Name: Accessing Early Differentiation of Virus-Specific Follicular Helper CD4+ T Cell in Acute LCMV-Infected Mice (Video) | JoVE Journal
Uploaded: 2024-04-26T15:00:00.000+00:00
Duration: 5 min 3 s
Description: Learn reproducible methods for assessing early virus-specific TFH cell commitment using acute LCMV-infected mouse models and flow cytometry. These techniques advance T-cell activation research and vaccine design optimization.

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5 minutes

This study was supported by grants from the National Natural Science Foundation of China (No. 32300785 to X.C.), the China National Postdoctoral Program for Innovative Talents (No. BX20230449 to X.C.), and the National Science and Technology Major Project (No. 2021YFC2300602 to L.Y.).

Deciphering Early Follicular Helper T-Cell Commitment in Acute LCMV Infection

<ol><li>O'shea, J. J., Paul, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+underlying+lineage+commitment+and+plasticity+of+helper+CD4%2B+T+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((T+follicular+helper+cell+biology%3A+A+decade+of+discovery+and+diseases%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">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/pubmed?term=((Follicular+helper+t+cells%5BTitle%5D)+AND+%22Annu+Rev+Immunol%22%5BJournal%5D)">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/pubmed?term=((ICOS+receptor+instructs+T+follicular+helper+cell+versus+effector+cell+differentiation+via+induction+of+the+transcriptional+repressor+bcl6%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">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/pubmed?term=((Bcl6+expressing+follicular+helper+CD4+T+cells+are+fate+committed+early+and+have+the+capacity+to+form+memory%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">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/pubmed?term=((Bcl6+and+BLIMP-1+are+reciprocal+and+antagonistic+regulators+of+T+follicular+helper+cell+differentiation%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Bcl6+mediates+the+development+of+T+follicular+helper+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((The+transcriptional+repressor+bcl-6+directs+T+follicular+helper+cell+lineage+commitment%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">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/pubmed?term=((LEF1+and+TCF1+orchestrate+T%28FH%29+differentiation+by+regulating+differentiation+circuits+upstream+of+the+transcriptional+repressor+bcl6%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">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/pubmed?term=((TCF1+is+required+for+the+t+follicular+helper+cell+response+to+viral+infection%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">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/pubmed?term=((The+transcription+factor+TCF-1+initiates+the+differentiation+of+T%28FH%29+cells+during+acute+viral+infection%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">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/pubmed?term=((Molecular+mechanisms+that+control+the+expression+and+activity+of+bcl-6+in+th1+cells+to+regulate+flexibility+with+a+TFH-like+gene+profile%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">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/pubmed?term=((Cutting+edge%3A+STAT1+is+required+for+IL-6-mediated+bcl6+induction+for+early+follicular+helper+cell+differentiation%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">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/pubmed?term=((The+histone+methyltransferase+ezh2+primes+the+early+differentiation+of+follicular+helper+T+cells+during+acute+viral+infection%5BTitle%5D)+AND+%22Cell+Mol+Immunol%22%5BJournal%5D)">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/pubmed?term=((Ezh2+programs+T%28FH%29+differentiation+by+integrating+phosphorylation-dependent+activation+of+bcl6+and+polycomb-dependent+repression+of+p19ARF%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">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/pubmed?term=((METTL3-dependent+m%286%29A+modification+programs+T+follicular+helper+cell+differentiation%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">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/pubmed?term=((STAT5+is+a+potent+negative+regulator+of+TFH+cell+differentiation%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">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/pubmed?term=((The+transcription+factor+KLF2+restrains+CD4%2B+T+follicular+helper+cell+differentiation%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">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/pubmed?term=((The+e3+ubiquitin+ligase+itch+is+required+for+the+differentiation+of+follicular+helper+t+cells%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">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/pubmed?term=((The+microRNA+cluster+mir-17%7E92+promotes+TFH+cell+differentiation+and+represses+subset-inappropriate+gene+expression%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">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/pubmed?term=((The+kinase+complex+mTORC2+promotes+the+longevity+of+virus-specific+memory+CD4%28%2B%29+T+cells+by+preventing+ferroptosis%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">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/pubmed?term=((Distinct+memory+CD4%2B+T+cells+with+commitment+to+T+follicular+helper-+and+T+helper+1-cell+lineages+are+generated+after+acute+viral+infection%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">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/pubmed?term=((Selenium-GPX4+axis+protects+follicular+helper+t+cells+from+ferroptosis%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">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/pubmed?term=((Virus-specific+MHC-class+II-restricted+TCR-transgenic+mice%3A+Effects+on+humoral+and+cellular+immune+responses+after+viral+infection%5BTitle%5D)+AND+%22Eur+J+Immunol%22%5BJournal%5D)">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/pubmed?term=((Isolation+and+enrichment+of+mouse+splenic+t+cells+for+ex+vivo+and+in+vivo+T+cell+receptor+stimulation+assays%5BTitle%5D)+AND+%22STAR+Protoc%22%5BJournal%5D)">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/pubmed?term=((Protocol+for+the+bottom-up+proteomic+analysis+of+mouse+spleen%5BTitle%5D)+AND+%22STAR+Protoc%22%5BJournal%5D)">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/pubmed?term=((Retroviral+vector+expression+in+TCR+transgenic+CD4%2B+T+cells%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">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/pubmed?term=((A+method+for+expansion+and+retroviral+transduction+of+mouse+regulatory+T+cells%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">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/pubmed?term=((Follicular+CXCR5-+expressing+CD8%28%2B%29+T+cells+curtail+chronic+viral+infection%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Follicular+helper+CD4+T+cells+%28TFH%29%5BTitle%5D)+AND+%22Annu+Rev+Immunol%22%5BJournal%5D)">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/pubmed?term=((Follicular+B+helper+T+cells+express+CXC+chemokine+receptor+5%2C+localize+to+B+cell+follicles%2C+and+support+immunoglobulin+production%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">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/pubmed?term=((The+kinase+mTORC1+promotes+the+generation+and+suppressive+function+of+follicular+regulatory+t+cells%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">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/pubmed?term=((The+phosphatase+pten+links+platelets+with+immune+regulatory+functions+of+mouse+T+follicular+helper+cells%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">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/pubmed?term=((Flow+cytometric+identification+of+T%28fh%2913+cells+in+mouse+and+human%5BTitle%5D)+AND+%22J+Allergy+Clin+Immunol%22%5BJournal%5D)">Flow cytometric identification of T(fh)13 cells in mouse and human.</a> J Allergy Clin Immunol. 147 (2), 470-483 (2021).</li>
</ol>

