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  Î³Î´ 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

26.6K 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

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.

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 ...

科研

免疫与感染

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

临床应用<em&gt;睡美人</em和人工抗原呈递细胞基因修饰T细胞从外设和脐带血中

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

评估的HIV-1感染的CD4的先天传感<sup&gt; +</sup&gt;性T细胞浆细胞样树突细胞使用<em&gt;体外</em&gt;共培养体系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

从外周血人类同种异体抗原特异性T细胞的生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

半自动化的方法准备抗体鸡尾酒人外周血中免疫表型分析

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

人类宫颈的γδT细胞的分离和流式细胞仪分析

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

扩展人外周血γδT细胞使用Zoledronate

摘要

探索更多视频

此视频中的章节

人外周血NK细胞的扩展，纯化和功能评估

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

临床应用

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

评估的HIV-1感染的CD4的先天传感

从外周血人类同种异体抗原特异性T细胞的生成

半自动化的方法准备抗体鸡尾酒人外周血中免疫表型分析

人类宫颈的γδT细胞的分离和流式细胞仪分析

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

使用madSTORM进行高分辨率，超分辨率的T细胞成像