1。隔离的PBMC <ol><li>绘制成一个BD真空采血管彩管的细胞与肝素钠的制备管血（7.5-8.0毫升）。管含有钠抗凝和聚蔗糖Hypaque密度流体，加上聚酯凝胶屏障，这两种液体分开。在一个水平旋翼为20分钟1800 × 克 （摇摆头），在室温（18℃至25℃）离心管/血液样本。切换离心机刹车关闭。 </li><li>离心后，层的顺序发生如下（从上到下看到）：一）等离子体 - B）外周血单个核细胞（PBMC）和血小板 - C）密度解决方案 - D）聚酯凝胶 - E），粒细胞 - F）红血细胞（ 图1）。 </li><li>收集的等离子层的一小部分，离开，而不会干扰细胞层的5至10毫米以上相间的血浆。血浆可用于文化（2.5节）。 </li><li>收获丰富的分数（​​PBMC），用吸管转移到一个15毫升的锥形管相间。 </li><li>洗净用10毫升的外周血单个核细胞的磷酸盐缓冲液（PBS），颠倒离心管5次，然后离心5分钟400 ×克。 </li><li>两次重复洗涤步骤，然后在5毫升的PBS重悬细胞沉淀。确定细胞数量。一般1.3 × 10 6个细胞，恢复从1毫升全血。 </li><li>确定γδT在外周血单个核细胞，通过流式细胞仪（第三部分）（ 图2）的频率和表型。 </li></ol> 2。扩大γδT细胞<ol><li>细胞悬浮液15 ml锥形管离心5 min，室温400 X克，丢弃上清液。 </li><li>准备加入人IL - 2（IL - 2）和zoledronate（择泰）1000 IU / ml和5微米的终浓度分别培养基（CM）。 ALyS203（细胞科技职业学院）或OpTmizer（Invitrogen公司）媒体支持γδT细胞（参考文献6日和9的更多信息）良好的扩展。择泰（4 mg/5-ml小瓶）以液态的形式提供。为了准备5微米的解决方案，30毫升培养液中添加50μL择泰。 </li><li>悬浮颗粒细胞在培养液中，并调整到1 × 10 6细胞/ ml 。 </li><li>吸取1毫升含有1 × 10 6个细胞，每孔24孔板的CM。大型文化，细胞可以接种0.5 × 10 6细胞/厘米2，根据板块井，盘，或烧瓶表面积。 </li><li>添加自体血浆（1.3节），汇集人类AB血清，或FCS因此，它是文化的体积（100μL每孔24孔板）的约10％。广场板块，在加湿37℃，5％二氧化碳培养箱中培养24-48小时。 </li><li>保持在0.5-2 × 10 6细胞/ ml的细胞密度的文化。添加新鲜培养基中含有人IL - 2（1000 IU / ml）的（不择泰）每2-3天，并转移到新井或在必要时烧瓶的培养细胞，根据细胞的增殖程度（ 图3） 。供应血浆或血清中的介质，使血药浓度可维持至少1％。 </li><li>每天12-14和确定的频率，表型和γδT的流式细胞仪检测细胞功能（见下文）收获细胞。 </li></ol> 3。表型分析流式细胞仪<ol><li>转移200μL的样品含有2 × 10 5细胞荧光激活细胞分选（FACS）管。 </li><li>加入2毫升冷PBS离心5分钟400 × 克 。然后，重新悬浮颗粒在50μL流式细胞仪缓冲液（PBS + 1％FCS + 0.1％的叠氮化钠）。添加5μL的每个抗体样品（ 表1中列出的单克隆抗体）。 </li><li>冰上放置在20分钟的黑暗。 </li><li>每个样品中加入2毫升流式细胞仪缓冲，然后涡。离心机样本为400 × 克在4 ° C 5分钟小心倒出上清液。 </li><li>细胞重悬于300μL流式细胞仪缓冲区和涡流。流式细胞仪（ 图2）分析样品。 </li></ol> 4。 