这项研究得到了中国国家自然科学基金（X.C. 的第 32300785 号）、中国国家创新人才博士后计划（第 1 号）的资助。BX20230449 至 X.C.），以及国家科技重大专项（编号 2021YFC2300602 至 L.Y.）。

所有动物实验均按照第三军医大学机构动物护理和使用委员会批准的程序进行。本研究使用了以下小鼠品系：C57BL/6J （B6） 小鼠（男女），年龄 6 至 8 周龄，体重 25-30 克;CD45.1+SM TCR转基因小鼠（B6 CD45.1 × SM TCR转基因），两性，年龄6-8周龄，体重25-30 g;和 CXCR5-GFP CD45.1+SM TCR 转基因小鼠 （B6 CD45.1 × SM TCR 转基因 × CXCR5-GFP 敲入），两性，年龄 6 至 8 周，体重 25-30 g。转基因物种是在先前发表的报告24 之后生成的。材料 表中列出了研究中使用的试剂、缓冲液和设备的详细信息。
1. 收获 CD45.1 + SM CD4 + T 细胞进行过继转移
<ol><li>对所需数量的 6 至 8 周龄 CD45.1 + TCR 转基因 SM 小鼠24 实施安乐死（遵循机构批准的方案）。用手术剪刀在安乐死小鼠的腹膜壁左侧切开 1 英寸，从腹膜解剖脾脏，撕开脾脏后面的结缔组织25,26。</li>
	<li>将每个脾脏放入培养皿（60 mm × 15 mm）内的 70 μm 细胞过滤器中，并使用 3 mL 注射器柱塞用 1 mL 红细胞 （RBC） 裂解缓冲液研磨脾脏。 注意：使用注射器柱塞的圆形侧面研磨脾脏，直到只剩下结缔组织。</li>
	<li>向每个培养皿中加入 6 mL RPMI + 2% FBS （RPMI 2%） 以稀释 3 mL RBC 裂解缓冲液。将细胞悬液从培养皿中移液到 15 mL 锥形管中。再加入 3 mL 2% RPMI 冲洗培养皿，然后转移到同一锥形管中。</li>
	<li>将细胞悬液在 4 °C 下以 800 × g 离心 5 分钟，然后倾析弃去上清液。用分离缓冲液重悬细胞，并将细胞密度调节至 ~1 × 108 个细胞/mL。将试管放在冰上。</li>
	<li>在分离缓冲液中制备针对 CD8a、CD19、NK1.1、F4/80、CD11c 和 Ter119 的 1x 生物素化抗体混合物，用于富集 CD4 + T 细胞。 注：如 材料表中所述，将生物素化抗体按指定稀释度添加到分离缓冲液中。保持抗体混合物的体积与步骤 1.4 中分离缓冲液的体积相同。</li>
	<li>将细胞悬液在 4 °C 下以 800 × g 离心 5 分钟，然后倾析弃去上清液。</li>
	<li>在锥形管中用指定的 1x 生物素化抗体混合物重悬细胞沉淀。</li>
	<li>将锥形管在摇床中的冰上以 45 rpm 的速度放置 30 分钟。</li>
	<li>在与生物素化抗体孵育期间，根据制造商的说明制备所需体积的链霉亲和素包被的珠子。 注：来自一只幼稚小鼠的总脾细胞需要 200 μL 链霉亲和素包被的珠子。</li>
	<li>通过在 4 °C 下用 800 x g 和 8-10 mL 分离缓冲液离心 5 分钟来洗涤细胞，然后弃去上清液。重复此步骤 2 次。</li>
	<li>用准备好的链霉亲和素包被的珠子重悬细胞沉淀。</li>
	<li>将试管放在冰上，在摇床中以 45 rpm 的速度放置 40 分钟。</li>
	<li>然后，将试管放在磁力架上 3-4 分钟，直到所有珠子都组装在磁性侧。</li>
	<li>小心收集上清液并将其转移到新的 15 mL 锥形管中。</li>
	<li>将细胞悬液在 4 °C 下以 800 × g 离心 5 分钟，然后倾析弃去上清液。 注：还有其他可用于 CD4 + T 细胞富集的商业试剂盒，可用作步骤 1.5-1.15 中描述的 CD4 + T 细胞富集程序的替代品。</li>
	<li>用 2-3 mL 的 2% RPMI 重悬细胞沉淀。吸出 50 μL 细胞悬液，用于流式细胞术分析 CD4 + T 细胞富集（CD4 和 Vα 2 ）的效率和细胞活力。</li>
	<li>将细胞悬液在 4 °C 下以 800 × g 离心 5 分钟，然后倾析弃去上清液。用 RPMI 重悬细胞沉淀，并将细胞密度调节至每 mL 1 ×7 个 CD4+Vα2+ SM 细胞。将试管放在冰上，并在下一步中进行过继细胞转移。</li>
</ol>2. 建立急性 LCMV 感染的 SM 嵌合体小鼠模型，用于获取早期分化的 SM TFH 细胞
<ol><li>用红外灯加热 6 至 8 周龄的 C57BL/6 受体小鼠，以扩张尾静脉进行静脉注射。</li>
	<li>用胰岛素注射器吸出 100 μL 细胞悬液，其中包含步骤 1.17 中制备的 1 × 106 个 SM 细胞。 注意：每位研究人员应修改注射量，并且不应超过机构和当地法律允许的最大注射量。</li>
	<li>用鼠标固定器将小鼠固定到位，拉直尾巴，然后用棉球用 75% 乙醇擦拭尾巴，使尾静脉可见。</li>
	<li>将胰岛素注射器针头插入尾静脉，平稳缓慢地注射细胞悬液。然后，让受体小鼠休息过夜。</li>
	<li>第二天，用 RPMI 培养基稀释 LCMV Armstrong 病毒原液，以达到 1 ×10 7 噬菌斑形成单位/mL 的最终浓度。</li>
	<li>将 100 μL 稀释的 LCMV Armstrong 溶液（含有 1 × 106 个噬菌斑形成单位）注入受体小鼠的尾静脉。 注意：每位研究人员都应谨慎对待机构和当地法律是否允许推荐的感染滴度。</li>
</ol>3. 早期分化 SM TFH 细胞的流式细胞术染色
<ol><li>在感染后第 3 天处死第 2.6 步的小鼠（遵循机构批准的方案），并按照第 1 步中的描述处理来自脾脏的淋巴细胞。将细胞密度调整为大约 2 × 107 个细胞/mL。</li>
	<li>将 50 μL 细胞悬液（~1 × 106 个细胞）加载到圆底 96 孔板中，并在每孔中加入 150 μL 染色缓冲液。将盘子放在冰上。</li>