The authors declare no competing interests.

The research in the field of TFH cells has been highlighted since the discovery of the specialized function of TFH cells in helping B cells. Accumulating studies indicated that TFH cell differentiation is a multistage and multifactorial process30, wherein the TFH cell fate commitment is determined in the early stage5. Therefore, a better understanding of the mechanisms underlying early-differentiated TFH cells is crucial for TFH cell biology and rational vaccine design. Herein, we provided methods to access the early fate commitment of virus-specific TFH cells and to manipulate gene expression in early-differentiated TFH cells by exploiting the LCMV Armstrong viral infection model, TCR-transgenic SM mouse, and retrovirus-mediated transduction.
The chemokine receptor CXCR5 is an important surface marker that distinguishes TFH cells from other CD4+ TH cell subsets6,7,8,31, thus necessitating the CXCR5 staining method of high specificity and sensitivity in TFH cell research. In the study, a three-step streptavidin-biotin CXCR5 staining method was showcased in identifying the TFH cell subset (Figure 1C). Though loss of specificity is one sticky problem for two- or three-strep antibody staining approaches of high sensitivity, it was found that the fluorescence signal by three-step CXCR5 antibody staining almost completely overlapped with the GFP signal in CXCR5-GFP29 reporter SM CD4+ T cells on day 3-post infection (Figure 1D), suggesting the achievement of both specificity and sensitivity by the three-step CXCR5 antibody staining method. Indeed, this three-strep CXCR5 staining strategy is tried-and-trusted and has helped the researchers to better investigate TFH cell biology11,14,15,21 as well as other CXCR5-expressing T cell types29,32.
Upon infection and immunization, the fate-commitment of TFH cells is generally modeled by a bifurcated differentiation of na&#239;ve CD4+ T cells into either TFH cells or non-TFH effector TH cells2,3,30. This study showcased the balanced bifurcation of TFH and TH1 cells in transferred SM CD4+ T cells. Moreover, these transferred SM CD4+ T cells are of adequate cell quantity in the early infection stage, thus acting as a suitable model for studying TFH cell differentiation. Assuredly, retrovirus-mediated ectopic Bcl-6 expression in transferred SM CD4+ T cells was strongly biased towards TFH cell differentiation as compared to that in control SM CD4+ T cells (86.6% vs. 53.8%) (Figure 3C), which is aligned with previous report6 and further proves the accuracy of the protocols described in the study. Alternatively, the aforementioned protocols can also be applied to retrovirus-mediated knock-down of interested genes (e.g., bcl6, Tcf7) in early-activated SM CD4+ T cells11 (data not shown).
One limitation in the protocol is the timing issue (more than 2 h in total) and the multiple-step process of the three-step CXCR5 staining method. Alternatively, one-step18,33 or two-step4,9,34 CXCR5 staining methods were also applied in TFH cell research. A comparison of the specificity/sensitivity of these CXCR5 staining methods is warranted.
In summary, the current study provided protocols to access early-differentiated virus-specific TFH cells and to investigate interested gene(s) that potentially regulate TFH cell differentiation. These protocols will fuel the investigations of early TFH cell fate commitment.