IFN -γ的生产检测10 <ol><li>检测前一天，准备刺激细胞培养3-5 X10 5 Daudi细胞/ ml的RPMI 1640培养液加10％FCS（RPMI - 10）择泰（5微米）（以下简称指定的Z -达乌迪姆瓦）过夜。 </li><li>在2 × 10 6细胞/ ml，收集在RPMI - 10的Z - Daudi和重悬。加入100μL的Z -达乌迪姆瓦（2X10 5）以及每一个圆底96孔板。 </li><li>准备在2 × 10 6细胞/ ml的RPMI - 10包含在20微克/毫升BrefeldinγδT细胞。转移100μLγδT细胞悬液（2 × 10 5）每孔含有的Z - Daudi细胞或控制井（100μLRPMI - 10，或20毫微克/毫升的佛波醇-12 -肉豆蔻13醋酸RPMI - 10[PMA]加2微克/毫升的离子霉素）。 </li><li>混合移液器向上和向下几次。在37℃，5％CO2培养箱孵育4小时。 </li><li>在4 ° C离心​​5分钟400 × 克板，并在200μL冷PBS重悬的颗粒。 </li><li>转移的样本，以流式细胞仪管。添加4毫升的冷PBS离心5分钟400 × 克管4 ° C。 </li><li>重悬在50μLFITC标记的抗TCRVγ9（5μL）和PE/Cy5-conjugated抗CD3 mAb（2.5μL）流式细胞仪缓冲颗粒。避光孵育室温为15分钟。 </li><li>加入100μL试剂1 IntraPrep和孵化在室温为15分钟。每管和离心5 min，室温400 X克加入4毫升的PBS。 </li><li>取出吸上清液，加100μLIntraPrep试剂2。为在室温下5分钟孵育无晃动。 </li><li>添加5μLPE标记的抗IFN -γ单克隆抗体的试管。避光孵育室温为15分钟。 </li><li>每管和离心5 min，室温400 X克加入4毫升的PBS。移除吸上清，重悬在0.5毫升流式细胞仪缓冲的细胞沉淀。 </li><li>流式细胞仪分析细胞。门上的CD3 +TCRVγ9+细胞研究干扰素-γ（ 图4）的表达。 </li></ol> 5。代表性的成果： 重要的是要确定在启动文化PBMC中γδT细胞百分比。 如图所示。 2，CD3 +TCRVγ9的百分比 +γδT在外周血单个核细胞为1.6％0天。优势种群CD27 + CD45RA +幼稚或CD27 + CD45RA -中央记忆体的表型。 γδT细胞的有效刺激时，便形成了第3-5天的集群（图3 A和 B） 。当集群的形成被推迟，增长的其他类型的细胞，如CD4 +或CD8 +αβT细胞或NK细胞可以主宰的γδT细胞（ 图3 C和D）的生长。经过14天的文化，γδT细胞的频率增加超过93.8％的培养细胞中成功的γδT细胞的文化（图 2五）。培养γδT细胞上调NKG2D及CD69的表达（ 图2 G和H）。他们显示CD27 - CD45RA -效应记忆体的表型（ 图2架F）。 γδT细胞的功能与细胞因子的产生和细胞毒性方面进行了评价。细胞内IFN -γ染色表明，γδT细胞产生的PMA /离子霉素治疗或Z - Daudi细胞中IFN -γ，积累的IPP zoledronate治疗后（ 图4）。这些结果表明，zoledronate可以有效地刺激和扩大功能γδT细胞。 <table border="1"><tbody><tr><td> 管 </td><td> FITC标记 </td><td> 体育 </td><td> 幼儿发展 </td><td> PE/Cy5 </td></tr><tr><td> 1 </td><td> CD3 </td><td> CD19 </td><td> CD45 </td><td> CD14的</td></tr><tr><td> 2 </td><td> CD3 </td><td> TCRαβ </td><td> CD4 + </td><td> CD8 + </td></tr><tr><td> 3 </td><td> CD3 </td><td></td><td></td><td> CD56 </td></tr><tr><td> 4 </td><td> TCRVγ9 </td><td> TCRαβ </td><td> CD45 </td><td> CD3 </td></tr><tr><td> 5 </td><td> TCRVγ9 </td><td> NKG2D </td><td></td><td></td></tr><tr><td> 6 </td><td> TCRVγ9 </td><td> CD69的</td><td></td><td></td></tr><tr><td> 7 </td><td> TCRVγ9 </td><td>鼠IgG1 </td><td></td><td></td></tr><tr><td> 8 </td><td> TCRVγ9 </td><td></td><td> CD45RA </td><td> CD27 </td></tr><tr><td> 9 </td><td> TCRVγ9 </td><td></td><td>鼠IgG1 </td><td>鼠IgG1 </td></tr></tbody></table> 表1。多色染色γδT细胞单克隆抗体。在我们的实验室进行的γδT细胞的表型分析的一个例子是如图所示。 2。 <img src="/files/ftp_upload/3182/3182fig1.jpg" alt="图1"/> 图1。PBMC的分离。绘制成一个BD真空采血管彩管的细胞与肝素钠的制备管血（7.5-8.0毫升），直接为20分钟，1800 X克离心。离心后，造成层从顶部博ttom：一）等离子体 - B）外周血单个核细胞和血小板 - C）密度的解决方案 - D）聚酯凝胶 - E），粒细胞 - F）红血细胞。 <img src="/files/ftp_upload/3182/3182fig2.jpg" alt="图2"/> 图2典型的表面γδT细胞表型。 PBMC的刺激zoledronate和IL - 2为14天。细胞染色，用FITC标记的抗- TCRVγ9PE/Cy5-labeled抗CD3监察γδT细胞（A和E）的扩展。 γδT细胞识别他们的表达TCRVγ9，CD27和CD45RA（B型和F），NKG2D的（C和G），或CD69的（D和H）的表达研究。 <img src="/files/ftp_upload/3182/3182fig3.jpg" alt="图3"/> 图3。代表γδT细胞的培养。 PBMC的刺激与IL - 2（1000国际单位/毫升）和zoledronate（5微米）。代表领域（IX71倒置显微镜[奥林巴斯] × 200）。集群和γδT细胞的聚集，可以观察到的第3天（A）和（二）5天，当γδT细胞，成功地扩大了。相比之下，没有集群或聚集γδT细胞的生长观察是不够的（C和D）。 <img src="/files/ftp_upload/3182/3182fig4.jpg" alt="图4"/> 图4。γ-干扰素的生产。 γδT细胞培养，RPMI - 10只（A）或PMA /离子霉素（B）或Z -达乌迪姆瓦（C）为4小时。首先，表面TCRVγ9表达染色，然后IFN -γ的生产是通过细胞内IFN -γ染色检查。 <img src="/files/ftp_upload/3182/3182fig5.jpg" alt="图5"/> 图5。γδT细胞文化的动力学。 （一）培养细胞的绝对数量，（二）γδT细胞，（三）在指定的时间点γδT细胞的绝对数量的百分比。

