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>
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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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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. 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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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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 crucia...

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

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, na&#239;ve 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 (<strong c...

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

Author Spotlight: Elucidating the Pathways of TFH Cell Differentiation in Acute LCMV Challenges

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

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, ...

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

Research

JoVE Journal

Immunology and Infection

596 Views.  Shenzhen University. Ons onderzoek beschrijft reproduceerbare methoden voor het beoordelen van de vroege lotsverbintenis van virusspecifieke TFH-cellen, waaronder het opzetten van een acuut LCMV-geïnfecteerd monoklonaal muismodel, het uitvoeren van flowcytometriekleuring en het uitvoeren van retrovirale vectorgebaseerde genmanipulatie. Vergeleken met in-vitro T-celstimulatie zou in-vivo-stimulatie T-cellen sneller kunnen activeren, zelfs in 12 uur, en het is volledig geactiveerd...

Video: Auteur in de schijnwerpers: opheldering van de routes van TFH-celdifferentiatie bij acute LCMV-uitdagingen

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector

Upon viral infection, antigen-specific CD8+ cytotoxic T cells (CTLs) arise and contribute to the elimination of infected cells to prevent the spread of pathogens. Therefore, the frequency of antigen-specific CTLs is indicative of the strength of the T cell response against a specific antigen. Such analysis is important in basic immunology, vaccine development, cancer immunobiology and the adaptive immunology. In the vaccine field, the CTL response directed against components of a viral vector co-determines how effective the generation of antigen-specific cells against the antigen of interest (i.e., transgene) is. Antigen-specific CTLs can either be detected by stimulation with specific peptides followed by intracellular cytokine staining or by the direct staining of antigen-specific T cell receptors (TCRs) and analysis by flow cytometry. The first method is rather time-consuming since it requires sacrificing of animals to isolate cells from organs. Also, it requires isolation of blood from small animals which is difficult to perform. The latter method is rather fast, can be easily done with small amounts of blood and is not dependent on specific effector functions, such as cytolytic activity. MHC tetramers are an ideal tool to detect antigen-specific TCRs.
Here, we describe a protocol to simultaneously detect antigen-specific CTLs for the immunodominant peptides of the viral vector VSV-GP (LCMV-GP, VSV-NP) and transgenes (OVA, HPV 16 E7, eGFP) by MHC I tetramer staining and flow cytometry. Staining is possible either directly from blood or from single cell suspensions of organs, such as spleen. Blood or single cell suspensions of organs are incubated with tetramers. After staining with antibodies against CD3 and CD8, antigen-specific CTLs are quantified by flow cytometry. Optionally, antibodies against CD43, CD44, CD62L or others can be included to determine the activation status of antigen-specific CD8+T cells and to discriminate between na&#239;ve and effector cells.

Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector

Here, we present a protocol for the ex vivo qualitative detection of antigen-specific CD8+ T cells. Analysis is possible with single cell suspensions from organs or from small amounts of blood. A broad range of studies require the analysis of cytotoxic T cell responses (vaccination and cancer immunotherapy studies).

Upon viral infection, antigen-specific CD8+ cytotoxic T cells (CTLs) arise and contribute to the elimination of infected cells to prevent the spread of ...

Chronic, Acute, and Reactivated HIV Infection in Humanized Immunodeficient Mouse Models

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical research on infectious diseases. Described here are three models of humanized immunodeficient mice for studying the dynamics of HIV infection. The first is based on the intrahepatic injection of CD34+ hematopoietic stem cells in newborn mice, which allows for the reconstitution of several blood and lymphoid tissue-confined cells, followed by infection with a reference HIV strain. This model allows monitoring for up to 36 weeks post-infection and is hence called the chronic model. The second and third models are referred to as the acute and reactivation models, in which peripheral blood mononuclear cells are intraperitoneally injected in adult mice. In the acute model, cells from a healthy donor are engrafted through the intraperitoneal route, followed by infection with a reference HIV strain. Finally, in the reactivation model, cells from an HIV-infected donor under antiretroviral therapy are engrafted via the intraperitoneal route. In this case, a drug-free environment in the mouse allows for virus reactivation and an increase in viral load. The protocols provided here describe the conventional experimental approach for humanized, immunodeficient mouse models of HIV infection.

