我们感谢佩内洛普. 加西亚的技术援助和这份手稿的准备工作。这项工作得到了一项研究创新补助金 (钻机) 从医学院在 PP1685 琳达大学 (681205-2967) 和试点补助金从国家多发性硬化症协会 () XT。

所有的实验都是遵照德克萨斯大学埃尔帕索分校和玛琳达大学的动物保育和使用委员会批准的机构动物保育和使用协议进行的。
1. OLP 库的生成覆盖了整个蛋白质的长度
<ol>
	<li>设计一个 OLP 库, 其中所有的多肽是15的长度, 和相邻的多肽重叠的11种氨基酸。 注: 在 MOG OLP 研究中, MOG 前体 [小家鼠] 的序列是从 NCBI 蛋白质数据库中检索到的, 遵循以下链接: https://www.ncbi.nlm.nih.gov/protein/NP_034944。MOG 前体 (247 种氨基酸), 其中包含信号和成熟的肽, 被使用, 因为大多数报告的 Qa-1 (HLA E) 表位是位于信号肽 (如 Qdm1)。从它的 N 端开始, 15-氨基肽的序列被辨认了, 因此二个毗邻肽重叠由11氨基酸 (图 1)。因此, 在247氨基酸 MOG 前体中, 准确地发现了59处。然而, 最后的 OLP 可以从12到15的范围, 取决于蛋白质的长度。此外, 我们选择 15-滨海图书馆, 因为我们和其他人已经表明, 15-滨海多肽, 当添加到树突状细胞 (DCs) 或巨噬, 可以有效地处理成表位, 以识别通过 CD8+ T 细胞14,15.</li>
	<li>在商业上购买每个单独的肽。5毫克每肽应该足够的筛选。处和截断的多肽 (步骤 6.1) 可盐水肽 (纯度约 50-70%)。最佳肽 (步骤 6.2) 的纯度将用于聚丙烯的生成和未来的生物分析应该和 #62; 90%。在无菌条件下重组多肽。</li>
	<li>在100% 亚砜中制作50毫克/毫升的单个肽类股。每多肽5毫克, 在每管中加入100μ l 的亚甲基亚砜, 混合, 并储存在-20 ° c 的多肽。这些股票将被用来做一个 OLP_pool 的股票, 生成的 CD8+ T 细胞系反应的 OLP_pool。此外, 这些股票还将用于10毫克/毫升个别肽类股, 以确定每个单独的肽的能力, 以刺激 Qa-1-restricted 反应在 OLP_pool 反应的 CD8+ T 细胞系。</li>
	<li>通过在新鲜的试管中加入等量的肽, 使 OLP_pool 的股票。这 OLP_pool 的股票含有100% 的亚甲基亚砜。在 MOG OLP 研究中, 有59处12。因此, OLP_pool 中各肽的浓度为847.46 微克/毫升 (50 毫克/毫升÷ 59)。</li>
	<li>通过稀释 (5x) 50 毫克/毫升在一个 V 底 96-井板中的不育的 H2O 中的储存量, 使10毫克/毫升的个体肽储存 (这股含有20% 亚砜)。使这些股票在一个96井板, 因为确定每一个单独的肽的 Qa-1-restricted 反应将在一个 96-井板使用8或12声道多通道吸管。 注: 所有肽, 包括处和截断多肽, 应稀释在一个 V 底96井板或96井样品机架充满1毫升管, 因为肽库的筛选是在96井板使用多通道吸管进行。如果是难以稀释的肽, 添加 1 N 氢氧化钠滴明智帮助解散肽。</li>
</ol>
2. 启动的 kb-/- db-/- CD8+ T 细胞与 OLP_pool 脉冲 Kb-/- b/-树突状细胞 (DCs)。
注意: 有两个主要的 Qa-1 等位基因: 一个是 Qa-1a, 另一个是 Qa-1b。由于通常用于学术研究的动物,例如C57BL/6 和 balb/c/c 小鼠, 携带 Qa-1b, 本协议描述了在蛋白质中映射 Qa-1b表位的过程。在本协议中使用的 CD8+ T 细胞从 Kb---Db-/小鼠 (C57BL/6 背景) 纯化, 其中 CD8+ t 细胞主要受经典 MHC-Ib 分子 (包括 Qa-1) 的限制。
<ol>
	<li>如前所述, 产生骨髓源性 DCs, 如12。
	<ol>
		<li>简言之, 培养骨髓单细胞悬浮液 (1 x 106细胞/毫升) 在 RPMI-10 培养基 (RPMI 1640 补充10% 胎牛血清, 5.5 x 10-5 M 2-基, 1 毫米钠丙酮酸盐, 和0.1 毫米不必要的氨基酸)包含 10 u/毫升 IL-4 和 100 u/毫升 GM-CSF 在一个6井板 (4 毫升/井) 在37° c, 5% CO2。</li>
		<li>两天后, 小心去除换药细胞, 添加新鲜培养基和细胞因子。</li>
		<li>在培养了两天的细胞后, 将含有新鲜培养基和细胞因子的换药细胞转移到一个新的6井板中。</li>
		<li>再培养两天的细胞, 并补充含有 LPS (0.1 µg/毫升) 的新鲜培养基和细胞因子, 以激活 DCs。</li>
		<li>24小时后, 收集 DCs 进行实验。</li>
	</ol>
	</li>
	<li>用3000拉德照射 DCs。
	<ol>
		<li>或者, 在 PBS 中, 在37° c 处将 DCs (5 x 107细胞/毫升) 与丝裂霉素 c-(50 微克/毫升) 进行处理, 20 分钟. 添加 RPMI-0 (RPMI 1640 无血清), 以填补管 (〜12毫升) 和旋转的细胞10分钟在一个表顶离心机在 300 x g. 丢弃清和重复再洗两次。这三洗是关键的, 因为任何痕量的丝裂霉素 C 可能抑制 CD8 的反应, 在 DC t 细胞共培养期间的 t 细胞.</li>
	</ol>
	</li>
	<li>在无血清培养基中将 DC 浓度调整为 5 x 106细胞/毫升 (目的-V 无血清培养基补充 5.5 x 10-5 M 2-基, 1 毫米丙酮酸钠, 和0.1 毫米不必要的氨基酸)。</li>
	<li>添加 OLP_pool 的股票解决方案, 其中包含100% 个亚甲基亚砜, 以使最终的亚砜浓度将是 0.5% (200x) 的 DCs。在 MOG OLP 研究中, OLP_pool 的每 OLP 浓度为847.46 微克/毫升;因此, 每个 OLP 在 DC 培养的最终浓度是4.2 微克/毫升 (847.46 微克/毫升÷ 200)。</li></ol>然而, 只要在细胞培养中的亚砜浓度小于 1%, 这种浓度就可以扩大到100微克/毫升。
	<li>室温下孵育 DCs 3 小时, 每15分钟轻轻摇动细胞。</li>
	<li>在这个潜伏期, 使用商用 CD8+ T 细胞纯化试剂盒, 从收获的脾脏或淋巴结中纯化 CD8 的+ T 细胞, 将细胞浓度调整到 10 x 106细胞/毫升无血清培养基含有 50 u/毫升白细胞介素 2 (IL-2) 和 100 u/毫升的 IL-7。</li>
	<li>现在, 将 CD8+ T 单元格添加到一个48井板中, 作为0.5 毫升/井 (在步骤2.8 中增加了0.5 毫升/OLP_pool 脉冲 DCs 后, CD8+ T 单元格的最终浓度将是 5 x 106单元格/mL)。 注意: 根据制造商提供的指示, 可以使用 CD8 阳性隔离试剂盒来纯化小鼠脾脏和淋巴结的 CD8+ T 细胞。获得的 CD8+ T 细胞没有珠子。</li>
	<li>旋下 OLP_pool 脉冲 DCs (300 x g, 10 分钟) 在 RT. 在无血清培养基中重建 OLP_pool 脉冲 DCs 至 2 x 106细胞/毫升, 并将0.5 毫升/井放入含有 CD8+ T 细胞的 48-井板中 (OLP 的最终浓度_pool 脉冲 DCs 是 1 x 106细胞/毫升, 最终浓度的 CD8+ T 细胞是 5 x 106细胞/毫升, 最终浓度的 IL-2 是 25 u/毫升, 最终浓度的 IL-7 是 50 u/毫升)。培养细胞在37° c 和 5% CO2。</li>
	<li>在4天, 删除和丢弃约400μ l 培养基, 并添加500μ l 的新鲜无血清培养基含有 100 u/毫升 IL-2 和 100 u/毫升的 IL-7 每井。孵育细胞在37° c 和 5% CO2。</li>
	<li>在7或8天, re-stimulate OLP_pool 的 CD8+ T 细胞与 OLP_pool。</li>

