تم دعم هذه الدراسة بمنح من المؤسسة الوطنية للعلوم الطبيعية في الصين (رقم 32300785 إلى X.C.) ، وبرنامج ما بعد الدكتوراه الوطني الصيني للمواهب المبتكرة (No. BX20230449 إلى X.C.) ، والمشروع الوطني الرئيسي للعلوم والتكنولوجيا (رقم 2021YFC2300602 إلى L.Y.).

فك رموز التزام الخلايا التائية المساعدة المسامية المبكرة في عدوى LCMV الحادة

<ol>
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تم تسليط الضوء على البحث في مجال الخلاياالتائية FH منذ اكتشاف الوظيفة المتخصصة للخلاياالتائية FH في مساعدة الخلايا البائية. أشارت الدراسات المتراكمة إلى أن تمايز الخلايا التائيةFH هو عملية متعددة المراحل ومتعددة العوامل30 ، حيث يتم تحديد التزام مصير الخلايا ا...

في مواجهة مسببات الأمراض أو التهديدات المختلفة ، تصمم الخلايا التائية CD4 + T الساذجة استجاباتها المناعية من خلال التمايز إلى مجموعات فرعية مختلفة من الخلايا التائية المساعدة (TH) ذات وظائف متخصصة1. في سيناريو العدوى الفيروسية الحادة ، يتمايز جزء كبير من الخلايا التائية CD4 + الساذجة إلى خلايا T (T FH) المساعدة المسامية التي توفر المساعدة للخلايا البائية 2,3. تختلف الخلايا TFH عن المجموعات الفرعية الأخرى لخلايا CD4 + T H (على سبيل المثال ، خلايا TH1 و TH2 و TH9 و TH17) ، وتعبر عن مستوى كبير من CXCR5 ، وهو مستقبل الكيموكين للكيموكين CXCL13 للخلايا البائية الموجهة ، مما يمكن الخلايا التائيةFH من الهجرة إلى بصيلات الخلايا البائية. في بصيلات الخلايا البائية ، تساعد الخلايا التائيةFH الخلايا البائية المشابهة في بدء تفاعلات المركز الجرثومي والحفاظ عليها ، مما يتيح الإنتاج السريع للأجسام المضادة عالية التقارب والذاكرة الخلطية طويلة المدى 2,3.
عند الإصابة الفيروسية الحادة ، يحدث الالتزام المبكر بالمصير لخلاياT FH الخاصة بالفيروس في غضون 72 ساعة 4,5 ويتم التحكم فيه بواسطة سرطان الغدد الليمفاوية للخلايا البائية المثبطة للنسخ -6 (Bcl-6) 5،6،7،8 ، والتي تعمل بمثابة "المنظم الرئيسي" الذي يحكم قرارات مصير الخلاياالتائية FH. يؤدي نقص Bcl-6 إلى إضعاف تمايز الخلايا التائيةFH بشدة ، بينما يعزز تعبير Bcl-6 خارج الرحم بشكل كبير الالتزام بمصير الخلايا التائيةFH. بالإضافة إلى Bcl-6 ، تشارك جزيئات متعددة في توجيه الالتزام المبكر بمصير الخلاياالتائية FH. تبدأ عوامل النسخ TCF-1 و LEF-1 تمايز الخلايا التائيةFH عن طريق تحريض Bcl-69،10،11. إن تثبيط Blimp1 ، بواسطة كل من Bcl-6 و TCF-1 ، مطلوب للالتزام المبكر بمصير الخلاياالتائية FH 11,12. STAT1 و STAT3 مطلوبان أيضا لتمايز الخلايا التائيةFH المبكرة 13. إلى جانب ذلك ، تساعد التعديلات اللاجينية بواسطة هيستون ميثيل ترانسفيراز EZH214،15و m6A methyltransferase METTL316 على استقرار برامج نسخ الخلايا التائيةFH (خاصة Bcl6 و Tcf7) وبالتالي الالتزام المبكر بمصير الخلاياالتائية FH. في حين تم إحراز تقدم ، بما في ذلك الجزيئات المذكورة أعلاه وغيرها ، والتي تم تلخيصها في مكان آخر3 ، في فهم اللوائح النسخية واللاجينية للالتزام المبكر بمصير الخلاياالتائية FH ، لا يزال يتعين تعلم الجزيئات غير المعروفة سابقا.
في نموذج الفأر لعدوى فيروس التهاب المشيمية اللمفاوي الحاد (LCMV) ، تنتقل بالتبني خلايا SMARTA (SM) CD4 + T المتجانسة TCR المعدلة وراثيا ، والتي تتعرف على وجه التحديد على خاتمة البروتين السكري LCMV I-AbGP66-77 ، إما تمايز الخلايا TFH أو TH1 أثناء العدوى الفيروسية. يدعم نمط تمايز التشعب TFH / TH1 تقدم نموذج عدوى SM / LCMV الحاد في دراسة بيولوجيا خلايا TFH الخاصة بالفيروس. في الواقع ، تم استخدام نموذج عدوى SM / LCMV الحاد على نطاق واسع في مجال أبحاث الخلايا التائيةFH ولعب دورا حاسما في الاكتشافات البارزة في بيولوجيا الخلايا TFH. يتضمن ذلك تحديد Bcl-6 المذكور أعلاه كعامل نسخ محدد للنسب لخلايا TFH 5,6 ، بالإضافة إلى عوامل النسخ المهمة الأخرى (على سبيل المثال ، Blimp-16 و TCF-1 / LEF9،10،11 و STAT1 / STAT313 و STAT517 و KLF218 و Itch19) توجيه تمايز الخلايا TFH ، لوائح ما بعد النسخ ( على سبيل المثال ، METTL316 و miR-17 ~ 9220) من تمايز الخلايا التائيةFH ، ذاكرة الخلايا التائيةFH واللدونة21،22 ، واستراتيجيات التطعيم العقلانية التي تستهدف خلاياT FH (على سبيل المثال ، السيلينيوم23).
تصف الدراسة الحالية طرقا قابلة للتكرار للوصول إلى التزام المصير المبكر لخلايا TFH الخاصة بالفيروس ، بما في ذلك (1) إنشاء نموذج فأر SM chimera مصاب ب LCMV بشكل حاد مناسب للوصول إلى خلايا TFH المتمايزة مبكرا ، (2) إجراء تلطيخ قياس التدفق الخلوي للجزيئات المتعلقة بخلايا TFH المتمايزة مبكرا ، و (3) إجراء معالجة الجينات القائمة على ناقلات الفيروسات القهقرية في SM CD4 + الخلايا التائية. ستكون هذه الطرق مفيدة للدراسات التي تبحث في الالتزام المبكر بالمصير لخلاياT FH الخاصة بالفيروس.

