Bu çalışma, Çin Ulusal Doğa Bilimleri Vakfı (No. 32300785 - X.C.), Çin Ulusal Yenilikçi Yetenekler Doktora Sonrası Programı (No. BX20230449'den X.C.'ye) ve Ulusal Bilim ve Teknoloji Büyük Projesi (No. 2021YFC2300602'den L.Y.'ye).

Akut LCMV Enfeksiyonunda Erken Foliküler Yardımcı T-Hücre Taahhüdünün Deşifre Edilmesi

<ol>
	<li>O'shea, J. J., Paul, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+underlying+lineage+commitment+and+plasticity+of+helper+CD4++T+cells.">Mechanisms underlying lineage commitment and plasticity of helper CD4+ T cells.</a> Science. 327 (5969), 1098-1102 (2010).</li><li>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=T+follicular+helper+cell+biology:+A+decade+of+discovery+and+diseases.">T follicular helper cell biology: A decade of discovery and diseases.</a> Immunity. 50 (5), 1132-1148 (2019).</li><li>Vinuesa, C. G., Linterman, M. A., Yu, D., Maclennan, I. 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. J., Mohn, S. E., Weinmann, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+that+control+the+expression+and+activity+of+bcl-6+in+th1+cells+to+regulate+flexibility+with+a+TFH-like+gene+profile.">Molecular mechanisms that control the expression and activity of bcl-6 in th1 cells to regulate flexibility with a TFH-like gene profile.</a> Nat Immunol. 13 (4), 405-411 (2012).</li><li>Choi, Y. S., Eto, D., Yang, J. A., Lao, C., 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=Cutting+edge:+STAT1+is+required+for+IL-6-mediated+bcl6+induction+for+early+follicular+helper+cell+differentiation.">Cutting edge: STAT1 is required for IL-6-mediated bcl6 induction for early follicular helper cell differentiation.</a> J Immunol. 190 (7), 3049-3053 (2013).</li><li>Chen, X., 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+histone+methyltransferase+ezh2+primes+the+early+differentiation+of+follicular+helper+T+cells+during+acute+viral+infection.">The histone methyltransferase ezh2 primes the early differentiation of follicular helper T cells during acute viral infection.</a> Cell Mol Immunol. 17 (3), 247-260 (2020).</li><li>Li, F., 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=Ezh2+programs+T(FH)+differentiation+by+integrating+phosphorylation-dependent+activation+of+bcl6+and+polycomb-dependent+repression+of+p19ARF.">Ezh2 programs T(FH) differentiation by integrating phosphorylation-dependent activation of bcl6 and polycomb-dependent repression of p19ARF.</a> Nat Commun. 9 (1), 5452 (2018).</li><li>Yao, 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=METTL3-dependent+m(6)A+modification+programs+T+follicular+helper+cell+differentiation.">METTL3-dependent m(6)A modification programs T follicular helper cell differentiation.</a> Nat Commun. 12 (1), 1333 (2021).</li><li>Johnston, R. 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. 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=Distinct+memory+CD4++T+cells+with+commitment+to+T+follicular+helper-+and+T+helper+1-cell+lineages+are+generated+after+acute+viral+infection.">Distinct memory CD4+ T cells with commitment to T follicular helper- and T helper 1-cell lineages are generated after acute viral infection.</a> Immunity. 38 (4), 805-817 (2013).</li><li>Yao, 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=Selenium-GPX4+axis+protects+follicular+helper+t+cells+from+ferroptosis.">Selenium-GPX4 axis protects follicular helper t cells from ferroptosis.