Described here are three experimental approaches for studying the dynamics of HIV infection in humanized mice. The first permits the study of chronic infection events, whereas the two latter allows for the study of acute events after primary infection or viral reactivation.

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical ...

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and ...

Optimized Whole Blood Immunoassays for Analyzing Fungal and Viral Immunity

Whole Blood Assay with Dual Co-Stimulation for Antigen-Specific Analysis of Host Immunity to Fungal and Viral Pathogens

Rapid and resource-efficient sample processing, high throughput, and high robustness are critical for effective scientific and clinical application of advanced antigen-specific immunoassays. Traditionally, such immunoassays, especially antigen-specific T-cell analysis by flow cytometry or enzyme-linked immunosorbent spot assays, often rely on the isolation of peripheral blood mononuclear cells. This process is time-consuming, subject to many pre-analytic confounders, and requires large blood volumes. Whole blood-based assays provide a facile alternative with increased pre-analytic robustness and lower blood volume requirements. Furthermore, whole blood-based assays allow for the preservation of inter-cellular interactions that are not captured by assays using isolated cell subsets. Recently, a refined whole blood immunoassay with dual anti-CD28 and anti-CD49d co-stimulation for comprehensive analysis of both antigen-specific T-cell functions and complex intercellular interactions in response to various fungal and viral antigens has been proposed. This protocol provides guidance for the preparation of stimulation tubes, blood stimulation, and downstream sample processing for flow cytometry, cytokine secretion assays, and transcriptional analyses. This includes a validated and functionally equivalent, previously unpublished, low-volume protocol (250 &#181;L) to make flow cytometric and cytokine-based T-cell monitoring more accessible for studies in pediatric patients or preclinical studies in small animals (e.g., mice). Altogether, these protocols provide a versatile toolbox for complex antigen-specific immune analysis in both clinical and translational research settings.

Author Spotlight: Advancing Immune Monitoring in Critical Care Patients Using Whole Blood Assays

Whole blood-based immunoassays provide a facile and resource-efficient tool to analyze antigen-specific immunity for diagnostic and research purposes. This article provides an optimized whole blood-based protocol with dual co-stimulation for comprehensive analysis of host immunity to fungal and viral pathogens, including a low-volume version for pediatric patients and small animals.

Rapid and resource-efficient sample processing, high throughput, and high robustness are critical for effective scientific and clinical application of ...

Th17 Cell Differentiation from Naive CD4+ T-Cells Using Flow Cytometry

In Vitro Differentiation of Naive CD4+ T Cells into Pathogenic Th17 Cells in Mouse

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have been linked to a wide range of diseases in recent times, including multiple sclerosis (MS), rheumatoid arthritis, acute respiratory distress syndrome (ARDS), sepsis, and other autoimmune disorders. However, the currently known in vitro differentiation protocols have difficulty achieving high purity of pathogenic Th17 cells, with the induction efficiency often below 50%, which is a key challenge in in vitro experiments. In this protocol, we propose an enhanced in vitro culture and differentiation protocol for pathogenic Th17 cells, which is used to directly differentiate naive CD4+ T cells isolated from mouse spleens into pathogenic Th17 cells. This protocol provides detailed instructions on splenocyte isolation, purification of naive CD4+ T cells, and differentiation of pathogenic Th17 cells. Through this protocol, we can achieve a differentiation purity of approximately 90% for pathogenic Th17 cells, which meets the basic needs of many cellular experiments.

Author Spotlight: Achieving High-Purity In Vitro Differentiation of Th17 Cells Using Cytokine Concentration Modulation

This protocol describes the differentiation of na&#239;ve CD4+ T cells into pathogenic Th17 cells in vitro. Specifically, when combined with a multi-parameter flow cytometry-based approach, 90% purity of pathogenic Th17 cells can be obtained from na&#239;ve CD4+ T cells using this differentiation method.

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have ...

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

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