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没有利益冲突的声明。

这里介绍的方法，使γδT细胞从外周血单个核细胞的有效扩张。 γδT细胞激活，并通过zoledronate扩大和IL - 2制定完整的效应功能，细胞因子的产生和细胞毒性反映。据报道，合成phosphoantigens bromohydrin焦（BrHPP）和2 - 甲基3 - 丁烯-1 - 焦磷酸（2M3B1PP）也扩大γδT细胞，但是，他们不市售。相比之下，zoledronate已经授权作为择泰的临床应用。因此，一个可靠的试剂是容易获得。 培养?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>择泰</td><td>诺华制药K. K </td><td></td><td> zoledronate </td></tr><tr><td> PROLEUKIN </td><td>诺华制药</td><td></td><td>重组人IL - 2 </td></tr><tr><td> BD真空采血管彩管细胞制备与肝素钠管</td><td>屋宇署</td><td> 362753 </td><td></td></tr><tr><td>的RPMI1640 </td><td> Invitrogen公司</td><td> 21870-076 </td><td></td></tr><tr><td> ALyS203介质</td><td>细胞科学与技术学院</td><td> 0301-7 </td><td></td></tr><tr><td> OpTmizer </td><td> Invitrogen公司</td><td> 0080022SA </td><td></td></tr><tr><td> brefeldin一个</td><td>西格玛</td><td> B5936 - 200UL </td><td></td></tr><tr><td>佛波醇-12 - 肉豆蔻13醋酸乙烯酯（PMA） </td><td>西格玛</td><td> P1585 - 1MG </td><td></td></tr><tr><td>离子霉素</td><td>西格玛</td><td> 13909 - 1ML </td><td></td></tr><tr><td> IntraPrep </td><td> Beckman Coulter公司</td><td> A07803 </td><td></td></tr><tr><td>抗人CD3 - FITC或PE/Cy5 </td><td> Beckman Coulter公司</td><td> A07746 FITC A07749 PE/Cy5 </td><td></td></tr><tr><td>抗人CD4 - ECD </td><td> Beckman Coulter公司</td><td> 6604727 </td><td></td></tr><tr><td>抗人类CD8-PE/Cy5 </td><td> Beckman Coulter公司</td><td> 6607011 </td><td></td></tr><tr><td>抗人类CD14-PE/Cy5 </td><td> Beckman Coulter公司</td><td> A07765 </td><td></td></tr><tr><td>抗人CD19 - PE </td><td> Beckman Coulter公司</td><td> A07769 </td><td></td></tr><tr><td>抗人CD45 - ECD </td><td> Beckman Coulter公司</td><td> A07784 </td><td></td></tr><tr><td>抗人类CD56-PE/Cy5 </td><td> Beckman Coulter公司</td><td> A07789 </td><td></td></tr><tr><td>抗人TCRαβ- PE </td><td> Beckman Coulter公司</td><td> A39499 </td><td></td></tr><tr><td>抗人TCRVγ9- FITC </td><td> Beckman Coulter公司</td><td> IM1463 </td><td></td></tr><tr><td>抗人类CD27-PE/Cy5 </td><td> Beckman Coulter公司</td><td> 6607107 </td><td></td></tr><tr><td>抗人CD45RA - ECD </td><td> Beckman Coulter公司</td><td> IM2711 </td><td></td></tr><tr><td>抗人CD69 - PE </td><td>屋宇署</td><td> 555531 </td><td></td></tr><tr><td>抗人NKG2D的聚乙烯</td><td> Beckman Coulter公司</td><td> A08934 </td><td></td></tr><tr><td>反humalIFNγ的聚乙烯</td><td> Beckman Coulter公司</td><td> IM2717U </td><td></td></tr><tr><td>鼠IgG1同型对照PE </td><td> Beckman Coulter公司</td><td> A07796 </td><td></td></tr><tr><td>鼠IgG1同型对照ECD或PE/Cy5 </td><td> Beckman Coulter公司</td><td> A07797 A07798 </td><td></td></tr></tbody></table>

expansion of human peripheral blood  &gamma;&delta; t cells using zoledronate

人类γδT细胞能够识别和响应到多种应激性抗原，从而开发先天广泛的抗肿瘤和抗感染活性。1外周血中大多数γδT细胞的Vγ9Vδ2T细胞受体。这些细胞识别抗原在主要组织相容性复合体独立的方式，并发展强大的杀伤和Th1样的效应功能。因此，γδT细胞是肿瘤免疫治疗的有吸引力的候选人的效应细胞。 Vγ9Vδ2T细胞响应，如phosphoantigens（E）4 -羟基- 3 -甲基- 2 -己烯焦磷酸（HMBPP），这是通过类异戊二烯生物合成的细菌合成;和异戊烯基焦磷酸（IPP），这是生产在真核细胞通过甲羟戊酸途径，在生理条件下 ，3中，IPP的nontransformed细胞的生成是没有足够的γδ T细胞的活化。甲羟戊酸途径在肿瘤细胞失调， 导致3，因为IPP的积累和γδT细胞的活化。aminobisphosphonates（如帕米膦酸或zoledronate）抑制法尼基焦磷酸合成酶（制剂），酶的作用IPP的甲羟戊酸途径下游，细胞内水平IPP和sensitibityγδT细胞识别，可以治疗由aminobisphosphonates增加。 IPP的积累是低效率的nontransfomred细胞比肿瘤细胞，使我们对癌症与aminobisphosphonatesγδT细胞通过激活免疫药理相关浓度的aminobisphosphonates，4有趣的是，IPP的积累在单核细胞的PBMC与aminobisphosphonates治疗，因为高效，这些细胞的药物摄取。积累的IPP成为抗原提呈细胞和Vγ9Vδ2T细胞刺激外周血单核细胞。基于这些机制，我们制定了一个大规模扩张γδT细胞zoledronate文化和白细胞介素的技术-2（IL - 2）7其他方法扩大γδT细胞利用合成phosphoantigens bromohydrin焦（BrHPP）8或2 -甲基-3 -丁烯- 1 -焦磷酸（2M3B1PP）9，所有这些方法允许前体外扩增，γδT细胞过继免疫治疗中使用的大量产生。然而，只有zoledronate是一个FDA批准的市售试剂。 Zoledronate扩大γδT细胞显示CD27 - CD45RA -效应记忆体的表型和它们的功能可以通过干扰素γ的生产检测7评估。

