摘要
研究背景和目的：
免疫治疗为肿瘤提供了一种有吸引力的疗法,其优势是在增强或重建患者的抗肿瘤免疫的同时副作用较小。目前已有两种主要策略被用于刺激抗肿瘤免疫。包括肿瘤治疗性疫苗(therapeutic vaccination)和过继性细胞治疗(adoptive cell therapy)。其中过继性细胞治疗主要是指向荷瘤宿主体内回输自体或异体免疫细胞,如以肿瘤反应性T细胞为主要成分的CTL细胞,肿瘤浸润性淋巴细胞(tumor-infiltrating lymphocyte, TIL)等。肿瘤反应性CTL细胞回输已被成功应用于黑色素瘤,EB病毒诱导淋巴瘤(Epstein-Barr Virus)等肿瘤的临床治疗。
然而目前将过继性细胞治疗广泛应用于大多数非病毒相关实体瘤中还存在不少问题亟待解决,包括：1)如何获得充分数量的肿瘤反应性T细胞是决定过继性细胞治疗效果的最关键因素。2)目前回输的免疫细胞尤其是克隆化CD8+T细胞在体内难以长期存活,极大的影响了过继性细胞治疗的临床疗效。
过继性T细胞治疗的一个重要限制是,由于肿瘤缺乏抗原性,仅能从部分患者体内分离获得高亲和力(highly avidity)T细胞。为解决此问题,研究者从患者TIL细胞中筛选获得了反应性T细胞克隆,继而鉴定了可特异性识别肿瘤抗原的T细胞受体(T Cell Receptor,TCR)基因,再将编码肿瘤特异性TCR的基因导入成熟T细胞,获得的TCR基因修饰T细胞(TCR gene engineered T cell)可在体外特异性识别抗原阳性肿瘤细胞,并在回输患者体内后建立抗肿瘤免疫。TCR基因修饰T细胞首先在黑色素瘤中开展了临床试验,将MART-1抗原特异的TCR基因转入黑色素瘤病人的外周血淋巴细胞(Peripheral Blood Lymphocyte,PBL),回输后可在患者体内建立针对抗原阳性肿瘤的免疫能力,部分患者可见肿瘤完全转归。用NY-ESO-1抗原特异性TCR基因修饰的T细胞(NY-ESO-1TCR gene engineered T cell)在以黑色素瘤和滑膜肉瘤(metastatic synovial cell sarcoma refractory)患者为对象的临床试验中也获得了类似结果。
决定TCR基因修饰T细胞体内抗肿瘤效果的关键问题是,鉴定可识别肿瘤抗原的TCR,并选择适用于过继性细胞治疗的T细胞亚群(subset)进行基因转染修饰。
从分化状态上可将T细胞分为幼稚性T细胞(naive T cell)、效应性T细胞(effecor T cell,TE)和记忆性T细胞(memory T cell)。为获得足够数量的效应细胞,T细胞通常需要在回输前经历体外刺激活化,在此过程中获得效应表型(effector phenotype)将极大的影响其回输体内后的存活能力,从而影响过继性免疫治疗的长期抗肿瘤效果。与TE细胞相比,记忆性T细胞具有的自我更新能力使其可在体内长期生存,并在再次遭遇相同抗原快速动员,有望在回输体内后建立更长期的抗肿瘤免疫。其中记忆性T细胞又可根据表型,归巢(homing)能力,和功能的差异分为效应型记忆性T细胞(effector memory T cell, TEM)和中枢型记忆性T细胞(central memory T cell, TCM)。TCM在细胞膜表面表达CD62L和CCR7分子,可促进TCM归巢至淋巴结,并在再次暴露于相同抗原时快速增殖。以豚尾猴(macaques)为模型的研究证实,来源于TCM活化产生的效应型T细胞可再次获得TCM表型,并在回输体内后长期存活。从而为机体提供长期免疫保护。
本研究将采用以下策略提供具有更强生存能力和免疫效应功能的肿瘤反应性T细胞用于过继性细胞治疗：
研究策略一：通过肿瘤特异性TCR转染T细胞赋予其肿瘤识别功能。
本实验室从一直从事肿瘤特异性TCR的筛选与功能研究。在前期研究中利用基因优势取用技术筛选获得了可特异性识别肝癌抗原的TCRV α12.2-Vβ7.1基因。我们将通过肝癌特异性的的TCRV α12.2-Vβ7.1基因赋予T细胞抗原识别特异性,使其有效识别杀伤肿瘤细胞。
研究策略二：肿瘤特异性TCR基因转染T细胞活化诱导记忆性T细胞分化。
研究肿瘤特异性TCR基因转染T细胞被肿瘤抗原充分激活后,记忆性T细胞的分化情况,并明确其表型和肿瘤反应性,为进一步获得TCR基因转染记忆性T细胞奠定基础。
研究策略三：肿瘤特异性TCR基因转染纯化后中枢型记忆性T细胞(TCM)
由于记忆性T细胞的抗肿瘤免疫能力和体内生存能力都强于混合成分的CD8+T细胞。而T细胞的内在性质决定了其回输体内后的生存能力和长期免疫功能。实验进一步探讨了直接用肿瘤特异性TCR基因转染纯化的TCM,并明确其表型特征与抗肿瘤免疫效应,以期为过继性回输提供更优化的免疫效应细胞。
第一部分：肿瘤特异性TCR基因转染诱导记忆性T细胞分化促进抗肿瘤免疫目的
研究TCR基因转染T细胞被肿瘤抗原激活后,记忆性T细胞的分化情况,并明确其表型和免疫功能,为进一步获得TCR基因转染记忆性T细胞奠定基础。
方法：
1.T细胞的培养、刺激与转染。
(1)密度梯度离心法分离人外周血单核细胞(peripheral blood monocyte, PBMC)。
(2)体外以协同刺激分子单抗／细胞因子初次刺激活化T细胞(培养后第1天)；(3)重组人腺病毒Ad5F35-TRAV-TRBV转染T细胞(培养后第3天)
(4)流式细胞术检测腺病毒转染后外源TCR基因表达效率(转染后3-14天)；
(5)人TAP缺陷型T2细胞负载HLA-A2+AFP218-226表位9肽(LLNQHACAV)再次刺激活化PBMC及TCR基因转染T细胞(培养后第5天)
2.TCR基因转染T细胞体外经肿瘤抗原刺激后表型变化流式细胞术检测TCR基因转染T细胞经肿瘤抗原刺激后CD45RO、CD62L、 CD44等标志性分子表达比例及动态变化(刺激后3-14天)。
3.TCR基因转染T细胞经肿瘤抗原刺激后体外抗肿瘤免疫功能