	<li>在 50 μL TFH 细胞染色缓冲液中，以 1：50 稀释度制备纯化的大鼠抗小鼠 CXCR5 抗体混合物（即 CXCR5 一抗），用于试管中的每个样品。彻底涡旋试管并在 4 °C 下以 15,000 × g 离心 3 分钟以聚集颗粒。将试管放在冰上。</li>
	<li>将 96 孔板在 4 °C 下以 800 × g 离心 1 分钟，倾析弃去上清液，短暂涡旋板，每孔加入 200 μL 染色缓冲液。</li>
	<li>重复步骤 3.4，每孔用 50 μL CXCR5 一抗重悬细胞。通过上下吹打充分混合，并在冰上孵育 1 小时。</li>
	<li>在新试管中的每个样品中，以 1：200 稀释度制备生物素化山羊抗大鼠抗体混合物（即 CXCR5 二抗），溶于 50 μL TFH 细胞染色缓冲液中。涡旋试管并在 4 °C 下以 15,000 × g 离心 3 分钟以聚集颗粒。将试管放在冰上。</li>
	<li>在 4 °C 下以 800 × g 离心板 1 分钟，倾析弃去上清液，短暂涡旋板，每孔加入 200 μL 染色缓冲液。</li>
	<li>重复步骤 3.7 两次，并将板保持在冰上。</li>
	<li>每孔向板中加入 50 μL CXCR5 二抗。通过上下吹打充分混合，并在冰上孵育 30 分钟。</li>
	<li>为每个样品在 50 μL TFH 细胞染色缓冲液中以 1：200 稀释度制备荧光偶联的链霉亲和素（即 CXCR5 三抗）。同时，添加其他针对表面标志物的抗体（例如，CD45.1、CD4、PD-1、CD25 和其他感兴趣的分子）和死细胞染色剂以及 CXCR5 三抗。</li>
	<li>涡旋试管并在 4 °C 下以 15,000 × g 离心 3 分钟以聚集颗粒。将试管放在冰上。</li>
	<li>在 4 °C 下以 800 × g 离心板 1 分钟，倾析弃去上清液，短暂涡旋板，每孔加入 200 μL 染色缓冲液。</li>
	<li>重复步骤 3.12 两次，并将板保持在冰上。</li>
	<li>每孔向板中加入 50 μL CXCR5 三级抗体混合物。通过上下吹打充分混合，并在冰上避光孵育 30 分钟。</li>
	<li>根据制造商的说明为每个孔准备 200 μL 固定/透化缓冲液。</li>
	<li>在 4 °C 下以 800 × g 离心板 1 分钟，倾析弃去上清液，短暂涡旋板，每孔加入 200 μL 染色缓冲液。</li>
	<li>重复步骤 3.16 两次，并将板放在冰上避光。</li>
	<li>向每个孔中加入 200 μL 固定/透化缓冲液。通过上下吹打并在室温下孵育 30 分钟（在黑暗中）充分混合。 注意：根据制造商的说明准备透化缓冲液 （1x）。</li>
	<li>在新试管中每个孔的 50 μL 透化缓冲液 （1x） 中制备针对转录因子的抗体混合物，包括 Bcl-6、TCF-1、T-bet 和其他感兴趣的分子。</li>
	<li>涡旋试管并在 4 °C 下以 15,000 x g 离心 3 分钟以聚集颗粒。</li>
	<li>在 4 °C 下以 800 × g 离心板 1 分钟，倾析弃去上清液，短暂涡旋板，每孔加入 50 μL 转录因子抗体混合物。充分混合并在冰上避光孵育 30 分钟。</li>
	<li>在 4 °C 下以 800 × g 离心板 1 分钟，弃去上清液，通过倾析短暂涡旋板，并每孔加入 200 μL 透化缓冲液 （1x）。</li>
	<li>重复步骤 3.22。</li>
	<li>向每个孔中加入 200 μL 染色缓冲液，在 4 °C 下以 800 × g 离心板 1 分钟，倾析弃去上清液，并短暂涡旋板。</li>
	<li>用 200 μL 染色缓冲液重悬每个孔中的细胞沉淀，并将细胞转移到流式细胞仪管中，以便立即进行流式细胞仪分析。 注：或者，在向每个试管中加入 200 μl 2% PFA 并在 4 °C 避光条件下储存的条件下，可以在 3 天内检测样品。</li>
</ol>4. 逆转录病毒生产
<ol><li>在 10 cm 培养皿中用 DMEM 10% 培养基培养 293T 细胞（传代次数少于 10 次），传代时间为 2-3 代。</li>
	<li>当细胞达到 90% 汇合时，丢弃旧培养基，用 PBS 轻轻洗涤细胞两次，然后使用 1 mL 0.25% 胰蛋白酶-EDTA 在室温下对 293T 细胞进行短暂胰蛋白酶消化 1 分钟。</li>
	<li>通过将细胞重悬于 2 mL 预热的 10% DMEM 培养基中，将其转移到 15 mL 锥形管中，并在 4 °C 下以 200 × g 离心 5 分钟来终止消化过程。</li>
	<li>弃去上清液，用 2 mL 预热的 10% DMEM 培养基上下吹打培养基，重悬细胞沉淀。</li>
	<li>定量细胞数量，并在 6 孔板中每孔 2.5 mL 10% DMEM 培养基中接种 5 × 10个 5 个细胞。 注：轻轻旋转培养皿，使 293T 细胞均匀分布在整个板中。</li>
	<li>当 293T 细胞汇合度超过 80% 时，按照步骤 4.7 至 4.9 中的说明制备转染试剂。</li>
	<li>对于 6 孔板的每个孔，将 250 μL 预热的 Opti-MEM 培养基添加到无菌 1.5 mL 试管中。然后，向 Opti-MEM 培养基中加入 3 μg 质粒 DNA（含有 2 μg 逆转录病毒载体和 1 μg pCL-Eco），然后轻轻上下吹打以确保完全混合。 注：本研究中用作示例的逆转录病毒载体是 MigR1 和修饰的 MigR1 载体， Bcl6 CDS 区域位于 IRES-GFP （MigR1-Bcl6） 的上游。</li>
	<li>接下来，向混合物中加入 7.5 μL 预热的转染试剂（市售），轻轻上下移液以确保充分混合。</li>
	<li>将混合物在室温下孵育 20 分钟。</li>
	<li>将混合物逐滴分配到 293T 细胞培养孔的不同区域，然后以来回和左右运动轻轻摇动培养容器，以确保混合物均匀分布。</li>
	<li>将 293T 细胞在含有 5% CO2 的 37 °C 培养箱中孵育 72 小时。</li>