Watch this Scientific Journal Video about Expansion of Human Peripheral Blood  ÃŽÂ³ÃŽÂ´ T Cells using Zoledronate at JoVE.com

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

扩大γδT细胞从外周血单个核细胞（PBMC）的一个方法来描述。外周血单核细胞衍生的γδT细胞受到刺激和扩大使用zoledronate和白细胞介素2（IL - 2）。 γδT细胞的大规模扩张，可应用于自体肿瘤细胞免疫疗法。

扩展人外周血γδT细胞使用Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

expansion-of-human-peripheral-blood-t-cells-using-zoledronate

Research

JoVE Journal

Immunology and Infection

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

26.8K 次观看.  University of Tokyo Hospital. 扩大γδT细胞从外周血单个核细胞（PBMC）的一个方法来描述。外周血单核细胞衍生的γδT细胞受到刺激和扩大使用zoledronate和白细胞介素2（IL - 2）。 γδT细胞的大规模扩张，可应用于自体肿瘤细胞免疫疗法。

视频: Expansion of Human Peripheral Blood  γδ T Cells using Zoledronate

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

前肿瘤反应性T细胞的体外扩增Bryostatin 1/Ionomycin手段和共同的γ链细胞因子配方

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

科研

免疫与感染

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology.

人地幔细胞淋巴瘤移植小鼠模型中肿瘤植入的研究

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

癌症研究

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

乳腺癌细胞系外周血单核细胞破的人前临床模型的建立

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells 

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

人外周血NK细胞的扩展，纯化和功能评估

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment challenges persist. Robust and disease-relevant animal models are vital resources for continued preclinical research and development in order to address a range of additional immune checkpoints. Here, we describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft model. BGB-A317 (Tislelizumab), an investigational humanized anti-PD-1 antibody in late-stage clinical development, is used as an example to discuss platform set-up, model characterization and drug efficacy evaluations. These humanized mice support the growth of most human tumors tested, thus allowing the assessment of I-O therapies in the context of both human immunity and human cancers. Once established, our model is comparatively time- and cost-effective, and usually yield highly reproducible results. We suggest that the protocol outlined in this article could serve as a general guideline for establishing mouse models reconstituted with human PBMC and tumors for I-O research.

A Human Peripheral Blood Mononuclear Cell PBMC Engrafted Humanized Xenograft Model for Translational Immuno-oncology I-O Research

We describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

用于转化免疫肿瘤学(I-O)研究的人类外周血单核细胞(PBMC)的人工化异种移植模型

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment ...

Expansion and Enrichment of Gamma-Delta (&#947;&#948;) T Cells from Apheresed Human Product

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major histocompatibility complex (MHC)-independent manner and develop strong cytolytic effector function that makes it an ideal candidate for cancer immunotherapy. Due to the low frequency of Gamma-Delta (&#947;&#948;) T cells in the peripheral blood, we developed an effective protocol to greatly expand a highly pure &#947;&#948; T cells drug product for first-in-human use of allogeneic &#947;&#948; T cells in patients with acute myeloid leukemia (AML). Using healthy donor apheresis as an allogenic cell source, the lymphocytes are isolated using a validated device for a counterflow centrifugation method of separating cells by size and density. 
The lymphocyte-rich fraction is utilized, and the &#947;&#948; T cells are preferentially activated with zoledronic acid (FDA-approved) and interleukin (IL)-2 for 7 days. Following the preferential expansion of &#947;&#948; T cells, a clinical-grade magnetic cell-separation device and TCR&#945;&#946; beads are used to deplete contaminating T-cell receptor (TCR)&#945;&#946; T cells. The highly enriched &#947;&#948; T cells then undergo a second expansion using engineered artificial antigen-presenting cells (aAPCs) derived from K562 cells-genetically engineered to express single-chain variable fragment (scFv) for CD3 and CD28, 41BBL (CD137L) and IL15-RA-together with zoledronic acid and IL-2. Seeding all day-7 enriched &#947;&#948; T cells in co-culture with the aAPCs facilitates the manufacture of highly pure &#947;&#948; T cells with an average fold expansion of &gt;229,000-fold from healthy donor blood.