(1)CTL活性：肿瘤抗原再次刺激后24小时,各组效应T细胞(PBMC对照组,经抗原刺激PBMC,TCR基因转染组,经抗原刺激TCR基因转染组)。以不同效靶比作用于各组肿瘤细胞HepG-2(HLA-A2+AFP+), SMMC-7721(HLA-A2+AFP+), MCF-74(HLA-A2+AFP-)4小时,MTT法检测肿瘤杀伤活性。
(2)诱导肿瘤细胞凋亡：再次刺激后24小时,各组效应T细胞以30：1的效靶比作用于靶细胞HepG-24小时,Annexin-V双染法检测靶细胞凋亡率。并以流式细胞术检测各组效应T细胞表面FasL表达比例。
(3)细胞因子分泌：各组效应T细胞以30：1的效靶比作用于靶细胞HepG-224小时后,ELISA法检测效应T细胞的细胞因子IFN-γ,IL-2分泌。
统计学处理
实验数据以均数±标准差来表示。应用SPSS(?) v16.0软件(SPSS Inc., Chicago, USA)进行统计分析。各指标先进行正态性及Levene's test方差齐性检验。两因素两水平数据比较采用2×2析因设计的方差分析(不同组细胞肿瘤杀伤活性比较,肿瘤细胞凋亡率比较,T细胞表面FasL表达比例分析,各组细胞的细胞因子分泌)。采用重复测量的方差分析完成各组细胞不同时间地点TCRVβ7表达比例分析。采用单因素方差分析对不同MOI值转染后TCR V β7阳性细胞比例进行分析。当P<0.05时,被认为差异具有统计学意义。
结果
1.嵌合型腺病毒载体Ad5F35-TRAV-TRBV可以有效转染T细胞,在感染复数(multiplicity of infection,MOI)为100时,具有最高的转染效率(17.120±0.983%),与其余各组相比差异有统计学意义(P<0.001)(n=5)外源TCR阳性T细胞比例在转染三天后约为30%。外源TCR阳性T细胞比例随转染后时间延长逐渐降低。
2.肿瘤抗原特异性TCR基因转染能有效赋予T细胞抗原识别特异性,TCR基因转染T细胞经肿瘤抗原刺激后充分活化,CD45RO+细胞比例逐渐上升。在活化后第14天,转染+刺激组CD45RO+细胞比例最高(39.150±4.005%),与其它各组相比差异有统计学意义(P<0.001)(n=4)。抗原有效刺激启动了记忆性T细胞的分化过程,CD62L、CD44抗体染色结果显示,CDRO+细胞中主要以CD62L-CD44+细胞(TE,TEM)为主。随着抗原刺激后时间的延长,CD62L+细胞在CD45RO+细胞中比例逐渐上升。最终出现明显的CD62L+CD44+TCM亚群。
3.肿瘤特异性TCR基因转染能够促进T细胞杀伤AFP+靶细胞HepG-2(转染组：25.479±8.574%,对照组：6.107±1.047%),SMMC-7721(转染组：24.368±8.411%,对照组：6.024±1.269%)。差异均具有统计学意义(P<0.001)(n=5)。通过AFP抗原预先刺激,进一步提高TCR基因转染T细胞的肿瘤杀伤活性(HepG-2:33.467±12.625%,SMMC-7721:32.027±12.309%)与单独转染组相比,差异均有统计学意义(P<0.001)。TCR基因转染和AFP刺激对TE细胞裂解AFP-的靶细胞MCF-7无明显影响(P>0.05)。提示TCR基因转染具有良好的抗原特异性。
4.肿瘤特异性TCR基因转染能够有效促进T细胞诱导靶细胞HepG-2凋亡(转染组：16.400±1.272%,对照组：1.375±0.573%),差异具有统计学意义(P<0.001)(n=4)。AFP抗原预先刺激使靶细胞凋亡比例进一步增加(转染+刺激组：28.750±4.160%),与单独转染组相比,差异具有统计学意义(P=0.001)。同时,TCR基因转染能够有效促进效应T细胞在作用于靶细胞后表达免疫效应分子FasL(转染组：14.700±1.706%,对照组：1.725±0.427%),差异具有统计学意义(P<0.001)(n=4)。抗原刺激能够进一步促进FasL表达(转染±刺激组：27.650±2.686%),与单独转染组相比差异具有统计学意义(P<0.001)。T细胞表面上调表达的FasL可通过FasL-Fas途径诱导靶细胞凋亡。
5.肿瘤特异性TCR基因转染能够促进T细胞作用于靶细胞HepG-2后分泌细胞因子IFN-γ(转染组：25.962±2.488ng/ml,对照组3.736±0.412ng/ml),差异具有统计学意义(P<0.001)(n=5)。特异性抗原的预先刺激活化使以上效果进一步增强(转染+刺激组：33.394±2.520ng/ml),与TCR基因转染T细胞组差异有统计学意义(P=0.002).。T细胞通过增加IFN-γ分泌介导抗肿瘤免疫反应。
结论
TCR基因转染能够有效赋予T细胞抗原特异性,使其有效识别肿瘤抗原后活化。经肿瘤抗原预先刺激充分活化的抗原特异性T细胞将启动记忆性T细胞分化进程,使效应T细胞逐渐获得TCM表型特征,并在再次遭遇表达相同抗原的肿瘤细胞时发挥更为强烈的免疫效应功能。
第二部分：TCR基因转染中枢型记忆性T细胞促进抗肿瘤免疫
目的
从健康人CD8+T细胞中鉴定并纯化TCM,研究体外活化后,CD8+T细胞与TCM来源的效应T细胞(TE)细胞分化表型的差异。并以肿瘤抗原特异性TCR基因修饰TCM细胞,明确TCR基因修饰TcM细胞的抗肿瘤免疫功能。
方法
1.TcM的分选、刺激与TCR基因转染
(1)磁珠负性筛选CD8+T细胞,二次阳性分选TCM
(2)协同刺激抗体／细胞因子初次刺激(培养后第1天),
(3)重组TCR腺病毒转染(培养后第3天)CD8+T细胞及TcM。
(4)HLA-A2+AFP218-226表位9肽(LLNQHACAV)再次刺激(培养后第5天)。
(5)台盼蓝染色法获取CD8+T细胞及TCM体外刺激活化后生长曲线(至体外培养第35天)
(6)流式细胞术-荧光原位杂交法(Flow-FISH)检测活化后细胞数量高峰期(第14天)CD8+T细胞与TCM来源TE端粒相对长度。