	<li>使用荧光显微镜或流式细胞术检测逆转录病毒载体的标签，作为转染 293T 细胞中转染功效的代理。 注：例如，我们在很大比例的 MigR1 载体转染的 293T 细胞中证实了 GFP 荧光信号，表明转染质量高。</li>
	<li>收集细胞培养基并在 4 °C 下以 2,500 × g 离心 10 分钟以去除细胞碎片。应仔细收集所得上清液并储存在 -80 °C 下长期保存或暂时保存在 4 °C 下。</li>
</ol>5. SM CD4+ T 细胞的体内活化、逆转录病毒转导和过继转移
<ol><li>在 100 μL RPMI 培养基中静脉注射一只 CD45.1 + SM 小鼠（6 至 8 周龄），其中含有 200 μg LCMV GP61-77 肽11,14。 注意：每位研究人员应修改进样量，且不得超过机构和当地法律允许的最大进样量。</li>
	<li>在肽注射后 16-18 小时对 SM 小鼠实施安乐死（遵循机构批准的方案）。按照步骤 1 中的上述程序获取脾细胞。 注：或者，体外抗 CD3/抗 CD28 刺激方法27,28 可用于激活 SM CD4 + T 细胞，以代替步骤 5.1 和 5.2 中描述的体内肽刺激。</li>
	<li>用 2% RPMI 悬浮脾细胞，并在 15 mL 锥形管中将细胞密度调节至大约 1 × 108 个细胞/mL。将试管放在冰上。</li>
	<li>将 50 μL 细胞悬液分配到圆底 96 孔板中，每孔补充 150 μL 染色缓冲液。</li>
	<li>将 96 孔板在 4 °C 下以 800 × g 离心 1 分钟以沉淀细胞，通过倾析小心去除上清液，短暂涡旋板，并每孔加入 200 μL 染色缓冲液。</li>
	<li>重复步骤 5.5 并用 50 μL 表面抗体混合物（包括 CD4、CD25 和 CD69）重悬细胞，以检测 SM CD4+ T 细胞的活化状态。通过上下吹打彻底混合，并在冰上避光孵育 30 分钟。</li>
	<li>用染色缓冲液加满每个孔至 200 μL，然后在 4 °C 下以 800 × g 离心板 1 分钟。 通过倾析弃去上清液，并短暂涡旋板。</li>
	<li>重复步骤 5.7 两次。</li>
	<li>将细胞重悬于 200 μL 染色缓冲液中，并将其转移到流式细胞仪管中。</li>
	<li>用流式细胞术分析荧光偶联抗体染色的细胞，以评估 SM CD4 + T 细胞的活化状态。 注：与初始细胞相比，活化的 SM CD4 + T 细胞表现出丰富的 CD25 和 CD69 表达，因此能够进行后续的转导程序。</li>
	<li>每孔加入 1 ×10 个 6 SM CD4 + T 细胞（来自步骤 5.3）到 24 孔板中。在 4 °C 下以 800 × g 离心细胞 3 分钟，然后通过倾析小心地去除培养基。</li>
	<li>如步骤 4.13 所述，向每个孔中加入 1 mL 逆转录病毒培养基和 8 μg 聚凝胺。</li>
	<li>在 37 °C 下以 800 × g 离心转导细胞 2 小时。</li>
	<li>通过移液小心丢弃逆转录病毒培养基，并向每个孔中加入 1 mL 预热的 10% RPMI，并补充有 10 ng/mL 重组鼠 IL-2。</li>
	<li>将细胞在含有 5% CO2 的 37 °C 培养箱中孵育 48 小时。</li>
	<li>将细胞悬液转移到 15 mL 锥形管中，并在 4 °C 下以 800 × g 的速度旋转细胞 6 分钟。</li>
	<li>通过倾析去除上清液，并用适当体积的 2% RPMI 重悬细胞，使细胞密度为 1 × 10 8个细胞/ mL，在 15 mL 锥形管中。将试管放在冰上。</li>
	<li>将 50 μL 细胞悬液分配到圆底 96 孔板中，每孔补充 150 μL 染色缓冲液。</li>
	<li>将 96 孔板在 4 °C 下以 800 × g 离心 1 分钟以沉淀细胞，通过倾析小心去除上清液，短暂涡旋板，并每孔加入 200 μL 染色缓冲液。</li>
	<li>重复步骤 5.19 并用 50 μL 表面抗体混合物（包括 CD4、Vα2 和载体标签相关抗体）重悬细胞。通过上下吹打彻底混合，并在冰上避光孵育 30 分钟。</li>
	<li>用染色缓冲液加满每个孔至 200 μL，然后以 800 × g 离心板 1 分钟。通过倾析弃去上清液，并短暂涡旋板。</li>
	<li>重复步骤 5.21 两次。</li>
	<li>将细胞重悬于 200 μL 染色缓冲液中，并将其转移到流式细胞仪管中。</li>
	<li>用流式细胞术分析荧光偶联抗体染色的细胞，以评估 SM CD4 + T 细胞的转导效率。 注意：在研究中，高效转导的 SM CD4 + T 细胞表现出逆转录病毒标签（如 GFP）的高表达。</li>
	<li>将步骤 5.17 中的细胞悬液在 4 °C 下以 800 × g 离心 5 分钟，然后倾析弃去上清液。用 RPMI 重悬细胞沉淀，并将细胞密度调节至 1 ×10 7 个 CD4+Vα2+ SM 细胞/mL。</li>
	<li>在步骤 5.25 中，通过静脉注射 100 μL 细胞悬液，将总共 1 ×10 个 6 个 CD45.1 + SM CD4 + T 细胞转移到每只 C57BL/6 受体小鼠（6 至 8 周龄）中。</li>
	<li>第二天，通过静脉注射用 1 ×10 6 个噬菌斑形成单位 LCMV Armstrong 感染 C57BL/6 受体小鼠。</li>
</ol>6. 逆转录病毒转导的 SM TFH 细胞在急性 LCMV 感染早期的流式细胞术分析
<ol><li>在感染后第 3 天从步骤 5.25 从 C57BL/6 小鼠获取脾细胞。</li>
	<li>如步骤 3 所述，对脾细胞中的表面标志物进行流式细胞术染色，包括 CD4、CD45.1、CXCR5、CD25 和其他感兴趣的分子。</li>
	<li>为了检测用荧光蛋白标记的逆转录病毒转导的 SM CD4 + T 细胞中的转录因子，向每个孔中加入 200 μL 2% PFA，并在室温下避光孵育 20 分钟。</li>
	<li>执行固定/透化，对感兴趣的转录因子进行流式细胞术染色，并按照步骤 3 中的说明进行流式细胞术分析。</li>
</ol>