<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

ينظر إلى الخلايا التائية المساعدة المسامية (TFH) على أنها سلالة خلايا CD4 + T مستقلة تساعد الخلايا البائية المشابهة في إنتاج أجسام مضادة عالية التقارب ، وبالتالي إنشاء مناعة خلطية طويلة المدى. أثناء العدوى الفيروسية الحادة ، يتم تحديد التزام مصير الخلاياالتائية FH الخاصة بالفيروس في مرحلة العدوى المبكرة ، والتحقيقاتفي الخلايا التائية FH المتمايزة المبكرة أمر بالغ الأهمية في فهم المناعة الخلطية المعتمدة على الخلايا التائية وتحسين تصميم اللقاح. في الدراسة ، باستخدام نموذج فأر لعدوى فيروس التهاب المشيمة اللمفاوي الحاد (LCMV) وفأر SMARTA (SM) المعدل وراثيا TCR مع خلايا CD4 + T التي تتعرف على وجه التحديد على خاتمة البروتين السكري LCMV I-AbGP66-77 ، وصفنا إجراءات للوصول إلى التزام المصير المبكر لخلاياT FH الخاصة بالفيروس بناء على تلطيخ قياس التدفق الخلوي. علاوة على ذلك ، من خلال استغلال نقل الفيروسات القهقرية لخلايا SM CD4 + T ، يتم أيضا توفير طرق لمعالجة التعبير الجينيفي خلايا T FH الخاصة بالفيروس المتمايزة مبكرا. ومن ثم ، ستساعد هذه الطرق في الدراسات التي تستكشف الآلية (الآليات) الكامنة وراء الالتزام المبكر لخلاياT FH الخاصة بالفيروس.

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.

تسليط الضوء على المؤلف: توضيح مسارات تمايز خلايا TFH في تحديات LCMV الحادة

الوصول إلى التمايز المبكر للخلية التائية المساعدة المسامية CD4 + الخاصة بالفيروس في الفئران المصابة بفيروس LCMV الحاد

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.

خصائص خلايا TFH الخاصة بالفيروس المتمايزة مبكرا أثناء عدوى LCMV الحادة للتحقق من التزام المصير المبكر لخلايا TFH الخاصة بالفيروس ، تم نقل خلايا SM CD4 + T الساذجة التي تتعرف على وجه التحديد على خاتمة LCMV GP I-AbGP66-77 بالتبني إلى مستلمي CD45.2 + C57BL / 6. في اليوم ا...

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

تعرض الدراسة الحالية بروتوكولات لتقييم الالتزام المبكر بالمصير لخلايا TFH الخاصة بالفيروس والتلاعب بالتعبير الجيني في هذه الخلايا.

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

438 Views.  Shenzhen University. يصف بحثنا طرقا قابلة للتكرار لتقييم التزام المصير المبكر لخلايا TFH الخاصة بالفيروس ، بما في ذلك إنشاء نموذج فأر أحادي النسيلة مصاب ب LCMV الحاد ، وإجراء تلطيخ قياس التدفق الخلوي ، وإجراء معالجة الجينات القائمة على ناقلات الفيروسات القهقرية. بالمقارنة مع تح...

Video: تسليط الضوء على المؤلف: توضيح مسارات تمايز خلايا TFH في تحديات LCMV الحادة

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.

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.