</a> Nat Immunol. 22 (9), 1127-1139 (2021).</li><li>Oxenius, A., Bachmann, M. F., Zinkernagel, R. M., Hengartner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-specific+MHC-class+II-restricted+TCR-transgenic+mice:+Effects+on+humoral+and+cellular+immune+responses+after+viral+infection.">Virus-specific MHC-class II-restricted TCR-transgenic mice: Effects on humoral and cellular immune responses after viral infection.</a> Eur J Immunol. 28 (1), 390-400 (1998).</li><li>Grosjean, 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=Isolation+and+enrichment+of+mouse+splenic+t+cells+for+ex+vivo+and+in+vivo+T+cell+receptor+stimulation+assays.">Isolation and enrichment of mouse splenic t cells for ex vivo and in vivo T cell receptor stimulation assays.</a> STAR Protoc. 2 (4), 100961 (2021).</li><li>Dowling, P., 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=Protocol+for+the+bottom-up+proteomic+analysis+of+mouse+spleen.">Protocol for the bottom-up proteomic analysis of mouse spleen.</a> STAR Protoc. 1 (3), 100196 (2020).</li><li>Choi, Y. S., 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=Retroviral+vector+expression+in+TCR+transgenic+CD4++T+cells.">Retroviral vector expression in TCR transgenic CD4+ T cells.</a> Methods Mol Biol. 1291, 49-61 (2015).</li><li>Wu, 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=A+method+for+expansion+and+retroviral+transduction+of+mouse+regulatory+T+cells.">A method for expansion and retroviral transduction of mouse regulatory T cells.</a> J Immunol Methods. 488, 112931 (2021).</li><li>He, 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=Follicular+CXCR5-+expressing+CD8(+)+T+cells+curtail+chronic+viral+infection.">Follicular CXCR5- expressing CD8(+) T cells curtail chronic viral infection.</a> Nature. 537 (7620), 412-428 (2016).</li><li>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=Follicular+helper+CD4+T+cells+(TFH).">Follicular helper CD4 T cells (TFH).</a> Annu Rev Immunol. 29, 621-663 (2011).</li><li>Breitfeld, 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=Follicular+B+helper+T+cells+express+CXC+chemokine+receptor+5,+localize+to+B+cell+follicles,+and+support+immunoglobulin+production.">Follicular B helper T cells express CXC chemokine receptor 5, localize to B cell follicles, and support immunoglobulin production.</a> J Exp Med. 192 (11), 1545-1552 (2000).</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+kinase+mTORC1+promotes+the+generation+and+suppressive+function+of+follicular+regulatory+t+cells.">The kinase mTORC1 promotes the generation and suppressive function of follicular regulatory t cells.</a> Immunity. 47 (3), 538-551 (2017).</li><li>Chen, X., 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+phosphatase+pten+links+platelets+with+immune+regulatory+functions+of+mouse+T+follicular+helper+cells.">The phosphatase pten links platelets with immune regulatory functions of mouse T follicular helper cells.</a> Nat Commun. 13 (1), 2762 (2022).</li><li>Chen, J. 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=Flow+cytometric+identification+of+T(fh)13+cells+in+mouse+and+human.">Flow cytometric identification of T(fh)13 cells in mouse and human.</a> J Allergy Clin Immunol. 147 (2), 470-483 (2021).</li></ol>