破译急性 LCMV 感染中的早期滤泡辅助性 T 细胞定型术

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follicular+helper+t+cells.">Follicular helper t cells.</a> Annu Rev Immunol. 34, 335-368 (2016).</li><li>Choi, Y. S., 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=ICOS+receptor+instructs+T+follicular+helper+cell+versus+effector+cell+differentiation+via+induction+of+the+transcriptional+repressor+bcl6.">ICOS receptor instructs T follicular helper cell versus effector cell differentiation via induction of the transcriptional repressor bcl6.</a> Immunity. 34 (6), 932-946 (2011).</li><li>Choi, Y. S., 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=Bcl6+expressing+follicular+helper+CD4+T+cells+are+fate+committed+early+and+have+the+capacity+to+form+memory.">Bcl6 expressing follicular helper CD4 T cells are fate committed early and have the capacity to form memory.</a> J Immunol. 190 (8), 4014-4026 (2013).</li><li>Johnston, R. J., 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=Bcl6+and+BLIMP-1+are+reciprocal+and+antagonistic+regulators+of+T+follicular+helper+cell+differentiation.">Bcl6 and BLIMP-1 are reciprocal and antagonistic regulators of T follicular helper cell differentiation.</a> Science. 325 (5943), 1006-1010 (2009).</li><li>Nurieva, R. I., 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=Bcl6+mediates+the+development+of+T+follicular+helper+cells.">Bcl6 mediates the development of T follicular helper cells.</a> Science. 325 (5943), 1001-1005 (2009).</li><li>Yu, D., 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+transcriptional+repressor+bcl-6+directs+T+follicular+helper+cell+lineage+commitment.">The transcriptional repressor bcl-6 directs T follicular helper cell lineage commitment.</a> Immunity. 31 (3), 457-468 (2009).</li><li>Choi, Y. S., 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=LEF1+and+TCF1+orchestrate+T(FH)+differentiation+by+regulating+differentiation+circuits+upstream+of+the+transcriptional+repressor+bcl6.">LEF1 and TCF1 orchestrate T(FH) differentiation by regulating differentiation circuits upstream of the transcriptional repressor bcl6.</a> Nat Immunol. 16 (9), 980-990 (2015).</li><li>Wu, 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=TCF1+is+required+for+the+t+follicular+helper+cell+response+to+viral+infection.">TCF1 is required for the t follicular helper cell response to viral infection.</a> Cell Rep. 12 (12), 2099-2110 (2015).</li><li>Xu, L., 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+transcription+factor+TCF-1+initiates+the+differentiation+of+T(FH)+cells+during+acute+viral+infection.">The transcription factor TCF-1 initiates the differentiation of T(FH) cells during acute viral infection.</a> Nat Immunol. 16 (9), 991-999 (2015).</li><li>Oestreich, K. 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J., Choi, Y. S., Diamond, J., Yang, J. A., Crotty, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STAT5+is+a+potent+negative+regulator+of+TFH+cell+differentiation.">STAT5 is a potent negative regulator of TFH cell differentiation.</a> J Exp Med. 209 (2), 243-250 (2012).</li><li>Lee, J. Y., 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+transcription+factor+KLF2+restrains+CD4++T+follicular+helper+cell+differentiation.">The transcription factor KLF2 restrains CD4+ T follicular helper cell differentiation.</a> Immunity. 42 (2), 252-264 (2015).</li><li>Xiao, 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=The+e3+ubiquitin+ligase+itch+is+required+for+the+differentiation+of+follicular+helper+t+cells.">The e3 ubiquitin ligase itch is required for the differentiation of follicular helper t cells.</a> Nat Immunol. 15 (7), 657-666 (2014).</li><li>Baumjohann, D., 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+microRNA+cluster+mir-17~92+promotes+TFH+cell+differentiation+and+represses+subset-inappropriate+gene+expression.">The microRNA cluster mir-17~92 promotes TFH cell differentiation and represses subset-inappropriate gene expression.</a> Nat Immunol. 14 (8), 840-848 (2013).</li><li>Wang, Y., 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+kinase+complex+mTORC2+promotes+the+longevity+of+virus-specific+memory+CD4(+)+T+cells+by+preventing+ferroptosis.">The kinase complex mTORC2 promotes the longevity of virus-specific memory CD4(+) T cells by preventing ferroptosis.</a> Nat Immunol. 23 (2), 303-317 (2022).</li><li>Hale, J. 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作者声明没有利益冲突。