3. Restimulation 的 CD8+ T 细胞的 OLP_pool
<ol>
	<li>四天前 re-stimulation 的 OLP_pool 引物 CD8 的+ T 细胞, 准备 2% (v/v) 聚丙烯酰胺珠解决方案: 洗涤2克聚丙烯酰胺珠两次在20毫升内毒素无 H2O 或 PBS。将聚丙烯酰胺微珠离心 (400 x g) 用于5分钟和重100毫升的 PBS。15磅/米的高压釜在室温下20分钟贮存。</li>
	<li>注射小鼠腹腔与1毫升/小鼠的无菌2% 聚丙烯酰胺珠溶液, 以吸引迁移的单核/巨噬细胞进入腹膜腔16。</li>
	<li>四天以后, 牺牲动物由 CO2药剂过量。
	<ol>
		<li>在无菌条件下, 在腹部中心切开一个小开口, 这样开口就足以传递5毫升的转移吸管。用 RPMI-0 填充转移吸管, 并通过开口将吸管插入腹腔。</li>
		<li>用移冲洗腹腔。从腹腔内取出尽可能多的液体到无菌管 (这些是腹膜巨噬细胞)。重复冲洗步骤 4-5 次。</li>
	</ol>
	</li>
	<li>用3000拉德照射腹膜巨噬细胞。
	<ol>
		<li>或者, 用丝裂霉素 C 治疗腹膜巨噬细胞 (遵循步骤2.2 中描述的步骤)。</li>
	</ol>
	</li>
	<li>在无血清培养基中调节腹腔巨噬细胞的浓度为 5 x 106 。添加 OLP_pool 的股票 (最后的亚砜浓度小于 1%) 和 M CSF (最终 concentration=100 U/毫升)。 注: 在本 MOG OLP 研究中, 我们使用了0.5% 的亚甲基亚砜。因此, MOG OLP_pool 的股票被稀释 200x (例如1 μ l 的 MOG OLP_pool 的股票被添加到199μ l 的腹膜巨噬细胞)。因此, 每个 OLP 的最终浓度是4.2 微克/毫升)。</li>
	<li>在48良好的组织培养板中添加200μ l/井 (1 x 106细胞/井), 并在37° c 处孵育该板块 4 h。</li>
	<li>用200μ l/5ml RPMI-0 的温和洗涤去除换药细胞和聚丙烯酰胺珠。</li>
	<li>收集并汇集步骤2.10 中的 OLP_pool 引物 CD8+ T 单元格。在含有 25 u/毫升 IL-2 和 50 u/毫升 IL-7 的无血清培养基中, 将 OLP_pool 引物 CD8+ T 细胞浓度调整为 1 x 106细胞/毫升。增加1毫升/井到 48-井板, 其中包含 OLP_pool 脉冲腹膜巨噬细胞。</li>
	<li>四天后, 在48井板中补充培养基。在48井培养皿中去除并丢弃大约400μ l 培养基。在每个井中加入500μ l 的新鲜无血清培养基, 含有100的 u/毫升 IL-2 和100毫升的 IL-7。培养细胞在37° c 和 5% CO2。</li>
	<li>三或四天后, 检查 OLP_pool-restimulated CD8+ T 细胞的 OLP_pool 特异, Qa-1-restricted 反应酶联 immunospot 检测 (ELISPOT)。在这一点之后, re-stimulate CD8 的+ T 单元格每 7-10 天。 注: 巨噬细胞是首选的 re-stimulation OLP_pool 引物的 CD8+ T 细胞。我们注意到, CD8 的+ T 细胞之后的增长比 DCs体外更好。</li>
</ol>
4. OLP_pool-restimulated CD8+ T 细胞系中的 OLP_pool 特异性、Qa-1-restricted 反应的测定
注: OLP_pool OLP_pool restimulated CD8+ T 细胞系中的特异性、Qa-1-restricted 反应是由 IFNγ在 OLP_pool 或 C1R 出现后由 C1R 分泌物所决定的。Qa-1b使用 IFNγ ELISPOT 检测的单元格。C1R 细胞可以在商业上获得。C1RQa-1b单元格可以通过换 C1R 单元格的 Qa-1 慢向量生成。
<ol>
	<li>添加100μ l/井的捕获抗干扰素γ抗体稀释在涂层缓冲 (PBS) 到 ELISPOT 板的井。</li></ol>在4° c 的夜间密封和孵化盘。
	<li>第二天, 丢弃含有捕获抗干扰素抗体的涂层缓冲液, 并添加200μ l/well-blocking 溶液 (无血清培养基), 并在室温下孵育2小时的板。</li>
	<li>照射 C1R 和 C1R。Qa-1b单元格, 具有9600拉德。
	<ol>
		<li>或者, 对待 C1R 和 C1R。Qa-1b细胞与丝裂霉素 C 如步骤2.2 所述, 除了治疗时间将是30分钟。</li>
	</ol>
	</li>
	<li>调整 C1R 和 C1R。Qa-1 在无血清培养基中的b细胞为 4 x 106细胞/毫升。</li>
	<li>丢弃板中的阻挡液, 添加 C1R 和 C1R。Qa-1b单元格位于50μ l/井 (20万细胞/井) 和混合。</li>
	<li>添加50μ l/100x 稀释 OLP_pool 库存 (在无血清培养基中), 并适当混合。在室温下孵育 2-3 小时。</li>
	<li>将 OLP_pool-restimulated CD8+ T 单元格调整为 1-2 x 106单元格/ml, 在含有 150 U/毫升 IL-7 的无血清培养基中, 并将50μ l/井 (5万-10万细胞/井) 添加到盘中。离心板 (58 x g) 为5分钟。</li>
	<li>在37° c 和 5% CO2的夜间孵育盘。</li>
	<li>吸气细胞悬浮。用去离子水 (250 μ l/井) 洗涤2次井。每洗一步, 允许水井浸泡5分钟。</li>
	<li>洗井3次与洗涤缓冲 I (PBS 包含 0.05% Tween20)。每洗一步, 允许水井浸泡1分钟。丢弃洗涤缓冲器。</li>
	<li>在稀释缓冲液中添加100μ l 的检测抗体 (PBS 含有10% 胎牛血清 (FBS))。</li>
	<li>在室温下孵育2小时。</li>
	<li>丢弃检测抗体溶液。洗涤井3次与250µL/井洗涤缓冲 i. 允许井在每个洗涤步骤浸泡1分钟。</li>
	<li>在稀释缓冲液中加入100μ l/好的酶共轭 (亲和-HRP) 稀释1:100 稀释。</li>
	<li>在室温下孵育1小时的板材。</li>
	<li>放弃酶共轭溶液。洗涤井4次与250µL/井洗涤缓冲 i. 允许井在每个洗涤步骤浸泡1分钟。</li>
	<li>洗井2次与250µL/洗井缓冲 II (PBS)。</li>
	<li>添加100µL 的衬底溶液 (混合 1 drop=20 µL 的原与1毫升的共体基底), 每井。监视5到60分钟的现场开发。</li>
	<li>当期望的结果开始出现时, 通过用蒸馏水冲洗水井来停止基底反应。</li>
	<li>在室温下晾干2小时或一夜, 直到完全干燥。去除板下的塑料托盘将有利于干燥。将盘子放在一个密封的塑料袋里, 直到被分析。</li>
	<li>通过在解剖显微镜下检查或使用 ELISPOT 板读取器来手动枚举斑点。请参见图 2中的代表结果。</li>

