组合使用。本领域技术人员可以进行尝试，确定合适的使用方式。
作为上述干细胞样记忆性T细胞体外扩增诱导方法的进一步改进，组胺H1受体拮抗剂为苯海拉明，其在培养基中的终浓度为10～50
μM，更佳为25μM。
下面结合附图和具体实验进一步详细说明本发明。除非特别说明，本发明采用的试剂、设备和方法为本技术领域常规市购的试剂、设备和常规使用的方法。
诱导剂对对TSCM细胞体外诱导扩增效果
1) 流式细胞仪分选出初始CD8+ T
细胞(CD8+、CD45RO-
、CD62L+)，将分选出来的CD8+T细胞铺在12孔板中，按照实验设计，每孔预期的细胞数为1×106个；
2) 对铺板后的CD8+T细胞加入1μg/ml（终浓度）Anti-CD3抗体 +
1μg/ml（终浓度）Anti-CD28抗体，以及不同的组胺H1受体拮抗剂，分别为25μM的苯海拉明（Diphenhydramine）、西替利嗪（Cetirizine）和氯雷他定（Loratadine）；对照组加入相应体积的PBS。每种处理设置三个复孔作为平行对照，并在细胞培养的第4天，第7天，第10天，第13天补加药物或PBS刺激；
3) 细胞培养14天后，用细胞计数仪进行计数，孵育检测CD8+
TSCM的流式抗体（CD8、CD45RA、CD45RO、CCR7、CD62L、CD95、CD122）运用流式细胞仪检测。对照组与实验组均为同一供体的CD8+
T细胞，每个时间点分别取不同供体的细胞进行检测。
实验结果如图1和2所示。从图1和2中可以看出，随机选择每一代的组胺受体拮抗剂均可在体外促进CD8+、CD45RA+、CD45RO-、CCR7+、CD62L+、CD95+、CD122+TSCM细胞的分化发育，最终显著抬高TSCM的比例，而其中苯海拉明的效果最为显著。
不同浓度诱导剂对TSCM细胞的体外诱导扩增效果
1) 流式细胞仪分选出初始CD8+ T
细胞(CD8+、CD45RO-
、CD62L+)，将分选出来的CD8+T细胞铺在12孔板中，按照实验设计，每孔预期的细胞数为1×106个；
2) 对铺板后的CD8+T细胞加入1μg/ml（终浓度）Anti-CD3抗体 +
1μg/ml（终浓度）Anti-CD28抗体，以及不同浓度的组胺H1受体拮抗剂苯海拉明，每种处理设置三个复孔作为平行对照，并在细胞培养的第4天，第7天，第10天，第13天补加药物以及细胞因子刺激；
3) 细胞培养14天后，用细胞计数仪进行计数，孵育检测CD8+
TSCM的流式抗体（CD8、CD45RA、CD45RO、CCR7、CD62L、CD95、CD122）运用流式细胞仪检测。对照组与实验组均为同一供体的CD8+
T细胞，每个时间点分别取不同供体的细胞进行检测。
实验结果如图3和图4所示。从图中可以看出，TSCM的体外诱导扩增和组胺H1受体拮抗剂的浓度成正相关关系，TSCM与苯海拉明相关系数r2=0.9913。结合药物浓度对细胞活性的影响，苯海拉明体外诱导扩增TSCM的最适浓度为25μM。
不同浓度的组胺H1受体拮抗剂苯海拉明对CD8+ T细胞的体外毒性的研究
流式细胞仪分选出初始CD8+ T
细胞(CD8+、CD45RO-
、CD62L+)，将分选出来的CD8+T细胞铺在96孔板中，按照实验设计，每孔预期的细胞数为1×105个；
对铺板后的CD8+T细胞加入1μg/ml（终浓度）Anti-CD3抗体 +
1μg/ml（终浓度）Anti-CD28抗体，以及不同浓度的组胺H1受体拮抗剂苯海拉明，每种处理设置三个复孔作为平行对照。培养72小时之后，使用CCK8细胞活性检测试剂盒，对细胞活性进行检测。
实验结果如图5所示，表明不同浓度的组胺H1受体拮抗剂苯海拉明对细胞活性没有影响。与之前文献报道可以在体外诱导TSCM的化合物TWS119相比，毒性非常低。
Claims (10)
- 组胺H1受体拮抗剂在制备干细胞样记忆性T细胞体外扩增诱导剂中的应用。
- 根据权利要求1所述的应用，其特征在于：组胺H1受体拮抗剂选自第一代组胺H1受体拮抗剂、第二代组胺H1受体拮抗剂和第三代组胺H1受体拮抗剂。
- 根据权利要求2所述的应用，其特征在于：组胺H1受体拮抗剂选自苯海拉明、异丙嗪、氯苯那敏、赛庚啶、去氯羟嗪、羟嗪、氯雷他定、西替利嗪、特非那丁、阿斯咪唑、艾巴斯丁、非索那丁、阿化斯丁、甲喹吩嗪、咪唑斯丁、依巴斯丁、非索非那丁、去甲基阿司咪唑、脱羧基氯雷他啶。
- 根据权利要求1所述的应用，其特征在于：干细胞样记忆性T细胞为CD8+ T细胞。
- 一种干细胞样记忆性T细胞体外扩增诱导培养基，其主体为T细胞培养基，其特征在于：培养基中添加有组胺H1受体拮抗剂。
- 根据权利要求5所述的干细胞样记忆性T细胞体外扩增诱导培养基，其特征在于：组胺H1受体拮抗剂选自苯海拉明、异丙嗪、氯雷他定、西替利嗪。
- 根据权利要求5所述的干细胞样记忆性T细胞体外扩增诱导培养基，其特征在于：组胺H1受体拮抗剂为苯海拉明，其在培养基中的终浓度为10～50 μM。
- 一种干细胞样记忆性T细胞体外扩增诱导方法，包括如下步骤：1) 分选出CD8+ T 细胞，置于T细胞培养液中培养；2) 在培养基中加入诱导剂Anti-CD3抗体、Anti-CD28抗体、组胺H1受体拮抗剂，继续诱导培养，在诱导培养的过程中，适时补充组胺H1受体拮抗剂；3) 培养结束，分选出需要的TSCM。
- 根据权利要求8所述的方法，其特征在于：组胺H1受体拮抗剂选自苯海拉明、异丙嗪、氯雷他定、西替利嗪。
- 根据权利要求8所述的方法，其特征在于：组胺H1受体拮抗剂为苯海拉明，其在培养基中的终浓度为10～50 μM。
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