自从发现 TFH 细胞在帮助 B 细胞方面的特殊功能以来，TFH 细胞领域的研究一直备受关注。累积的研究表明，TFH 细胞分化是一个多阶段和多因素的过程30，其中 TFH 细胞命运承诺在早期确定5。因此，更好地了解早期分化 TFH 细胞的潜在机制对于 TFH 细胞生物学和合理的疫苗设计至关重要。在此，我们提供了通过利用...

遇到不同的病原体或威胁，初始 CD4+ T 细胞通过分化为具有特殊功能的各种辅助性 T （TH） 细胞亚群来定制其免疫反应1。在急性病毒感染的情况下，很大一部分幼稚 CD4+ T 细胞分化为滤泡辅助性 T （TFH） 细胞，为 B 细胞提供帮助 2,3。与其他 CD4+ TH 细胞亚群（例如，TH1、TH2、TH9 和 TH17 细胞）不同，TFH 细胞表达大量 CXCR5，这是 B 细胞归巢趋化因子 CXCL13 的趋化因子受体，使 TFH 细胞能够迁移到 B 细胞滤泡中。在 B 细胞滤泡中，TFH 细胞协助同源 B 细胞启动和维持生发中心反应，从而能够快速产生高亲和力抗体和长期体液记忆 2,3。
急性病毒感染后，病毒特异性 TFH 细胞的早期命运承诺发生在 72 小时内 4,5，并受转录抑制因子 B 细胞淋巴瘤-6 （Bcl-6）5,6,7,8 的控制，该细胞是控制 TFH 细胞命运决定的“主调节因子”。Bcl-6 的缺乏会严重减弱 TFH 细胞的分化，而异位 Bcl-6 的表达会显著促进 TFH 细胞的命运决定。除 Bcl-6 外，多个分子还参与指导早期 TFH 细胞命运定型。转录因子 TCF-1 和 LEF-1 通过诱导 Bcl-6 启动 TFH 细胞分化 9,10,11。Bcl-6 和 TCF-1 对 Blimp1 的抑制是早期 TFH 细胞命运承诺所必需的11,12。STAT1 和 STAT3 也是早期 TFH 细胞分化所必需的13。此外，组蛋白甲基转移酶 EZH214,15 和 m6A 甲基转移酶 METTL316 的表观遗传修饰有助于稳定 TFH 细胞转录程序（尤其是 Bcl6 和 Tcf7），从而引发早期 TFH 细胞命运承诺。虽然在理解早期 TFH 细胞命运承诺的转录和表观遗传调控方面取得了进展，包括上述分子和其他分子，在别处3 中总结，但以前未知的分子仍有待了解。
在急性淋巴细胞性脉络丛脑膜炎病毒 （LCMV） 感染的小鼠模型中，过继转移的同基因 TCR 转基因 SMARTA （SM） CD4+ T 细胞，特异性识别 LCMV 糖蛋白表位 I-AbGP66-77，在病毒感染期间经历 TFH 或 TH1 细胞分化。这种 TFH/TH1 分叉分化模式支持 SM/急性 LCMV 感染模型在研究病毒特异性 TFH 细胞生物学方面的进步。事实上，SM/急性 LCMV 感染模型已广泛应用于 TFH 细胞研究领域，并在 TFH 细胞生物学的里程碑性发现中发挥了至关重要的作用。这包括将上述 Bcl-6 鉴定为 TFH 细胞的谱系定义转录因子 5,6，以及其他重要的转录因子（例如，Blimp-16、TCF-1/LEF9、10、11、STAT1/STAT313、STAT517、KLF218 和 Itch19）指导 TFH 细胞分化、转录后调节 （例如，TFH 细胞分化的 METTL316 和 miR-17~9220），TFH 细胞记忆和可塑性 21,22，以及针对 T FH 细胞的合理疫苗接种策略（例如，硒23）。
目前的研究描述了获取病毒特异性 TFH 细胞早期命运承诺的可重复方法，包括 （1） 建立适用于访问早期分化 TFH 细胞的急性 LCMV 感染的 SM 嵌合体小鼠模型，（2） 对与早期分化 TFH 细胞相关的分子进行流式细胞术染色，以及 （3） 在 SM CD4+ 中进行基于逆转录病毒载体的基因操作T 细胞。这些方法将有助于调查病毒特异性 TFH 细胞的早期命运承诺的研究。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Corning</td>
			<td>25-052-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde Fix Solution, 4% PFA</td>
			<td>Beyotime</td>
			<td>P0099-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m cell strainer</td>
			<td>Merck</td>
			<td>CLS431751</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 anti-mouse TCR V&#945;2 (clone B20.1)</td>
			<td>Biolegend</td>
			<td>127812</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor 700 anti-mouse CD45.1 (clone A20)</td>
			<td>Biolegend</td>
			<td>110724</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>APC anti-mouse CD25 (clone PC61)</td>
			<td>Biolegend</td>
			<td>101910</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>B6 CD45.1 (B6.SJL-Ptprca Pepcb/BoyJ) mouse</td>
			<td>The Jackson Laboratory</td>
			<td>002014</td>
			<td></td>
		</tr>
		<tr>
			<td>BeaverBeads Streptavidin</td>
			<td>Beaver</td>
			<td>22321-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin anti-mouse F4/80 Antibody (clone BM8)</td>
			<td>Biolegend</td>
			<td>123106</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Biotin Rat anti-mouse CD11c (clone N418)</td>
			<td>Biolegend</td>
			<td>117304</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Biotin Rat anti-Mouse CD19 (clone 6D5)</td>
			<td>Biolegend</td>
			<td>115504</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Biotin Rat anti-Mouse CD8a (clone 53-6.7)</td>
			<td>Biolegend</td>
			<td>100704</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Biotin Rat anti-mouse NK-1.1 (clone PK136)</td>
			<td>Biolegend</td>
			<td>108704</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Biotin Rat anti-mouse TER-119/Erythroid Cells (clone TER-119)</td>
			<td>Biolegend</td>
			<td>116204</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>bovine serum albumin, BSA</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 421 anti-T-bet (clone 4B10)</td>
			<td>Biolegend</td>
			<td>644816</td>
			<td>1:100 dilution</td>
		</tr>
		<tr>
			<td>Brilliant Violet 605 anti-mouse CD279 (PD-1) (clone 29F.1A12)</td>
			<td>Biolegend</td>
			<td>135220</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>C57BL/6J (B6) mouse</td>
			<td>The Jackson Laboratory</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>CXCR5-GFP knock-in reporter mouse</td>
			<td></td>
			<td></td>