5. 在 OLP_pool 特异的 CD8+ T 细胞系中, 测定 OLP_pool 中的单个肽, 刺激 Qa-1 限制的干扰素-γ分泌
<ol>
	<li>使用上述的干扰素-γ ELISPOT 检测方法, 确定刺激 OLP_pool 特异的、Qa-1-restrictred 的干扰素分泌物的单个肽, 在 OLP_pool 特定的 CD8 的 T 细胞系中 (每个单独的肽的最终浓度为 ELISOPT 试验是10微克/毫升)。请参见图 3中的代表性结果。 注意: OLP 特定的、Qa-1-restircted 的响应被定义为响应, 它在 C1R 的存在中至少是响应的三倍。Qa-1b单元格, 但没有 OLP。在 MOG OLP 研究中, OLP68、OLP96 和 OLP105 都达到了这个标准。因此, 所有三处可能包含 Qa-1 表位12 (图 3)。然而, OLP105 始终给予最强烈的回应;因此, 我们对 OLP105 (图 4和图 5)12进行了详细的分析。</li>
</ol>
6. 在一个15度的 OLP 中确定最佳的 Qa-1 表位, 它在 OLP_pool 特定的 CD8+ T 细胞系中激发特定的、Qa-1-restricted 的反应
<ol>
	<li>合成了一个 15 OLP 的 C 和 N-末期截断肽, 如图 4所示。</li>
	<li>在 OLP 特定的 CD8+ t 细胞系中, 通过刺激 CD8 的+ t 细胞与每个被截断的肽, 以及在 C1R 或 C1R 的存在下的父母15的 OLP。Qa-1b使用以上描述的干扰素 ELISPOT 检测细胞。每个单独的肽的最终浓度用于 ELISPOT 测定是10微克/毫升。肽被定义为最佳的 Qa-1 表位, 如果肽: 1) 一致地给予相似或更强的干扰素-γ反应, 与原始的 15-在 C1R 的存在相比。Qa-1b单元格;2) 介于 8-10-滨海 (9-滨海肽的首选) 之间的肽长度绑定到 MHC I 分子15,17。请参见图 5中的代表性结果。</li>
</ol>