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.

<table><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 &amp;mu;m cell strainer</td><td>Merck</td><td>CLS431751</td><td></td></tr><tr><td>Alexa Fluor 647 anti-mouse TCR V&amp;alpha;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)&amp;nbsp;</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

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

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.

Characteristics of early-differentiated virus-specific TFH cells during acute LCMV infection 
To probe the early fate commitment of virus-specific TFH cells, naïve congenic SM CD4+ T cells that specifically recognize LCMV GP epitope I-AbGP66-77 were adoptively transferred into CD45.2+ C57BL/6 recipients. The next day, these recipients were intravenously infected with a high dosage of the acutely resolved LCMV Armstrong (Figure 1A). On day 3 after infection, splenocytes of C57BL/6 recipients were analyzed by flow cytometry, and the results indicated a large quantity of transferred CD45.1+ SM CD4+ T cells (~10% of total CD4+ T cells) (Figure 1B), which provided sufficient number of virus-specific CD4+ T cells for further investigation. Further analyses of transferred SM CD4+ T cells showed co-expression of CXCR5 and Bcl-6/TCF-1 and reciprocal expression of CXCR5 and CD25/T-bet, thus defining TFH (CXCR5+Bcl-6+ or CXCR5+TCF-1+) and TH1 (CXCR5-CD25+ or CXCR5-T-bet+) lineages (Figure 1C). To evaluate the specificity and sensitivity of CXCR5 antibody staining presented in the study, we further crossed the aforementioned CD45.1+ SM mouse line with the CXCR5-GFP knock-in mouse line, which was generated by the insertion of an IRES-GFP construct after the open reading frame of Cxcr529. Then, CD45.1+ CXCR5-GFP reporter SM cells were transferred into C57BL/6 recipients. These recipients were infected with LCMV Armstrong, and their splenocytes were analyzed on day 3-post infection. Indeed, the fluorescence signal by the CXCR5 antibody staining method in the study finely overlapped with the GFP signal in transferred CXCR5-GFP reporter29 SM CD4+ T cells on day 3-post infection (Figure 1D), indicating the high specificity of the three-step CXCR5 antibody staining method.
Retrovirus-based Bcl-6 overexpression in SM CD4+ T cells 
Bcl-6 is one of the most important molecules in fostering TFH cell differentiation6,7,8. To ascertain the reliability of the current protocol in accessing early-differentiated TFH cells, CD45.1+ SM CD4+ T cells were in vivo activated and transduced with retrovirus supernatant from MigR1 plasmid- or MigR1-Bcl6 overexpression plasmid-transfected 293T cells (Figure 2 and Figure 3A). Retrovirus-transduced SM CD4+ T cells were next adoptively transferred into C57BL/6 recipients, which were then infected with LCMV Armstrong (Figure 3B). On day 3 after infection, we found a balanced bifurcation of TFH and TH1 cells in MigR1-SM CD4+ T cells, while a predominant TFH cell-directed differentiation in MigR1-Bcl6-SM CD4+ T cells (Figure 3C). Consistently, the Bcl-6 expression level was enhanced in MigR1-Bcl6-SM CD4+ T cells as compared to that in MigR1-SM CD4+ T cells (Figure 3D). Therefore, the phenotype that ectopic Bcl-6 expression-driven TFH cell differentiation6 was finely reproduced following the current protocol.
<img alt="Virus-specific T\(_{FH}\) cell differentiation process, flow cytometry, gating strategy, co-staining analysis." class="xfigimg" src="/files/ftp_upload/66752/66752fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Flow cytometry analyses of early fate commitment of SM TFH cells during LCMV Armstrong infection. (A) Experimental design. Splenic CD4+ T cells were harvested from CD45.1+ SM mice and then adoptively transferred into C57BL/6 recipients (CD45.2+). The next day, these recipients were infected with LCMV Armstrong. On day 3 after infection, transferred CD45.1+ SM cells from the spleens of recipients were analyzed by flow cytometry. (B) Representative flow cytometry data