			<td>In house; the CXCR5-GFP knock-in mouse line was generated by the insertion of an IRES-GFP construct after the open reading frame of Cxcr5.</td>
		</tr>
		<tr>
			<td>DMEM 10% medium</td>
			<td></td>
			<td></td>
			<td>DMEM medium containing 10% FBS</td>
		</tr>
		<tr>
			<td>DMEM medium</td>
			<td>Gibco</td>
			<td>11885092</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSFortesa</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum, FBS</td>
			<td>Sigma</td>
			<td>F8318</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo (version 10.4.0)</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Foxp3/Transcription Factor Staining Buffer Set</td>
			<td>Invitrogen</td>
			<td>00-5523-00</td>
			<td>The kit contains three reagents: a. Fixation/Permeabilization Concentrate (4X); b. Fixation / Permeabilization Diluent; c. Permeabilization Buffer.</td>
		</tr>
		<tr>
			<td>Goat Anti-Rat IgG Antibody (H+L), Biotinylated</td>
			<td>Vector laboratories</td>
			<td>BA-9400-1.5</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Invitrogen EVOS FL Auto Cell Imaging System</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isolation buffer</td>
			<td></td>
			<td></td>
			<td>FACS buffer containing 0.5% BSA and 2mM EDTA</td>
		</tr>
		<tr>
			<td>LCMV GP61-77 peptide (GLKGPDIYKGVYQFKSV)</td>
			<td>Chinese Peptide Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit, for 633 or 635 nm excitation</td>
			<td>Life Technologies</td>
			<td>L10199</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>MigR1</td>
			<td>addgene</td>
			<td>#27490</td>
			<td></td>
		</tr>
		<tr>
			<td>NaN3</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM medium</td>
			<td>Gibco</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>pCL-Eco</td>
			<td>addgene</td>
			<td>#12371</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-mouse CD69 (clone H1.2F3)</td>
			<td>Biolegend</td>
			<td>104508</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>PE Mouse anti-Bcl-6 (clone K112-91)</td>
			<td>BD Biosciences</td>
			<td>561522</td>
			<td>1:50 dilution</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline, PBS</td>
			<td>Gibco</td>
			<td>10010072</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Solarbio</td>
			<td>H8761</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified Rat Anti-Mouse CXCR5 (clone 2G8)</td>
			<td>BD Biosciences</td>
			<td>551961</td>
			<td>1:50 dilution</td>
		</tr>
		<tr>
			<td>Rat monoclonal PerCP anti-mouse CD4 (clone RM4-5)</td>
			<td>Biolegend</td>
			<td>100538</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>recombinant murine IL-2</td>
			<td>Gibco</td>
			<td>212-12-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysis Buffer</td>
			<td>Beyotime</td>
			<td>C3702-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Sigma</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 2%</td>
			<td></td>
			<td></td>
			<td>RPMI 1640 medium containing 2% FBS</td>
		</tr>
		<tr>
			<td>SMARTA (SM) TCR transgenic mouse</td>
			<td></td>
			<td></td>
			<td>SM TCR transgenic line in our lab is a gift from Dr. Rafi Ahmed (Emory University). Additionally, this mouse line can also be obtained from The Jackson Laboratory (stain#: 030450).</td>
		</tr>
		<tr>
			<td>Staining buffer</td>
			<td></td>
			<td></td>
			<td>PBS containing 2% FBS and 0.01% NaN3</td>
		</tr>
		<tr>
			<td>Streptavidin PE-Cyanine7</td>
			<td>eBioscience</td>
			<td>25-4317-82</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>TCF1/TCF7 (C63D9) Rabbit mAb (Alexa Fluor 488 Conjugate)&#160;</td>
			<td>Cell signaling technology</td>
			<td>6444S</td>
			<td>1:400 dilution</td>
		</tr>
		<tr>
			<td>TFH cell staining buffer</td>
			<td></td>
			<td></td>
			<td>FACS buffer containing 1% BSA and 2% mouse serum</td>
		</tr>
		<tr>
			<td>TransIT-293 reagent</td>
			<td>Mirus Bio</td>
			<td>MIRUMIR2700</td>
			<td></td>
		</tr>
	</tbody>
</table>

accessing early differentiation of virus-specific follicular helper cd4+ t cell in acute lcmv-infected mice