<ol>
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W., Tang, X., Kim, H. 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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+selection+by+MHC+class+I+molecules.">Peptide selection by MHC class I molecules.</a> Nature. 350 (6320), 703-706 (1991).</li><li>Zhang, X., Goncalves, R., Mosser, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+and+characterization+of+murine+macrophages.">The isolation and characterization of murine macrophages.</a> Curr Protoc Immunol. , (2008).</li><li>Guo, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+length+peptides+bind+to+HLA-Aw68+similarly+at+their+ends+but+bulge+out+in+the+middle.">Different length peptides bind to HLA-Aw68 similarly at their ends but bulge out in the middle.</a> Nature. 360 (6402), 364-366 (1992).</li><li>Soloski, M. J., Metcalf, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+involvement+of+class+Ib+molecules+in+the+host+response+to+infection+with+Salmonella+and+its+relevance+to+autoimmunity.">The involvement of class Ib molecules in the host response to infection with Salmonella and its relevance to autoimmunity.</a> Microbes Infect. 3 (14-15), 1249-1259 (2001).</li><li>Smith, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+use+endocytic+pathway+for+cross-priming+class+Ib+MHC-restricted+CD8alphaalpha+TCRalphabeta++T+cells+with+regulatory+properties.">Dendritic cells use endocytic pathway for cross-priming class Ib MHC-restricted CD8alphaalpha+TCRalphabeta+ T cells with regulatory properties.</a> J Immunol. 182 (11), 6959-6968 (2009).</li><li>Kalra, A., Mukherjee, P., Chauhan, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+fine+specificity+of+the+immune+response+to+a+Plasmodium+falciparum+rhoptry+neck+protein,+PfAARP.">Characterization of fine specificity of the immune response to a Plasmodium falciparum rhoptry neck protein, PfAARP.</a> Malar J. 15, 457 (2016).</li><li>Kambayashi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nonclassical+MHC+class+I+molecule+Qa-1+forms+unstable+peptide+complexes.">The nonclassical MHC class I molecule Qa-1 forms unstable peptide complexes.</a> J Immunol. 172 (3), 1661-1669 (2004).</li></ol>

作者声明没有利益冲突。

在这里, 我们描述了一个用于分析蛋白质中 Qa-1 表位的协议。关于该议定书, 以前还报告了其他一些战略。首先, 异基因 CD8+ T 单元格线和克隆用于标识 Qdm1。其次, 一个假定的 Qa-1-binding 主题从分析 Qdm 用于识别 HSP60p216-224 和 TCRBV8.1 表位9,18。第三, 从蛋白质中单独重叠的肽被用于免疫动物。随后, 从免疫动物中分离出来的 CD8+ T 细胞被用来识别 TCRBV8.2 肽 p42-50, 后者随后被确认为绑定到 Qa-16、10、19。第四, 功能性 CD8+ T 细胞系结合 cDNA 显示库用于鉴定 FL9 Qa-1 表位的4。
参照以前的技术, 使用异基因 CD8 的+ T 细胞系和克隆可能不适合分析在生理免疫应答过程中提出的 Qa-1 表位。此外, 积累的数据表明, 先前描述的结合主题可能只部分代表了 Qa-1 分子的肽结合能力。因此, 确切的 Qa-1-binding 主题仍然不知道4。此外, 以单个肽为 Qa-1 抗原表位的免疫接种是费力的。最后, cDNA 显示库策略涉及在细胞转染过程中多肽长度载体的构建。相比之下, 这里描述的 OLP 库策略很简单, 并且已知的映射多肽可以刺激 Qa-1-restricted CD8 的+ T 细胞。因此, 我们的战略具有独特的优势。
这里描述的策略使用了 OLP_pool 特定的 CD8+ T 细胞, 它们是由体外启动和 restimulation 生成的。CD8+ T 细胞的另一个来源可以是从引流的淋巴结的 kb-/-Db-/-鼠标, 免疫与源蛋白20。这样的免疫 CD8+ T 细胞可以检查干扰素γ反应的处和截断多肽, 以确定 Qa-1 表位。从理论上讲,体内免疫 CD8 的+ t 细胞比体外生成的 CD8+ t 细胞更接近生理条件。然而, 我们发现免疫 CD8+ T 细胞从免疫小鼠直接纯化的反应较弱, 往往需要体外restimulations 以获得满意的肽筛选结果。此外, 该协议的目的是为了将来的翻译人类研究的 HLA-E 表位, 其中在体内免疫是不实际的。
从技术上讲, 此协议的关键部分是生成特定于 OLP_pool 或单个 OLP 的 CD8+ T 单元格行。由于 Qa-1/peptide 配合物的不稳定与经典的 MHC I/肽配合物21, 在抗原呈递细胞与处或肽脉冲后, 脉冲细胞不应被广泛冲洗。我们发现, 多肽脉冲细胞, 如果被广泛冲洗, 会丢失或显著降低刺激 CD8 的+ T 细胞的能力。因此, 我们建议在共使用 CD8+ T 细胞之前, 立即清洗多肽脉冲抗原呈递细胞。
此外, 由于 Qa-1/peptide 配合物的不稳定性, 我们建议使用无血清培养基进行肽脉冲和刺激 CD8+ T 细胞。此外, 我们经常增加 50 U/毫升的 IL-7 在 CD8+ T 细胞培养期间, 以及在干扰素γ ELISPOT 检测。IL-7 的目的是维护 CD8+ T 单元的可行性和功能。IL-7 本身并不刺激 CD8 的+ T 细胞产生干扰素γ。
与所有策略相似, Qa-1 表位映射的这一策略也需要进一步研究生物学意义。例如, 一个重要的问题是, 是否可以在生理学上提出映射的表位。为了解决这个问题, DCs 可以转慢向量, 表达表位源蛋白 (如 MOG 在 MOG OLP 研究)。随后, 研究了特定于表位的 CD8+ T 细胞对转 DCs 的响应。阳性反应表明, 该表位可由 DCs 进行生理处理和呈现。此外, 应进一步研究 CD8+ T 细胞的表位识别结果的功能性后果。在这方面, 对 Qdm 和 FL9 Qa-1 表位的分析导致了 Qa-1 分子对免疫监测非常重要的结论1,4。此外, 对 HSP60p216、TCRBV8.1 肽和 p42-50 的研究也导致了 Qa-1-restricted CD8+ t 细胞通过靶向致病 CD4+ t 细胞调节免疫应答的结论6,7 ,9,19。此外, 对 MOG196的研究首次显示, Qa-1-restricted CD8+ T 细胞可以通过直接靶向组织来调节免疫应答, 以防止潜在致病性自身免疫细胞的损伤,12.最后, 对表位特定的、Qa-1-restricted 的 CD8+ T 细胞的功能分析可由聚丙烯辅助。这种聚丙烯可以在 NIH 聚丙烯核心设施 (http://tetramer.yerkes.emory.edu/) 或公司生产。为了请求聚丙烯的生成, 您可以发送支持新映射的最佳 Qa-1 表位的绑定的功能数据, 以 Qa-1 蛋白质为其生成 Qa-1 聚丙烯的批准。
最后, 先前的研究表明, Qa-1-restricted CD8+ T 细胞在调节局部免疫应答中扮演重要角色12。在这方面, 组织损害可能是由直接针对组织的免疫反应引起的。此外, 间接损害可能是由于免疫反应的目标是入侵的病原体。因此, 了解 Qa-1-restricted CD8 在这两种不同组织损伤中的作用, 对其临床应用非常重要.为了达到这个目标, 需要对 self-tissue 或入侵病原体的 Qa-1 表位进行透彻的分析。本议定书将适用于这些分析。