2.CD8+T细胞与TCM来源TE细胞分化表型流式细胞术检测CD8+T细胞与TCM经刺激活化后CD45RO、CD62L、CD44、 CCR7、CD28等标志性分子表达及动态变化(至体外培养后第33天)。
3.TCR基因转染TCM来源TE的抗肿瘤免疫功能
(1)流式细胞术检测腺病毒转染后外源TCR基因表达效率(转染后3-14天)；
(2)CTL活性：肿瘤抗原再刺激后一天,以钙黄绿素标记靶细胞(HepG-2, SMMC-7721,MCF-7)。检测分别来源于CD8+T细胞,TCM,TCR基因转染CD8+T细胞,TCR基因转染TCM的各组TE不同效靶比对靶细胞的特异性裂解百分比。
(3)抗体阻断实验：抗人HLA-A2抗体与HLA-A2阳性靶细胞HepG-2和SMMC-7721共孵育后,钙黄绿素释放法检测抗体阻断对TCR基因转染TcM来源TE的CTL活性影响。
(4)细胞裂解核蛋白分泌：肿瘤抗原再刺激后一天,各组TE细胞作用于靶细胞Hep-G2,SMMC7721后4小时。胞内细胞因子染色法检测T细胞内新合成的穿孔素与颗粒酶B分泌。
(5)细胞因子分泌：肿瘤抗原再刺激后一天,各组TE细胞作用于靶细胞HepG-2, SMMC7721,MCF-724小时后,ELISA法检测细胞培养液上清中IFN-γ, IL-2含量。
统计学处理
实验数据以均数±标准差来表示。应用SPSS(?) v16.0软件(SPSS Inc., Chicago, USA)进行统计分析。各指标先进行正态性及Levene's test方差齐性检验。两因素两水平数据比较采用2×2析因设计的方差分析(不同组细胞肿瘤杀伤活性比较,抗体抑制试验,各组细胞的细胞因子分泌)。采用重复测量的方差分析完成各组细胞不同时间地点细胞数量分析。采用单因素方差分析对不同组细胞端粒长度进行分析。当P<0.05时,被认为差异具有统计学意义。
结果
1.本研究分析了来自12个健康人样本的CD8+T细胞,其CD62L+CD44+TCM比例在13-35%之间。选择6个HLA-A2+样本,分选获得了纯度高于97%的TCM
2.体外培养过程中,通过协同刺激分子/细胞因子和肿瘤抗原的两轮刺激,诱导了TCM的效应性分化,细胞数量在体外培养14天左右达到峰值。在此过程中,与CD8+T细胞相比,TCM来源TE具有更高的增殖倍数(第14天,TCM组细胞数量19.421±3.003×106/m1),与CD8+T细胞组相比(第14天时细胞数目为14.533±1.874×106/m1),细胞数目差异有统计学意义(t=-5.349,P<0.001)(n=15)。在细胞数到达峰值后,TCM来源TE与CD8+T来源TE相比保持了更为稳定的细胞数量,更低的活化后细胞死亡比例。在第21天(t=-12.226,P<0.001),28天(t=-16.472,P<0.001),35天(t=-20.203,P<0.001),细胞数目差异均有统计学意义。TCM来源TE与CD8+T来源TE相比显示了更长的端粒长度(TCM来源TE：1.005±0.053,CD8+T来源TE：0.968±0.017),但端粒长度差异尚无统计学意义(P=0.228)(n=4)。
3.在体外效应分化过程中,TCM来源的TE细胞始终保持了一定程度的CD62L表达(>10%),CD44表达比例始终保持较高水平(85%)。在培养中晚期(21天后),所有CD62L+细胞均为CD44high表型。从而重新获得明显的CD62L+CD44highTCM分群。而CD8+T细胞来源TE的CD62L+阳性细胞比例逐渐下降至无法检测。CD44+细胞比例逐渐降低至20%以下。
4.作为T细胞活化标志,CD45RO在TCM来源TE中保持较稳定的高水平表达(>40%),并在各时间点均高于CD8+T细胞来源TE。同时,CD28与CCR7分子在TCM来源TE中始终保持了一定比例的表达(>19%),而在CD8+T细胞来源TE中表达逐渐降至较低水平(<10%)。
5.通过嵌合型腺病毒载体Ad5F35-TRAV-TRBV,成功的将肿瘤特异性TCR转染至TCM。外源性TCR阳性T细胞比例在转染后3天超过30%。此后随着转染后时间的延长,外源性TCR阳性T细胞比例逐渐下降。
6.TCR基因转染有效促进了TCM识别杀伤HLA-A2+AFP+靶细胞HepG-2(TCR基因转染TCM组：32.382±14.311%,TCM组：10.566±2.034%)(n=6)和SMMC-7721(TCR基因转染TCM组：29.512±12.246%， TcM组：10.878±2.114%)(n=6)。与TCR基因转染CD8+T细胞相比(HepG-2:24.424±9.340%,SMMC-7721:21.368±6.948%),以HepG-2为靶细胞时,TCR基因转染TCM在效靶比为10：1(P=0.04)和30：1组(P=0.015),特异性裂解百分比与基因转染CD8+T细胞间差异有统计学意义。以SMMC-7721为靶细胞时,TCR基因转染TcM在效靶比为10：1(P=0.007)和30：1组(P=0.037),特异性裂解百分比与基因转染CD8+T细胞间差异有统计学意义。
7.TCR基因转染及AFP抗原预先刺激对TcM来源TE裂解HLA-A2+AFP-靶细胞MCF-7的能力没有显著影响(P<0.05)(n=6)。提示TCR基因转染TCM来源TE的CTL活性具有良好的抗原特异性
8.通过HLA-A2抗体预先孵育HLA-A2+AFP+靶细胞HepG-2和SMMC-7721,有效阻断了转染TCR基因转染YcM来源TE的CTL活性。以HepG-2为靶细胞时,抗体封闭后,在效靶比3：1,10：1,30：1时靶细胞裂解百分比与抗体封闭前靶细胞裂解百分比差异有统计学意义(t=-3.406, t=-20.945, t=-15.188; P=0.003, P<0.001, P<0.001)(n=10)。以SMMC7721为靶细胞时,抗体封闭后,在效靶比3：1,10：1,30：1时靶细胞裂解百分比与抗体封闭前靶细胞裂解百分比差异有统计学意义(t=-4.021, t=-15.640, t=-12.443; P=0.001, P<0.001, P<0.001)(n=10)。提示TCR基因转染TcM来源TE的CTL效应具有MHC限制性。