滤泡辅助性 T （TFH） 细胞被认为是一种独立的 CD4+ T 细胞谱系，可帮助同源 B 细胞产生高亲和力抗体，从而建立长期体液免疫。在急性病毒感染期间，病毒特异性 TFH 细胞的命运承诺在感染早期就确定了，早期分化的 TFH 细胞的研究对于了解 T 细胞依赖性体液免疫和优化疫苗设计至关重要。在这项研究中，使用急性淋巴细胞性脉络丛脑膜炎病毒 （LCMV） 感染的小鼠模型和具有特异性识别 LCMV 糖蛋白表位 I-AbGP66-77 的 CD4+ T 细胞的 TCR 转基因 SMARTA （SM） 小鼠，我们描述了基于流式细胞术染色的病毒特异性 TFH 细胞的早期命运承诺的程序。此外，通过利用 SM CD4 + T 细胞的逆转录病毒转导，还提供了操纵早期分化病毒特异性 TFH 细胞中基因表达的方法。因此，这些方法将有助于探索病毒特异性 TFH 细胞早期定型机制的研究。

Encountering different pathogens or threats, na&#239;ve CD4+ T cells tailor their immune responses by differentiating into various helper T (TH) cell subsets with specialized functions1. In the scenario of acute viral infection, a large portion of na&#239;ve CD4+ T cells differentiate into follicular helper T (TFH) cells that provide help to B cells2,3. Distinct from other CD4+ TH cell subsets (e.g., TH1, TH2, TH9, and TH17 cells), TFH cells express a substantial level of CXCR5, which is the chemokine receptor for the B cell homing chemokine CXCL13, enabling TFH cells to migrate into B cell follicles. In the B cell follicles, TFH cells assist cognate B cells in initiating and maintaining germinal center reactions, thus enabling rapid high-affinity antibody production and long-term humoral memory2,3.
Upon acute viral infection, the early fate commitment of virus-specific TFH cells occurs within 72 h4,5and is controlled by the transcriptional repressor B cell lymphoma-6 (Bcl-6)5,6,7,8, which acts as the &#34;master regulator&#34; governing TFH cell fate decisions. Deficiency of Bcl-6 severely blunts TFH cell differentiation, while ectopic Bcl-6 expression substantially promotes TFH cell fate commitment. In addition to Bcl-6, multiple molecules are involved in instructing early TFH cell fate commitment. Transcription factors TCF-1 and LEF-1 initiate TFH cell differentiation via the induction of Bcl-69,10,11. The inhibition of Blimp1, by both Bcl-6 and TCF-1, is required for early TFH cell fate commitment11,12. STAT1 and STAT3 are also required for early TFH cell differentiation13. Besides, epigenetic modifications by histone methyltransferase EZH214,15and m6A methyltransferase METTL316 help to stabilize TFH cell transcriptional programs (especially Bcl6 and Tcf7) and thus prime early TFH cell fate commitment. While advances, including the aforementioned molecules and others, summarized elsewhere3, have been made in understanding the transcriptional and epigenetic regulations of early TFH&#160;cell fate commitment, previously unknown molecules remain to be learned.
In the mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection, adoptively transferred congenic TCR-transgenic SMARTA (SM) CD4+ T cells, which specifically recognize the LCMV glycoprotein epitope I-AbGP66-77, undergo either TFH or TH1 cell differentiation during viral infection. This TFH/TH1 bifurcation differentiation pattern supports the advancement of the SM/acute LCMV infection model in studying the biology of virus-specific TFH cells. Indeed, the SM/acute LCMV infection model has been widely used in the TFH cell research field and has played a crucial role in milestone discoveries in TFH cell biology. This includes the identification of the aforementioned Bcl-6 as the lineage-defining transcription factor of TFH cells5,6, as well as other important transcriptional factors (e.g., Blimp-16, TCF-1/LEF9,10,11, STAT1/STAT313, STAT517, KLF218, and Itch19) guiding TFH cell differentiation, post-transcriptional regulations (e.g., METTL316 and miR-17~9220) of TFH cell differentiation, TFH cell memory and plasticity21,22, and rational vaccination strategies targeting TFH cells (e.g., selenium23).
The current study describes reproducible methods for accessing the early fate commitment of virus-specific TFH cells, including (1) establishing an acute LCMV-infected SM chimera mouse model suitable for accessing early-differentiated TFH cells, (2) conducting flow cytometry stainings of molecules related to early-differentiated TFH cells, and (3) performing retroviral vector-based gene manipulation in SM CD4+ T cells. These methods will be useful for studies investigating the early fate commitment of virus-specific TFH cells.

作者聚焦：阐明急性 LCMV 挑战中 TFH 细胞分化的途径

在急性 LCMV 感染小鼠中获得病毒特异性滤泡辅助性 CD4 + T 细胞的早期分化

Our research describes reproducible methods for assessing the early fate commitment of virus-specific TFH cells, including establishing acute LCMV-infected monoclonal mouse model, conducting flow cytometry staining, and performing retroviral-vector-based gene manipulation. Compared to in-vitro T-cell stimulation, in-vivo stimulation could activate T cells more quickly, even in 12 hours, and it's fully activated for the next retrovirus transduction. Our findings will help in studies exploring the mechanisms underlying the early commitment of various specific TFH cells.Furthermore, our research will contribute to understanding T-cell-dependent humoral immunity and optimizing vaccine design.