Yazarlar hiçbir rekabet çıkarı beyan etmezler.

TFH hücreleri alanındaki araştırmalar, TFH hücrelerinin B hücrelerine yardımcı olmada özel işlevinin keşfedilmesinden bu yana vurgulanmıştır. Biriken çalışmalar, TFH hücre farklılaşmasının çok aşamalı ve çok faktörlü bir süreçolduğunu göstermiştir 30, burada TFH hücre kaderi taahhüdü erken evre5'te belirlenir. Bu nedenle, erken diferansiye TFH hücrelerinin altında yatan mekanizmalar...

Farklı patojenler veya tehditlerle karşılaşan saf CD4 + T hücreleri, özel işlevlere sahip çeşitli yardımcı T (TH ) hücre alt kümelerine farklılaşarak bağışıklık tepkilerini uyarlar1. Akut viral enfeksiyon senaryosunda, naif CD4+ T hücrelerinin büyük bir kısmı, B hücrelerine yardım sağlayan foliküler yardımcı T (TFH) hücrelerine farklılaşır 2,3. Diğer CD4 + T H hücre alt kümelerinden farklı olarak (örneğin, TH1, TH2, TH9 ve TH17 hücreleri), TFH hücreleri, B hücresi homing kemokin CXCL13 için kemokin reseptörü olan önemli bir CXCR5 seviyesini eksprese eder ve TFH hücrelerinin B hücresi foliküllerine göç etmesini sağlar. B hücre foliküllerinde, TFH hücreleri, germinal merkez reaksiyonlarını başlatmada ve sürdürmede aynı kökenli B hücrelerine yardımcı olur, böylece hızlı yüksek afiniteli antikor üretimi ve uzun süreli humoral hafızasağlar 2,3.
Akut viral enfeksiyon üzerine, virüse özgü TFH hücrelerinin erken kader taahhüdü 72 saat 4,5 içinde gerçekleşir ve TFH hücresi kader kararlarını yöneten "ana düzenleyici" olarak görev yapan transkripsiyonel baskılayıcı B hücreli lenfoma-6 (Bcl-6)5,6,7,8 tarafından kontrol edilir. Bcl-6 eksikliği, TFH hücre farklılaşmasını ciddi şekilde köreltirken, ektopik Bcl-6 ekspresyonu, TFH hücre kaderi bağlılığını önemli ölçüde arttırır. Bcl-6'ya ek olarak, erken TFH hücre kaderi taahhüdünün talimatında birden fazla molekül yer alır. Transkripsiyon faktörleri TCF-1 ve LEF-1, Bcl-6 9,10,11'in indüksiyonu yoluyla TFH hücre farklılaşmasını başlatır. Blimp1'in hem Bcl-6 hem de TCF-1 tarafından inhibisyonu, erken TFH hücre kader taahhüdü11,12 için gereklidir. Erken TFH hücre farklılaşması için STAT1 ve STAT3 de gereklidir13. Ayrıca, histon metiltransferaz EZH214,15 ve m6A metiltransferaz METTL316 ile yapılan epigenetik modifikasyonlar, TFH hücre transkripsiyonel programlarını (özellikle Bcl6 ve Tcf7) stabilize etmeye ve böylece erken TFH hücre kader bağlılığını artırmaya yardımcı olur. Yukarıda bahsedilen moleküller ve diğerleri de dahil olmak üzere, başka bir yerdeözetlenen ilerlemeler kaydedilmiş olsa da, erken TFH hücre kaderi bağlılığının transkripsiyonel ve epigenetik düzenlemelerini anlamada ilerlemeler kaydedilmiş olsa da, daha önce bilinmeyen moleküller öğrenilmeyi beklemektedir.
Akut lenfositik koryomenenjit virüsü (LCMV) enfeksiyonunun fare modelinde, LCMV glikoprotein epitopu I-AbGP66-77'yi spesifik olarak tanıyan evlat edinilmiş konjenik TCR-transgenik SMARTA (SM) CD4 + T hücreleri, viral enfeksiyon sırasında TFH veya TH1 hücre farklılaşmasına uğrar. Bu TFH / TH1 çatallanma farklılaşma modeli, virüse özgü TFH hücrelerinin biyolojisini incelemede SM / akut LCMV enfeksiyon modelinin ilerlemesini destekler. Gerçekten de, SM / akut LCMV enfeksiyon modeli, TFH hücre araştırma alanında yaygın olarak kullanılmaktadır ve TFH hücre biyolojisindeki kilometre taşı keşiflerinde çok önemli bir rol oynamıştır. Bu, yukarıda bahsedilen Bcl-6'nın TFH hücrelerinin 5,6 soy tanımlayıcı transkripsiyon faktörü olarak tanımlanmasını ve ayrıca diğer önemli transkripsiyonel faktörleri (örneğin, Blimp-16, TCF-1 / LEF 9,10,11, STAT1 / STAT313, STAT517, KLF218 ve Itch19) TFH hücre farklılaşmasına, transkripsiyon sonrası düzenlemelere ( örneğin, METTL316 ve miR-17 ~ 9220) TFH hücre farklılaşması, TFH hücre hafızası ve plastisite21,22 ve TFH hücrelerini hedefleyen rasyonel aşılama stratejileri (örneğin, selenyum23).
Mevcut çalışma, (1) erken farklılaşmış TFH hücrelerine erişmek için uygun akut LCMV ile enfekte SM kimera fare modelinin oluşturulması, (2) erken farklılaşmış TFH hücrelerine ilişkin moleküllerin akış sitometrisi boyamalarının yürütülmesi ve (3) SM CD4 + 'da retroviral vektör tabanlı gen manipülasyonu gerçekleştirme dahil olmak üzere, virüse özgü TFH hücrelerinin erken kader taahhüdüne erişmek için tekrarlanabilir yöntemleri açıklamaktadır T hücreleri. Bu yöntemler, virüse özgü TFH hücrelerinin erken kader bağlılığını araştıran çalışmalar için faydalı olacaktır.