9.TCR基因转染有效促进了TCM来源TE作用于靶细胞HepG-2和SMMC-7721后裂解核蛋白穿孔素与颗粒酶B的分泌。与TCR基因转染CD8+T细胞相比,经AFP抗原预先刺激的TCR基因转染TCM再次遭遇抗原阳性肿瘤细胞时裂解核蛋白分泌明显上升。
10.TCR基因转染有效促进了TCM来源TE作用于靶细胞HepG-2和SMMC-7721后细胞因子IFN-γ分泌(作用于HepG-2时,TCR基因转染TCM:35.761±7.311ng/ml,TCM3.288±0.740ng/ml;作用于SMMC-7721时,TCR基因转染TCM:27.310±3.672ng/ml,TCM3.211±1.181ng/ml。)。差异具有统计学意义(P<0.001)与TCR基因转染CD8+T细胞相比(作用于HepG-2时,27.920±3.722ng/ml;作用于SMMC-7721时,24.673±2.279ng/ml),经AFP抗原预先刺激的TCR基因转染TCM再次遭遇抗原阳性肿瘤细胞时IFN-γ分泌差异具有统计学意义(作用于HepG-2,样本1：P=0.003,样本2：P=0.017；作用于SMMC-7721时,样本1：P=0.005,样本2：P=0.001)。
结论
与CD8+T细胞相比,分离TCM并通过TCR基因转染有望提供具有更强生存能力和效应功能的肿瘤反应性T细胞用于过继性T细胞治疗。
Background and Objection:
Immunotherapy provides an attractive treatment of cancer, the advantage of which is that it can enhance or reconstruct the patients' anti-tumor immunity with weak side effect. Currently, there are mainly two strategies for tumor immunotherapy, namely, therapeutic vaccination and adoptive cell therapy, and the latter normally refers to the transfusion of autologous or allogenic immune cells into tumor-bearing hosts. The immune cells include Cytotoxic Lymphocytes (CTLs) with tumor-reactive T cells as its major ingredient and tumor-infiltrating lymphocytes (TILs) etc. The transfusion of tumor-reactive CTLs has been employed in treatment of metastatic melanoma, Epstein-Barr virus Induced Lymphoma and other tumors.
But the clinical application of adoptive cell therapy in most solid tumor is dependent on the solution of two major problems.1) How to obtain sufficient numbers of tumor-reactive T cells.2) The efficacy of adoptive immunotherapy in humans is often limited by the failure of cultured T cells, particularly cloned CD8+T cells, to persist in vivo.
A major restriction of adoptive T cell therapy is that highly avid T cells can only be separated from some particular patients, because tumor lacks of antigenicity [4]. To solve this problem, reactive T cell clones were selected from TILs. Through this approach, researchers identified the genes of T Cell Receptors (TCRs), which can specifically recognize tumor antigen, and then transduced the genes encoding tumor-specific TCRs into mature T cells to acquire TCR gene engineered T cells that can specifically recognize antigen positive tumor cells in vitro and can mediate anti-tumor immunity after being re-injected into the patient. TCR gene engineered T cells were first employed