Expansion and Enrichment of Gamma-Delta &#947;&#948; T Cells from Apheresed Human Product

Presented is a protocol for the expansion of Gamma-Delta (&#947;&#948;) T cell drug product. Lymphocytes are isolated by elutriation and &#947;&#948;-enriched with zoledronic acid and interleukin-2. Alpha-beta T cells are depleted using a clinical-grade magnetic separation device. The &#947;&#948; cells are co-cultured with K562-derived, artificial antigen-presenting cells and expanded.

来自分离人产品的γ-Delta（γδ）T细胞的扩增和富集

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

一种新型的<em&gt;在体内</em&gt;基因转移技术和<em&gt;在体外</em&gt;细胞分析为肌肉骨骼疾病骨质流失的研究

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

医学

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological deficiencies and malignancies. Acute GVHD occurs when donor T cells recognize host tissues as a foreign antigen and mount an immune response to the host. Current treatments involve toxic immunosuppressive drugs that render patients susceptible to infection and recurrence. Thus, there is ongoing research to provide an acute GVHD therapy that can effectively target donor T cells and reduce side effects. Much of this pre-clinical work uses the xenogenic GVHD (xenoGVHD) murine model that allows for testing of immunosuppressive therapies on human cells rather than murine cells in an in vivo system. This protocol outlines how to induce xenoGVHD and how to blind and standardize clinical scoring to ensure consistent results. Additionally, this protocol describes how to use digital PCR to detect human T cells in mouse tissues, which can subsequently be used to quantify efficacy of tested therapies. The xenoGVHD model not only provides a model to test GVHD therapies but any therapy that can suppress human T cells, which could then be applied to many inflammatory diseases.

Here, we present a protocol to induce and score disease in a xenogeneic graft-versus-host disease (xenoGVHD) model. xenoGVHD provides an in vivo model to study immunosuppression of human T cells. Additionally, we describe how to detect human T cells in tissues with digital PCR as a tool to quantify immunosuppression.

在异种性鼠模型中的移植-无源疾病的诱导和评分,以及使用数字PCR对小鼠组织中人类T细胞的定量

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological ...

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

骨髓移植平台研究树突状细胞在移植与宿主疾病中的作用

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...

<meta charset="utf8"></head><body>骨类器官衍生细胞在辐照小鼠中进行有效 HSCT

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an adequate source of HSPCs, we developed an in vivo osteo-organoid by implanting scaffolds loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) into an internal muscle pouch near the femur in mice. After 12 weeks of implantation, we retrieved the in vivo osteo-organoids and conducted flow cytometry analysis on HPSCs, revealing a significant presence of HSPC subsets within the in vivo osteo-organoids.
We then established a sublethal model of hematopoietic/immune system injury in mice through radiation and performed hematopoietic stem cell transplantation (HSCT) by injecting the extracted osteo-organoid-derived cells into the peripheral blood of radiated mice. The effect of hematopoietic recovery was evaluated through hematological, peripheral blood chimerism, and solid organ chimerism analyses. The results confirmed that in vivo osteo-organoid-derived cells can rapidly and efficiently reconstruct damaged peripheral and solid immune organs in irradiated mice. This approach holds potential as an alternative source of HSPCs for HSCT, offering benefits to a larger number of patients.

<meta charset="utf8"></head><body>用于收获治疗性造血干细胞/祖细胞的体内骨类器官方法

Here, we established in vivo osteo-organoids triggered by bone morphogenetic protein-2-loaded gelatin scaffolds to harvest therapeutic hematopoietic stem/progenitor cells for the reconstruction of a damaged hematopoietic and immune system. Overall, this approach can provide a promising cell source for cell therapies.

用于收获治疗性造血干细胞/祖细胞的体内骨类器官方法

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an ...