Qa-1 属于一组经典主要组织相容性复合体 1b (MHC-Ib) 分子的小鼠。它的人类同源是 HLA E。以前的证据表明 Qa-1 分子具有重要的生物学功能。首先, Qa-1 分子在测量细胞结构和功能完整性方面起着重要作用。在这方面, Qa-1 分子已经进化了一些策略来监测细胞的正常功能。一个这样的策略使 Qa-1 分子能够形成与加工的领导肽 (表位),即Qa-1 行列式修饰 (Qdm), 从经典的 MHC Ia 分子在内质网1处理的复合物。这些 Qa-1/Qdm 配合物随后在细胞表面显示, 并结合 nk 细胞抑制性 NKG2A 受体, 抑制 nk 杀伤活性2。如果 MHC-Ia 分子的表达丢失, 细胞 (如恶性细胞) 就会对 NK 的杀伤2敏感。另一种策略使 Qa-1 分子能够在细胞表面形成新的 Qa-1/epitope 配合物 (与抗原处理相关的运输载体)3和/或 ERAAP (内质网肽相关抗原处理)4 (这两种缺陷经常发生在恶性细胞中)。表达这些新的 Qa-1/epitope 复合体的细胞可以通过表位特异的 Qa-1-restricted CD8+ T 细胞识别和消除。其次, Qa-1 分子诱导免疫调节5。在这方面, Qa-1/epitope 配合物已被证明刺激 CD8+调节 T (t) 细胞, 这是重要的预防免疫介导的损害 self-tissues67,8 ,9,10。第三, Qa-1-restricted CD8+ t 细胞已被证明可以限制免疫应答病毒感染11。
因此, 具体增加表位特定的 Qa-1-restrictred CD8+ T 细胞是一个潜在的可行的策略, 以消除异常细胞, 增强免疫调节, 并为控制的大小病毒诱导的免疫应答。虽然尚未确定是否增加表位特定的 Qa-1-restricted CD8+ T 细胞可以增强免疫监测和限制病毒诱导的免疫应答, 但我们的实验室和其他人已经清楚地表明免疫 Qa-1 表位可增强 Qa-1-restricted CD8 t 细胞特异性自身免疫 CD4 + t 细胞的功能, 从而有效控制 CD4+ t 细胞介导的自身免疫性疾病实验性反应性脑脊髓炎动物模型的多样性 ( 人多发性硬化动物模型 )6,10, 胶原诱导关节炎 ( 人类类风湿关节炎动物模型 )7, 以及肥胖糖尿病 (人类1型糖尿病动物模型)8。此外, 我们发现, 免疫与组织特定的 Qa-1 表位导致特定的控制免疫介导的炎症, 在该组织通过增强 CD8+ t 细胞12。上述临床前研究的成功表明, 需要对 Qa-1 表位免疫进行全面评估, 以治疗组织特异性免疫介导的疾病, 并有可能治疗与自来水缺乏症相关的其他疾病, 并ERAAP
因此, 有一个技术的需求, 可以可靠和快速地分析 Qa-1 表位的蛋白质。在这方面, 描述了数量有限的生物重要的 Qa-1 表位。大多数这些 Qa-1 表位被确定偶然在研究 CD8+ T 细胞对细菌的反应13, 细胞缺乏在 TAP3, 细胞缺陷在 ERAAP4, 和细胞, 导致 EAE6, 9。因此, 一个高吞吐量的技术是可取的, 以确定生物重要的 Qa-1 表位在一个定义的蛋白质。在下面, 我们描述一个重叠的肽 (OLP) 图书馆策略, 映射功能的 Qa-1 表位在一个蛋白质使用 Qa-1-resrticted CD8+ T 细胞系特定的 OLP 池 (OLP_pool) 的蛋白质。