in clinical trials treating melanoma, in which MART-1antigen specific TCR genes were transduced into melanoma patients'peripheral blood lymphocytes (PBLs). The transfused TCR gene engineered T cell then directly destroy antigen positive tumor in vivo. By this means, complete tumor regression were observed in some cases. Similar results have been achieved in clinical trials in which NY-ESO-1antigen-specific TCR gene engineered T cells were utilized to treat melanoma and synovial sarcoma patients.
A critical point, which determines the antitumor efficacy of TCR gene engineered T cells, is to identify the TCRs that can recognize tumor antigens and select T cell subsets that are suitable for adoptive cell therapy.
Based on differentiations, T cells fall into three categories, naive T cells, effector T cells (TE), and memory T cells. In order to obtain sufficient cell numbers for adoptive transfer, T cell usually need to be stimulated in vitro. A vital reason for the short-term survival of transfused T cells is that effector phenotypes are acquired after T cells being activated in vitro. Compared with effector T cell, there are reasons to believe that transfer of cells with memory properties, including enhanced recall response and the ability to undergo self-renewal, may be superior mediators of an antitumor response in vivo.
According difference of phenotype, homing ability and function, memory T cells can be divided into effector memory T cells (TEM) and central memory T cells (TCM)-Tcm express CD62L and CCR7, which can promote the homing of them to lymph nodes and enable them to proliferate rapidly upon re-exposure to the same antigen. Berger's landmark research modeled on macaques has proved that effector T cells (TE) derived from Tcm can persist long-term in vivo, and reacquired phenotypic and functional properties of memory T cells.
We sought to obtain tumor-reactive T cells population with improved capacity to persist and heightened anti tumor reactivity by the following strategy.
1) T cell modified by tumor antigen specific TCR gene.
In previous research, we had screened AFP peptide binding specific TCR genes-TCRV α12.2-V β7.1, by gene preferential usage of TILs isolated from hepatocellular carcinoma patients. The antigen specificity of T cell is redirected by the modification of tumor specific TCRV α12.2-V β7.1, which enable T cell to recognize antigen-positive tumor cell.
2) The activation of tumor specific TCR gene modified T cell induces the differentiation of memory T cell.