急性 LCMV 感染期间早期分化病毒特异性 TFH 细胞的特征 为了探测病毒特异性 TFH 细胞的早期命运承诺，将特异性识别 LCMV GP 表位 I-AbGP66-77 的幼稚同源 SM CD4 + T 细胞过继转移到 CD45.2 + C57BL/6 受体中。第二天，这些接受者被静脉注射高剂量的急性缓解的 LCMV Armstrong 感染（图 1A）。感染后第 3 天，通过流式细胞术分?...

Read this Scientific Article Protocol about Accessing Early Differentiation of Virus-Specific Follicular Helper CD4+ T Cell in Acute LCMV-Infected Mice at JoVE.com

目前的研究展示了评估病毒特异性 TFH 细胞的早期命运承诺和操纵这些细胞中基因表达的方案。

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, ...

accessing-early-differentiation-virus-specific-follicular-helper-cd4

Research

JoVE Journal

Immunology and Infection

Accessing Early Differentiation of Virus-Specific Follicular Helper CD4+ T Cell in Acute LCMV-Infected Mice

436 次观看.  Shenzhen University. 我们的研究描述了评估病毒特异性 TFH 细胞早期命运承诺的可重复方法，包括建立急性 LCMV 感染的单克隆小鼠模型、进行流式细胞术染色和进行基于逆转录病毒载体的基因操作。与体外 T 细胞刺激相比，体内刺激可以更快地激活 T 细胞，甚至可以在 12 小时内激活，并且它被完全激活以进行下一次逆转录病毒转导。我们的研究结果将有助于研究探索各?...

视频: 作者聚焦：阐明急性 LCMV 挑战中 TFH 细胞分化的途径

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, thus establishing long-term humoral immunity. During acute viral infection, the fate commitment of virus-specific TFH cells is determined in the early infection phase, and investigations of the early-differentiated TFH cells are crucial in understanding T cell-dependent humoral immunity and optimizing vaccine design. In the study, using a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection and the TCR-transgenic SMARTA (SM) mouse with CD4+ T cells specifically recognizing LCMV glycoprotein epitope I-AbGP66-77, we described procedures to access the early fate commitment of virus-specific TFH cells based on flow cytometry stainings. Furthermore, by exploiting retroviral transduction of SM CD4+ T cells, methods to manipulate gene expression in early-differentiated virus-specific TFH cells are also provided. Hence, these methods will help in studies exploring the mechanism(s) underlying the early commitment of virus-specific TFH cells.

The current study showcases protocols for assessing the early fate commitment of virus-specific TFH cells and manipulating gene expression in these cells.

科研

免疫与感染

Mapping the Early Development of LCMV-Specific Follicular Helper T-Cells in Mice

To begin, obtain a six to eight week old CD 45.1 positive SMARTA mouse and inject it with 200 micrograms of LCMV GP 61-77 peptide in 100 microliters of RPMI medium intravenously. After acquiring the splenocytes from the mouse, suspend them in 2%RPMI to achieve a cell density of one times 10 to the eighT-cells per milliliter and place the tube with cells on ice. Dispense 50 microliters of cell suspension along with 150 microliters of staining buffer to each well of a round-bottom 96-well plate.Centrifuge the 96-well plate at 800 G for one minute at four degrees Celsius and carefully decant the supernatant. Vortex the plate and add 200 microliters of staining buffer to each well. After pelleting the cells, resuspend and incubate them with surface antibody cocktail on ice for 30 minutes in the dark.After washing two times, resuspend the cells in 200 microliters of staining buffer and transfer them into a flow cytometry tube. Perform flow cytometry analysis to check the activation status of SMARTA CD4 positive T-cells. Then seed one times 10 the six SMARTA CD4 positive T-cells per well into a 24 well plate.Spin down the cells at 800 G for three minutes at four degrees Celsius and carefully remove the supernatant medium. Next, add one milliliter of retrovirus medium and eight micrograms of polybrene to each well. Spin transduce the cells at 800 G for two hours at 37 degrees Celsius.After discarding the retrovirus medium, incubate the cells with one milliliter of prewarmed 10%RPMI supplemented with interleukin 2. Transfer the cell suspension into a 15 milliliter conical tube and centrifuge it. After discarding the supernatant, resuspend the cells with 2%RPMI to achieve a cell density of one times 10 to the eighth cells per milliliter.For assessing the transduction efficiency, aliquot 50 microliters of cell suspension into a round bottom 96-well plate. Incubate them on ice with the surface antibody cocktail including CD4, V alpha two, and vector tag-associated antibody. Wash the cells and perform flow cytometry as demonstrated earlier.Then inject one times 10 of the six CD 45.1 positive SMARTA CD4 positive T-cells to a C57BL/6 recipient mouse intravenously followed by one times 10 to the six plaque forming units of LCMV Armstrong the next day. After acquiring the splenocytes from the mouse, stain them to check the desired markers. To detect transcriptional factors, incubate the cells with 200 microliters of 2%paraformaldehyde at room temperature for 20 minutes in the dark.Finally, perform flow cytometry to detect factors at the early acute LCMV infection stage. On day three after infection, a balanced bifurcation of follicular helper T-cells and T-helper 1 cells Among MIGR1 SMARTA CD4 positive T-cells was found. A predominant follicular helper T-cell directed differentiation and enhanced BCL6 expression levels were observed in MIGR1 BCL6 SMARTA CD4 positive T-cells.