<table>
	<tbody>
		<tr>
			<td>The protein to be analyzed</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Sequence of the protein can be obtained from NCBI</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>Cat#: D2650 SIGMA</td>
			<td>DMSO should be sterile and cell culture tested.</td>
		</tr>
		<tr>
			<td>Kb-/-Db-/- mice</td>
			<td>The Lackson Laboratory</td>
			<td>Stock#: 019995</td>
			<td>We used Taconic H2-KbH2-Db doube knockout mice (Cat#: 4215-F and 4215-M) which however are not available anymore.</td>
		</tr>
		<tr>
			<td>AIM V Serum Free Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat#: 12055091</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat#: 21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat#: 11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential Amino Acids</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat#: 11140076</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads CD8 Positive Isolation Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat#: 11333D</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Gel P-100</td>
			<td>Bio-Rad</td>
			<td>Cat#: 150-4171</td>
			<td></td>
		</tr>
		<tr>
			<td>Phoshate Balanced Solution (PBS)</td>
			<td>ThermoFisher Scientific</td>
			<td>Cat#: 20012027</td>
			<td></td>
		</tr>
		<tr>
			<td>Trasfer pipette</td>
			<td>Globe Scientific</td>
			<td>Mfg#: 137238</td>
			<td></td>
		</tr>
		<tr>
			<td>Murine M-CSF</td>
			<td>PeproTech</td>
			<td>Cat#: 315-02</td>
			<td></td>
		</tr>
		<tr>
			<td>48-well tissue culture plates</td>
			<td>USA Scientific</td>
			<td>Cat#: CC7682-7548</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar TC-treated Multiple well Plates, 96-well, V-shaped bottom</td>
			<td>Sigma-Aldrich</td>
			<td>Cat#: Z372129 Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>1ml deep 06-well PP plate, sterile</td>
			<td>USA Scientific</td>
			<td>Item#: 1896-1110</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant murine IL-2</td>
			<td>PeproTech</td>
			<td>Cat#: 212-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant murine IL-7</td>
			<td>PeproTech</td>
			<td>Cat#L: 217-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Capture anti-IFN-&#947; antibody</td>
			<td>BD Biosciences</td>
			<td>Cat#: 551881</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISPOT plate</td>
			<td>Sigma-Aldrich</td>
			<td>Cat#: S2EM004M99</td>
			<td></td>
		</tr>
		<tr>
			<td>C1R</td>
			<td>ATCC</td>
			<td>Cat#: ATCC CRL-1993</td>
			<td></td>
		</tr>
		<tr>
			<td>C1R.Qa-1b</td>
			<td></td>
			<td></td>
			<td>Custom made (GenBank access#: NM_010398.3)</td>
		</tr>
		<tr>
			<td>Qa-1 lentiviral vector</td>
			<td>GeneCopoeia</td>
			<td>Product#: Mm02955</td>
			<td></td>
		</tr>
		<tr>
			<td>Detection anti-IFN-&#947; antibody</td>
			<td>BD Biosciences</td>
			<td>Cat#: 551881</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Sigma-Aldrich</td>
			<td>Cat#: P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-HRP</td>
			<td>BD Biosciences</td>
			<td>Cat#: BD557630</td>
			<td></td>
		</tr>
		<tr>
			<td>AEC substrate</td>
			<td>BD Biosciences</td>
			<td>Cat#: 551951</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmunoSpot Analyzer</td>
			<td>ImmunoSpot</td>
			<td></td>
			<td>Any immunoSpot analyer should work for this purpose.</td>
		</tr>
	</tbody>
</table>

overlapping peptide library to map qa-1 epitopes in a protein

Qa-1 (HLA E 在人) 属于一组经典主要组织相容性复合体 1b (MHC-Ib) 分子。最近的数据表明, Qa-1 分子在测量细胞的结构和功能完整性, 诱导免疫调节, 并限制免疫应答病毒感染的重要作用。此外, 通过表位免疫功能增强 Qa-1-restricted CD8 的+ T 细胞, 在几种自身免疫性疾病动物模型中显示了治疗效果,例如: 实验过敏脑脊髓炎,胶原诱导的关节炎, 肥胖糖尿病。因此, 迫切需要一种方法, 可以有效和快速地确定功能 Qa-1 表位的蛋白质。在这里, 我们描述一个协议, 利用 Qa-1-restricted CD8+ T 细胞系特定的重叠肽 (OLP) 库, 以确定 Qa-1 表位的蛋白质。这个 OLP 库包含15的重叠多肽, 覆盖了整个蛋白质的长度, 相邻的多肽与11种氨基酸重叠。使用该协议, 我们最近确定了一个 9 Qa-1 表位的髓鞘胶质糖蛋白 (MOG)。这一新映射的 MOG Qa-1 表位被证明是为了诱导特定的抗原, Qa-1-restricted CD8+ T 细胞增强髓鞘特异的免疫调节。因此, 该协议对于将来研究 Qa-1-restricted CD8+ T 单元格的新目标和功能是有用的。