To investigate the differentiation of memory T cell, after activation of TCR gene modified T cell stimulated by tumor antigen, and determine the phenotype and antitumor reactivity of TCR transferred T cell induced memory T cell.
3) TCR gene transferred central memory T cell (Tcm).
Since memory T cell is superior to CD8+T cells in the persistence and antitumor efficiency, and intrinsic properties of T cells that are isolated for adoptive therapy determine their fate in vivo. We than attempt to obtain a superior tumor-reactive T cell population by isolating CD8+central memory T cells for transferring with tumor specific TCR gene.
Chapter1Enhanced anti tumor reactivity by TCR gene transferred T cell induced memory T cell
Objective
To investigate the differentiation of memory T cell after the activation of TCR gene transferred T cell stimulated by tumor antigen in vitro. And determine the phenotype and antitumor reactivity of TCR transferred T cell induced memory T cell.
Methods and materials
1. T cell culture, stimulation and transduction
1) Human peripheral monocytes (PBMC) were isolated by Ficoll-plaque plus (GE Healthcare) density gradient centrifugation of buffy coats from healthy donors.
2) Primary stimulation was accomplished by McAb to costimulatory molecule and cytokine on day1
3) Transduction was performed2days after initial stimulation using Ad5F35-TRAV-TRBV adenovirus supernatant produced in HEK293packaging cells.
4) The relative efficiency of transgene expression in T cells was detected by flow cytometry (3-14days after transduction).
5) Restimulation was performed on day5, using T2cell pulsed with9-amino HLA-A2+epitope from AFP2i8-226(LLNQHACAV).
2. Phenotype changes of TCR gene transferred T cell stimulated by tumor antigen.
Expression of maker molecule CD45RO、CD62L、CD44on TCR gene transferred T cells was detected by FACS from3-14days after stimulation
3. Anti tumor reactivity of TCR gene ttansferred T cell after stimulated by tumor antigen
1) CTL activatity. After coculture24hours with different target cells:HepG-2(HLA-A2+AFP+), SMMC-7721(HLA-A2+AFP+), and MCF-7(HLA-A2+AFP-) at different E/T ranging from30:1to3:1, tumor specific lysis of TE from PBMC control, Stimulated PBMC, TCR gene transferred T cell and Stimulated TCR gene ttansferred T cell was evaluated by MTT assays.
2) Induced apoptosis. HepG-2was coculture4hours with TE of each group (E/T30:1). The frequency of apoptosis target cell was detected by Annexin V/PI double-labeled FACS.The proportion of FasL+cell in TE of each group was detected by FACS.
3)Cytokine secretion. The secretion of cytokine IFN-γ and IL-2of TE coculture with HepG-2(E/T30:1) were quantified by ELISA assays
Statistical analysis
The experimental data are expressed as mean (x)±standard deviation (s). Statistical analysis was carried out by SPSS v13.0software (SPSS Inc, Chicago, USA).After checking for normal distribution and homogeneity of variances, Factorial design variance analysis was used to detect the main effects and interaction of factors. Analysis of one fact was evaluated using Independent samples T test or One way Anova. Probabilities (P)<0.05was considered to be statistical difference and P<0.01was considered to be significantly statistical difference.
Results
1. Ad5F35chimeric adenoviral vector transduced T cell effectively. The highest tranduce efficiency was obtained at MOI100(17.120±0.983%, which was significantly higher than other groups(P<0.001) The frequency of exogenous TCR positive cell is about30%3days after transduction. The frequency of exogenous TCR decreased over the time.
2. Tumor specific TCR gene modification redirected the antigen specificity of T cell effectively; TCR gene transferred T cells were activated by stimulation of tumor antigen AFP. The frequency of CD45RO+cell increased after activation, reached (39.150±4.005%)14days after stimulation in TCR gene transferred and stimulated with antigen group, which is significantly higher than other groups. Antigen stimulation initiated the differentiation process of memory T cell. As indicated by CD62L, CD44fluorescent McAb labeling, phenotype of most CD45RO+cells are CD62L-CD44-. The frequency of CD62L positive cell increased as time went on after stimulation. Cell clusters with CD62L+CD44+Tcm phenotype appeared in TE cells eventually.
3. Tumor specific TCR gene modification enhanced the ability of T cells to lyse HLA-A2+AFP+target cells:HepG-2(TCR gene transferred group:25.479±8.574%, control group:6.107±1.047%), SMMC-7721(TCR gene transferred group: 24.368±8.411%, control group:6.024±1.269%).The difference was statistically significant (P<0.001)(n=5). Stimulating by tumor antigen could improve specific lysis of TCR gene transferred T cells (HepG-2:33.467±12.625%, SMMC-7721:32.027±12.309%), showing significant difference with TCR gene transferred group (P<0.001). The percent specific lysis of antigen stimulated TCR gene transferred T cell was significantly higher than that of TCR gene transferred T cell (P<0.001). TCR gene modification and antigen stimulation had no significant effect on the specific lysis of T cell against AFP-target cell MCF-7(P>0.05), which indicates antigen specificity of TCR gene modification.