<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

Foliküler Yardımcı T (TFH) hücreleri, aynı kökenli B hücrelerinin yüksek afiniteli antikorlar üretmesine yardımcı olan ve böylece uzun süreli humoral bağışıklık sağlayan bağımsız bir CD4+ T hücre soyu olarak algılanır. Akut viral enfeksiyon sırasında, virüse özgü TFH hücrelerinin kader taahhüdü erken enfeksiyon fazında belirlenir ve erken diferansiye TFH hücrelerinin araştırılması, T hücresine bağımlı humoral bağışıklığın anlaşılmasında ve aşı tasarımının optimize edilmesinde çok önemlidir. Çalışmada, akut lenfositik koriomenenjit virüsü (LCMV) enfeksiyonunun bir fare modeli ve LCMV glikoprotein epitopu I-AbGP66-77'yi spesifik olarak tanıyan CD4 + T hücrelerine sahip TCR-transgenik SMARTA (SM) faresi kullanılarak, akış sitometrisi boyamalarına dayalı olarak virüse özgü TFH hücrelerinin erken kader taahhüdüne erişmek için prosedürleri tanımladık. Ayrıca, SM CD4 + T hücrelerinin retroviral transdüksiyonundan yararlanılarak, erken farklılaşmış virüse özgü TFH hücrelerinde gen ekspresyonunu manipüle etmek için yöntemler de sağlanmaktadır. Bu nedenle, bu yöntemler, virüse özgü TFH hücrelerinin erken bağlılığının altında yatan mekanizma(lar)ı araştıran çalışmalara yardımcı olacaktır.

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.

Yazar Gündemi: Akut LCMV Zorluklarında TFH Hücre Farklılaşmasının Yollarının Aydınlatılması

Akut LCMV ile Enfekte Farelerde Virüse Özgü Foliküler Yardımcı CD4+ T Hücresinin Erken Farklılaşmasına Erişim

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.

Akut LCMV enfeksiyonu sırasında erken diferansiye virüse özgü TFH hücrelerinin özellikleri Virüse özgü TFH hücrelerinin erken kader bağlılığını araştırmak için, LCMV GP epitopu IAbGP66-77'yi spesifik olarak tanıyan naif konjenik SM CD4+ T hücreleri, CD45.2+ C57BL / 6 alıcılarına evlat edinerek aktarıldı. Ertesi gün, bu alıcılara intravenöz olarak yüksek dozda akut rezolüsyonlu LCMV Armstrong enjekte edil...

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

Mevcut çalışma, virüse özgü TFH hücrelerinin erken kader bağlılığını değerlendirmek ve bu hücrelerde gen ekspresyonunu manipüle etmek için protokoller göstermektedir.

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

434 Görüntüleme.  Shenzhen University. Araştırmamız, akut LCMV ile enfekte monoklonal fare modeli oluşturma, akış sitometrisi boyama yapma ve retroviral vektör tabanlı gen manipülasyonu gerçekleştirme dahil olmak üzere virüse özgü TFH hücrelerinin erken kader taahhüdünü değerlendirmek için tekrarlanabilir yöntemleri açıklamaktadır. İn vitro T hücresi stimülasyonu ile karşılaştırıldığında, in vivo stimülasyon, T hücrelerini 12 saat içinde bile daha hızlı akt...