一种覆盖整个蛋白质长度的 OLP 库的设计
从蛋白质的 N 末端开始, 每个肽是15氨基酸 (15)。因此, 第一肽跨越位置1到位置15。第二肽的 N 末端与第一个肽的 C 末端重叠由11氨基酸。因此, 第二肽跨越位置5到位置19。将其余的肽设计到蛋白质的 C 端末端 (图 1)。我们选择了 15-滨海图书馆, 因为我们和其他人已经表明, 15-滨海肽, 当添加到树突状细胞 (DCs) 或巨噬, 可以有效地处理成表位, 以识别通过 CD8+ T 细胞14,15。库中处的数量取决于蛋白质的长度。另外, 最后的 OLP 可以从12到15的范围, 取决于蛋白质的长度。例如, 髓鞘胶质糖蛋白 (MOG) 的长度为247氨基酸。MOG OLP 库包含59处12。本文的代表性成果来自于对 MOG OLP 图书馆的分析。
OLP_pool 刺激 CD8+ T 细胞系中 OLP_pool 特异、Qa-1-restricted 反应的测定
我们生成了一个 CD8 的+ T 单元格, 该行由 Kb---Db/ -DCs 脉冲 MOG OLP_pool (MOG_pool) 和 restimulated 由 MOG_pool 脉冲 Kb-Db-腹腔巨噬细胞的描述上述议定书 (议定书1、2和 3)。为了确定此 CD8+ t 单元格行中潜在的 MOG_pool 特定的 Qa-1-restricted 响应, 我们检查了此 CD8+ t 单元格线对 MOG_pool 的响应, 在 C1R 或 C1R 的存在。使用干扰素-γ ELISPOT 检测 (图 2) (协议步骤 4) Qa-1b单元格。我们的数据显示, MOG_pool 刺激的 CD8+ T 细胞系在 C1R 的存在下分泌了低水平的干扰素γ。Qa-1b单元格 (32 点形成单元格或 SFCs), 但不 C1R 单元格 (2 SFCs), 提示 CD8 细胞 (Qa-1-binding 细胞是人类 b C1R 细胞) 中出现了对 C1R 抗原表位作出反应的+ T 细胞。当 MOG_pool 被添加到包含 C1R 细胞 (78 SFCs) 的井中时, SFCs 增加了, 这表明 CD8 的+ T 细胞的存在对 non-Qa-1 表位有反应。当 MOG_pool 被添加到含有 C1R 的井中时, SFCs 急剧增加。Qa-1b单元格 (320 SFCs), 表明存在 CD8+ T 细胞的反应 Qa-1-binding 肽。数据还表明, MOG_pool 包含 Qa-1-binding 表位 (s)。
OLP_pool 中单个肽的测定在 OLP_pool 反应 CD8 细胞系中对 Qa-1-restricted 干扰素分泌的贡献 
为了确定在 MOG_pool 反应 CD8+ t 细胞系中刺激 Qa-1-restricted 干扰素分泌的 OLP_pool 中的多肽, 我们刺激了 MOG_pool-反应 CD8+ t 细胞, 其中有处的个体 59 C1R或 C1R。Qa-1b单元格 (图 3) (协议步骤 5)。我们的数据显示, 在含有 C1R 细胞和处的水井中观察到的 SFCs 很少。相比之下, SFCs 在所有含 C1R 的水井中都增加了。Qa-1b单元格和处。从图 2中, 我们了解到在存在 C1R 的情况下 CD8+ T 单元格。Qa-1b也单独形成了 SFCs。因此, 在大多数水井中增加的 SFCs 可能是由于非特异性的刺激, 这是由于 Qa-1 分子在 C1R 细胞内的胞浆蛋白的 Qa-1 表位的表达。但是, 在图 3C (B8、D12 和 E9) 中的3帧井中的处在图 3A (处、OLP68 和 OLP96) 中与3突出显示的 OLP105 相匹配, 满足了定义 OLP 特定、Qa-1-restricted 干扰素-γ响应的标准, 作为在协议步骤 5.112中描述。数据表明, 3 处含有 Qa-1 表位 (s)。由于 OLP105 始终产生最高的响应, 我们对 OLP10512进行了详细的分析。
一种 15 OLP 的截断肽库的设计
一个 15 OLP, 已决定刺激 Qa-1-restricted 干扰素分泌在 OLP_pool 反应 CD8+ T 细胞系, 需要分析的最佳表位使用截断肽库。这种截断肽库的设计是通过在 N 和 C (图 4) 中逐步截断 15 OLP 的1氨基酸。类似于其他 MHC I 类分子, Qa-1 分子主要绑定到 8-10-滨海肽。因此, 我们建议最短截短肽的长度为6。
在 OLP_pool 反应 CD8+ T 细胞系中激发 Qa-1-restricted 干扰素分泌物的 15 OLP 中最佳 Qa-1 表位的鉴定
上述 N 和 C-末期截短肽测试的强度, 以刺激干扰素γ分泌在一个 CD8 的+ T 细胞线特定的 OLP_pool 或 15 OLP 使用的干扰素γ ELISPOT 如上所述。在 MOG12的 Qa-1 表位的研究中, 我们生成了一个 CD8+ T 单元格, 该行是为 MOG OLP105 (图 5A) 指定的。进一步的分析表明, N-终末截断的 9-滨海肽满足了协议步骤 6.2 (图 5B) 中所述的最佳表位的两个标准。因此, 我们得出结论, n-终末期截断 9-滨海肽是最佳的 Qa-1 表位。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56401/56401fig1.jpg"> 
图 1: 设计一个覆盖整个蛋白质长度的 OLP 库.从 N 末端开始, 确定了15氨基酸 (15-长度) 的多肽。除第一肽外, 每个肽的 N 末端与前肽的 C 末端重叠, 由11氨基酸组成。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56401/56401fig2.jpg"> 
图 2: MOG_pool 激发的 CD8+ T 单元格是部分 MOG_pool 特定和 Qa-1-restricted12.体外CD8 MOG OLP_pool (MOG_pool) 刺激的 T 细胞在 MOG_pool 或 C1R 的存在下对 C1R 的反应进行了检查.Qa-1b细胞作为抗原呈递细胞使用干扰素γ ELISPOT 检测。显示了具有代表性的 ELISPOT 图像。在每一个井的左上角都显示了成 SFCs 细胞的数量。CD8+ T 单元格: 5万细胞/井。C1R 或 C1R。Qa-1b单元格: 20万细胞/井。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56401/56401fig3.jpg"> 
图 3: 分析 MOG_pool 中负责 Qa-1 限制响应的处.(A) V 底 96-井板的布局, 其中包含10毫克/毫升个人 MOG 处。井中的数字代表 OLP id。突出显示的3口井包含符合 OLP 特定的 Qa-1-restricted 干扰素响应标准的处, 如协议步骤 5.112所述。(B)代表 SFCs 在每个井中包含一个 OLP (最终浓度 = 4.2 微克/毫升, OLP ID 匹配在 "A"), MOG_pool 反应 CD8+ T 细胞 (5万细胞/井), 和 C1R 细胞 (20万细胞/井)。(C)代表 SFCs 在每个井中包含一个 OLP (最终浓度 = 4.2 微克/毫升, OLP ID 匹配在 "A"), MOG_pool 反应 CD8+ T 细胞 (5万细胞/井), 和 C1R。Qa-1b单元格 (20万单元格/井)。3框架井包含了符合标准的处 OLP 特异性, Qa-1-restricted 干扰素γ响应12。该图已从引用12中进行了改编。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56401/56401fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56401/56401fig4.jpg"> 
图 4:15 肽的截断肽的设计.15-氨基肽在其 N 和 C 的终点被1的氨基酸逐渐截断为 6-滨海。然后合成了15的蛋白及其截短肽。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56401/56401fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56401/56401fig5.jpg"> 
图 5: OLP105 中最佳 Qa-1 表位的分析。(A)在 OLP105 或 C1R 的存在下, 检查 OLP105-stimulated CD8+ T 细胞系, 以响应 OLP68、OLP96 和 C1R。使用干扰素-γ ELISPOT 法 Qa-1b细胞。代表干扰素γ SFCs (数字在左上角) 显示。(B) OLP105-specific CD8+ T 单元格线, 如 (A) 所示, 被检查以响应单个 N 和 C-末期截断肽, 如图 4所示, 在 C1R 的存在。使用干扰素-γ ELISPOT 法 Qa-1b细胞。OLP105: 一个 15 OLP。N6-14: n-终末期截断 OLP105 肽。C6-14: c-终末期截断 OLP105 肽。CD8+ T 单元格: 5万细胞/井。C1RQa-1b单元格: 20万细胞/井。左上角的数字为每 5万 CD8+ T 单元格 SFCs。红色矩形标记的井符合定义的标准, 在一个 OLP 中的最佳 Qa-1 表位, 如协议步骤6.2 所述。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56401/56401fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein at JoVE.com

Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein

重叠肽库在蛋白质 Qa-1 表位中的映射

Qa-1 (HLA) 属于一组经典主要组织相容性复杂的1b 分子。免疫 Qa-1-binding 表位已表明, 以加强组织特异性免疫调节和改善一些自身免疫性疾病。在此, 我们描述一个重叠的肽库策略, 以确定 Qa-1 表位的蛋白质。

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules ...

overlapping-peptide-library-to-map-qa-1-epitopes-in-a-protein

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

6.8K 次观看.  Loma Linda University. Qa-1 (HLA) 属于一组经典主要组织相容性复杂的1b 分子。免疫 Qa-1-binding 表位已表明, 以加强组织特异性免疫调节和改善一些自身免疫性疾病。在此, 我们描述一个重叠的肽库策略, 以确定 Qa-1 表位的蛋白质。

视频: Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

使用γ-干扰素酶酶联免疫检测的特点人类乳头状瘤病毒的新型T-细胞表位

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

科研

免疫与感染

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

使用人工痰介质测试抗生素药效试验<em&gt;绿脓杆菌</em&gt;更多相关的肺囊肿性纤维化的条件

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

比较方法来表征景观的宿主 - 病原体蛋白 - 蛋白相互作用

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

总蛋白提取和二维凝胶电泳方法<em&gt;鼻疽</em&gt;物种

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

对于血液处理快速的方法，以提高血浆肽水平的人血产量

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents directly bind to T-cell receptors on the surface of CD8+ T-lymphocytes, fluorochrome-labeled pMHC-I tetramers enable the accurate detection of antigen (Ag)-specific CD8+ T-cells without the need for in vitro re-stimulation. Moreover, when combined with multi-color flow cytometry, pMHC-I tetramer staining can reveal key aspects of Ag-specific CD8+ T-cells, including differentiation stage, memory phenotype, and activation status. These types of analyses have been especially useful in the field of HIV immunology where CD8+ T-cells can affect progression to AIDS. Experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) provides an invaluable tool to study cellular immunity against the AIDS virus. As a result, considerable progress has been made in defining and characterizing T-cell responses in this animal model. Here we present an optimized protocol for enumerating SIV-specific CD8+ T-cells in rhesus macaques by pMHC-I tetramer staining. Our assay permits the simultaneous quantification and memory phenotyping of two pMHC-I tetramer+ CD8+ T-cell populations per test, which might be useful for tracking SIV-specific CD8+ T-cell responses generated by vaccination or SIV infection. Considering the relevance of nonhuman primates in biomedical research, this methodology is applicable for studying CD8+ T-cell responses in multiple disease settings.

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Here, we present an optimized protocol for enumerating and characterizing rhesus macaque CD8+ T cells against the AIDS virus. This article is useful not only to the field of HIV immunology, but also to other areas of biomedical research where CD8+ T cell responses are known to affect disease outcome.

猴免疫缺陷病毒特异性CD8分析<sup&gt; +</sup&gt; T细胞猕猴由肽MHC-I四聚体染色

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

使用细菌共轭的 Enterobacterial 菌株 (如大肠杆菌或志贺菌福氏) 创建稠密的转插入库

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

分子互补亲和纯化 (BiCAP) 对多蛋白信号络合物的解剖

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...