4. Tumor specific TCR gene modification enhanced the ability of T cells to induce HepG-2apoptosis (TCR gene transferred group:16.400±1.272%, control group:1.375±0.573%), showing significant difference(P<0.001)(n=4). The frequency of apoptosis target cell induced by antigen stimulated TCR gene transferred T cell (28.750±4.160%) was significantly higher than that of TCR gene transferred T cell (P=0.001). TCR gene modification and antigen stimulation promoted the expression of FasL on TE cells which mediate apoptosis in Fas-FasL pathway. The frequency of FasL+T cells in TCR gene transferred group (14.700±1.706%) was significantly higher than that of control group(1.725±0.427%)(P<0.001)(n=4). The frequency of FasL+T cells in antigen stimulated TCR gene transferred T cell (27.650±2.686%) was significantly higher than that of TCR gene transferred group(P<0.001).
5. Tumor specific TCR gene modification enhanced T cells to secret IFN-γ which mediate anti tumor response((TCR gene transferred group:25.962±2.488ng/ml, control group:3.736±0.412ng/ml, P<0.001, n=5). The production of IFN-γ by antigen stimulated TCR gene transferred T cell (33.394±2.520ng/ml) was significantly higher than that of TCR gene transferred T cell (P=0.002).
Conclusion
Tumor specific TCR gene modification redirected antigen specificity of T cell effectively. Antigen specific T cell stimulated by tumor antigen initiated the differentiation of memory T cell, which enabled TE acquire phenotypic character of Tcm and mediated enhanced response if re-exposure to tumor antigen.
Chapter2Enhanced anti tumor reactivity of Tcm modified by TCR gene transferred.
Objective
To investigate phenotypic character and anti tumor reactivity of TE derived from sorted CD8+Tcm modified by tumor specific TCR gene.
Methods and materials
1. TCM sorting, stimulation and transduction
1) CD8+T cells were isolated from PBLs of HLA-A2+healthy donors by magnetic bead negative selection, CD62L+CD44+cells were then separated by positive selection.
2) Primary stimulation was accomplished by McAb to costimulatory molecule and cytokine on day1
3) Transduction was performed2days after initial stimulation using Ad5F35-TRAV-TRBV adenovirus supernatant produced in HEK293packaging cells.
4) Restimulation was performed on day5, using T2cell pulsed with9-amino HLA-A2+epitope from AFP218-226(LLNQHACAV).
5) In vitro growth of TE derived from CD8+T cell and Tcm was measured by counting viable cells using trypan blue dye exclusion.
6) Telomera length of CD8+T cell and TCM was measured by in situ hybridization and flow cytometry (Flow-FISH) on day14which is climax of cell numer after stimulation.
2. Phenotype changes of TE derived from TCM stimulated by tumor antigen. Expression of maker molecule CD45RO, CD62L, CD44, CD28and CCR7on TE derived from Tcm and CD8+T cells were detected by FACS from7-28days after stimulation.
3. Anti tumor reactivity of TE derived from TCR gene transferred TCM.
1) The relative efficiency of transgene expression in Tcm and CD8+T cells was detected by FACS (3-14days after transduction).
2) CTL activity,1days after restimulation, Cytotoxicity of TE (derived from TCR gene transferred or untransferred Tcm and CD8+T cell) against target cells (HepG-2, SMMC-7721and MCF-7) at different E/T ranging from30:1to3:1was evaluated by the calcein release assay.
3) Antibody blocking experiments. Target cells (HepG2, SMMC-7721) were incubated with anti-human HLA-A2mAb before coculture. Cytotoxicity of TE derived from TCR gene transferred Tcm against HLA-A2+target cells (HepG-2, SMMC-7721) was evaluated by the calcein release assay.
4) Cytolytic granule protein secretion, ldays after restimulation, TE from TCR gene transferred or untransferred Tcm and CD8+T cells were cocultured4h with target cells (HepG2, SMMC-7721) at30:1(E/T). The frequency of perforin+and Granzyme B+cells in TE was detected by intracellular staining with FACS.
5) Cytokine secretion.1days after restimulation, TE from TCR gene transferred or untransferred Tcm and CD8+T cell were cocultured overnight with target cells (HepG2, SMMC-7721, and MCF-7) at30:1(E/T). IFN-γ and IL-2contents in the supernatants were determined by ELISA assays.
Statistical analysis
The experimental data are expressed as mean (x)±standard deviation (s). Statistical analysis was carried out by SPSS v13.0software (SPSS Inc., Chicago, USA). After checking for normal distribution and homogeneity of variances, Factorial design variance analysis was used to detect the main effects and interaction of factors. Analysis of one fact was evaluated using Independent samples T test and One way Anova. Probabilities (P)<0.05was considered to be statistical difference and P<0.01was considered to be significantly statistical difference.
Result
1. The frequency of Tcm was13%-35%of CD8+T cells, which was variable among12different donors. CD62L+CD44+TCM were sorted from6HLA-A2+samples. Isolating efficiency reached97%or higher.