showing the gating strategy of transferred CD45.1+ SM CD4+ T cells from the spleens of recipients on day 3-post LCMV Armstrong infection. (C) Representative flow cytometry data showing co-stainings of CXCR5 and other TFH cell-associated molecules (Bcl-6, TCF-1, CD25, and T-bet) in transferred CD45.1+ SM cells from the spleens of recipients on day 3-post LCMV Armstrong infection. (D) Representative flow cytometry data showing co-stainings of fluorescence-conjugated antibody against CXCR5 and GFP signal in transferred CD45.1+ CXCR5-GFP reporter SM cells from the spleens of recipients on day 3-post LCMV Armstrong infection. <a href="https://www.jove.com/files/ftp_upload/66752/66752fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Retrovirus viral transduction in SM CD4+ T cells; diagram, GFP signal, flow cytometry analysis." class="xfigimg" src="/files/ftp_upload/66752/66752fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Retroviral transduction in SM CD4+ T cells. (A) Schematic diagram of retrovirus production and retroviral transduction in SM CD4+ T cells. For retrovirus production, 293T cells seeded in a 6-well plate were transfected with target DNA plasmids when cell density reaches ≥80% confluence. Then, cell culture supernatant containing retroviruses was collected 72 h after transfection and immediately used or stored at -80 °C. For retroviral transduction in SM CD4+ T cells, a CD45.1+ SM mouse was intravenously injected with 200 µg of LCMV GP61-77 peptide. Then, the spleen was dissected from SM mouse 16-18 h after peptide injection, and the activation status of splenic CD4+ T cells was detected. Activated SM CD4+ T cells were transduced with culture supernatant containing retroviruses and cultured in vitro for 48 h before accessing the transduction efficiency. (B) Fluorescence microscopy imaging of GFP in plasmid DNA-transfected 293T cells. Scale bar: 400 µm. (C) Flow cytometry analysis of CD25 and CD69 expression levels in splenic naïve CD4+ T cells from naïve mouse (naïve, left) or splenic SM CD4+ T cells from LCMV GP61-77 peptide-injected SM mouse (activated, right). (D) Flow cytometry analysis of GFP expression in retrovirus-transduced SM CD4+ T cells (transduced, left) or retrovirus-non-transduced SM CD4+ T cells (non-transduced, left). <a href="https://www.jove.com/files/ftp_upload/66752/66752fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Schematic of MigR1 and MigR1-Bcl6 plasmids, flow cytometry, T cell differentiation analysis." class="xfigimg" src="/files/ftp_upload/66752/66752fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: Effects of Bcl-6 overexpression in SM TFH cell fate commitment. (A) Linear maps of retroviral MigR1 backbones (up panel) and modified MigR1 vector with Bcl6 CDS region at the upstream of IRES-GFP (MigR1-Bcl6) (bottom panel). (B) Experimental design. MigR1-transduced or MigR1-Bcl6-transduced SM CD4+ T cells were adoptively transferred into C57BL/6 recipients (CD45.2+) one day prior to LCMV Armstrong. On day 3 after infection, transferred CD45.1+ SM CD4+ T cells from recipients' spleens were analyzed by flow cytometry. (C) Flow cytometry analysis of CXCR5 and CD25 in MigR1-transduced or MigR1-Bcl6-transduced GFP+ SM CD4+ T cells. The numbers adjacent to the gate indicate the frequencies of TFH cells in SM CD4+ T cells. (D) Flow cytometry analysis of Bcl-6 expression level in MigR1-transduced or MigR1-Bcl6-transduced SM CD4+ T cells. MFI, mean fluorescence intensity. <a href="https://www.jove.com/files/ftp_upload/66752/66752fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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

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Learn reproducible methods for assessing early virus-specific TFH cell commitment using acute LCMV-infected mouse models and flow cytometry. These techniques advance T-cell activation research and vaccine design optimization.

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

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