2. TE from each subset displayed rapid expansion after stimulation, and significantly greater number of cells was obtained from TE derived from TcM(day14,19.421±3.003×106/ml) compared with that from CD8+T cells(day14,14.533±1.874×106/ml)(t=-5.349, P<0.001)(n=15). After reaching the peak at day14, number of TE from Tcm displayed slower decline than that from CD8+T cells. The number of TE derived from Tcm was significantly higher than that of TE derived from CD8+T cells on day21(t=-12.226, P<0.001), day28(t=-16.472, P<0.001) and day 35(t=-20.203, P<0.001). The relative telomere length of TE derived from TCM(1.005±0.053) was longer than that from CD8+T cells(0.968±0.017). However, the difference was not significant (P=0.228)(n=4).
3. After being stimulated, the frequency of CD62L+cell in TE derived from CD8+T cells gradually decreased to the extent that cannot be detected. On the contrary, the frequency of CD62L+cell in TE derived from Tcm maintained certain level (>10%). The frequency of CD44+cells in TE derived from CD8+T cells gradually decreased, reaching20%at the end of culture. In contrast, the frequency of CD44+cells in TE derived from Tcm constantly keeps relatively high, accounting for85%during the whole culture period. Especially in mid-late period of culture (21days later), cell clusters with high CD44expression appeared in TE from TCM,all CD62L positive cells were CD44high cells, i.e., CD62L+CD44high cluster.
4. The expression of CD45RO in TE cells derived from Tcm was higher than that in TE cells derived from CD8+T cells during the culture period. Similar to the trend of CD62L+T cells, the frequency of CD28+cells and CCR7+cells in TE cells derived from Tcm maintained certain level during the culture period (>19%), whereas the frequency of CD28+cells and CCR7+cells in TE cells derived from CD8+T cells gradually decreased to lower extent (<10%).
5. TCM and CD8+T cell were efficiently transduced by Ad5F35chimeric adenoviral vector. The frequency of TCRa12+-TCRVβ7+cells is30-36%of T cells3days after transduction, the frequency of TCRa12+-TCRVβ7+cells declined gradually over time after transduction.
6. TCR gene transfer enhanced the ability of T cells to lyse HLA-A2+AFP+target cells:HepG-2(TCR gene transferred TCM:32.382±14.311%, TCM:10.566±2.034%(n=6). SMMC-7721(TCR gene transferred TCM:29.512±12.246%, TCM:10.878±2.114%)(n=6). AFP antigen stimulation enhanced specific lysis of TCR gene transfer T cm, which was significantly higher than TCR gene transfer CD8+T cell (HepG-2:24.424±9.340%, SMMC-7721:21.368±6.948%) in10:1(P=0.04) and30:1(P=0.015) coculture with HepG-2, and in10:1(P=0.007) and30:1(P=0.037) coculture with SMMC-7721.
7There are no statistical differences in level of specific lysis between TCR gene transferred T cells and untransferred control groups in the CTL assays using AFP-MCF-7as target cells, which indicates antigen specificity of TCR gene modification.
8. After blocking the HLA-A2sites on the surface of HepG-2and SMMC-7721with HLA-A2mAb, the specific lysis effects of TCR gene transferred T cells could be significantly eliminated. The specific lysis was significantly lower after blocking by HLA-A2mAb in3:1,10:1and30:1using HepG-2(t=-3.406, t=-20.945, t=-15.188. P=0.003, P<0.001, P<0.001)(n=10) or SMMC-7721(t=-4.021, t=-15.640, t=-12.443. P=0.001, P<0.001,P<0.001)(n=10) as target cells. These results indicate that TCR gene transferred CTL recognized target tumor cells in an HLA-A2-restricted manner.
9. After coculture with HLA-A2+AFP+target cells, the frequency of perforin+and granzyme B+cell in TE derived from TCR gene transferred TCM are higher than that in untransferred TCM-The frequency of perforin+and granzyme B+cell in TE derived from TCR gene transferred CD8+T cells and untransferred CD8+T cells are both lower than that of corresponding TCM cell groups.
10. After coculture with HLA-A2+AFP+target cells, TCR gene transferred TCM produced greater quantities of IFN-y (coculture with HepG-2:35.761±7.311ng/ml, coculture with SMMC-7721:27.310±3.672ng/ml) than untransferred TCM (coculture with HepG-2:3.288±0.740ng/ml, coculture with SMMC-7721:3.211±1.181ng/ml), showing significant difference(P<0.001). Furthermore, Content of IFN-y secretion in TCR gene transferred Tcm was higher than that of TCR gene transferred CD8+T cell (coculture with HepG-2:27.920±3.722ng/ml, coculture with SMMC-7721:24.673±2.279ng/ml). The difference was statistically significant (HepG-2, donor1: P=0.003, donor2:P=0.017. SMMC-7721, donor1:P=0.005, donor2:P=0.001)
Conclusion
Isolating central memory T cells rather than CD8+T cells for insertion of gene encoding tumor-specific TCR may provide a superior tumor-reactive T cell population for adoptive transfer.
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