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Accessing Early Differentiation of Virus-Specific Follicular Helper CD4+ T Cell in Acute LCMV-Infected Mice
* These authors contributed equally
In This Article
Summary
The current study showcases protocols for assessing the early fate commitment of virus-specific TFH cells and manipulating gene expression in these cells.
Abstract
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.
Introduction
Encountering different pathogens or threats, naïve 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 naïve 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 leve....
Protocol
All animal experiments were conducted following procedures approved by the Institutional Animal Care and Use Committees of the Third Military Medical University. The following mouse strains were used in the present study: C57BL/6J (B6) mouse (both genders), aged 6 to 8 weeks, weighing 25-30 g; CD45.1+SM TCR transgenic mouse (B6 CD45.1 × SM TCR transgenic), both genders, aged 6 to 8 weeks, weighing 25-30 g; and CXCR5-GFP CD45.1+SM TCR transgenic mouse (B6 CD45.1 × SM TCR transgenic × C.......
Representative Results
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 (
Discussion
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.......
Acknowledgements
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.).
....Materials
| Name | Company | Catalog Number | Comments |
| 0.25% Trypsin-EDTA | Corning | 25-052-CI | |
| 4% Paraformaldehyde Fix Solution, 4% PFA | Beyotime | P0099-500mL | |
| 70 μm cell strainer | Merck | CLS431751 | |
| Alexa Fluor 647 anti-mouse TCR Vα2 (clone B20.1) | Biolegend | 127812 | 1:200 dilution |
| Alexa Fluor 700 anti-mouse CD45.1 (clone A20) | Biolegend | 110724 | 1:200 dilution |
| APC anti-mouse CD25 (clone PC61) | Biolegend | 101910 | 1:200 dilution |
| B6 CD45.1 (B6.SJL-Ptprca Pepcb/BoyJ) mouse | The Jackson Laboratory | 002014 | |
| BeaverBeads Streptavidin | Beaver | 22321-10 | |
| Biotin anti-mouse F4/80 Antibody (clone BM8) | Biolegend | 123106 | 1:200 dilution |
| Biotin Rat anti-mouse CD11c (clone N418) | Biolegend | 117304 | 1:200 dilution |
| Biotin Rat anti-Mouse CD19 (clone 6D5) | Biolegend | 115504 | 1:200 dilution |
| Biotin Rat anti-Mouse CD8a (clone 53-6.7) | Biolegend | 100704 | 1:200 dilution |
| Biotin Rat anti-mouse NK-1.1 (clone PK136) | Biolegend | 108704 | 1:200 dilution |
| Biotin Rat anti-mouse TER-119/Erythroid Cells (clone TER-119) | Biolegend | 116204 | 1:200 dilution |
| bovine serum albumin, BSA | Sigma | A7906 | |
| Brilliant Violet 421 anti-T-bet (clone 4B10) | Biolegend | 644816 | 1:100 dilution |
| Brilliant Violet 605 anti-mouse CD279 (PD-1) (clone 29F.1A12) | Biolegend | 135220 | 1:200 dilution |
| C57BL/6J (B6) mouse | The Jackson Laboratory | 000664 | |
| CXCR5-GFP knock-in reporter mouse | 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. | ||
| DMEM 10% medium | DMEM medium containing 10% FBS | ||
| DMEM medium | Gibco | 11885092 | |
| EDTA | Sigma | E9884 | |
| FACSFortesa | BD Biosciences | ||
| Fetal bovine serum, FBS | Sigma | F8318 | |
| FlowJo (version 10.4.0) | BD Biosciences | ||
| Foxp3/Transcription Factor Staining Buffer Set | Invitrogen | 00-5523-00 | The kit contains three reagents: a. Fixation/Permeabilization Concentrate (4X); b. Fixation / Permeabilization Diluent; c. Permeabilization Buffer. |
| Goat Anti-Rat IgG Antibody (H+L), Biotinylated | Vector laboratories | BA-9400-1.5 | 1:200 dilution |
| Invitrogen EVOS FL Auto Cell Imaging System | ThermoFisher Scientific | ||
| Isolation buffer | FACS buffer containing 0.5% BSA and 2mM EDTA | ||
| LCMV GP61-77 peptide (GLKGPDIYKGVYQFKSV) | Chinese Peptide Company | ||
| LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit, for 633 or 635 nm excitation | Life Technologies | L10199 | 1:200 dilution |
| MigR1 | addgene | #27490 | |
| NaN3 | Sigma | S2002 | |
| Opti-MEM medium | Gibco | 31985070 | |
| pCL-Eco | addgene | #12371 | |
| PE anti-mouse CD69 (clone H1.2F3) | Biolegend | 104508 | 1:200 dilution |
| PE Mouse anti-Bcl-6 (clone K112-91) | BD Biosciences | 561522 | 1:50 dilution |
| Phosphate buffered saline, PBS | Gibco | 10010072 | |
| Polybrene | Solarbio | H8761 | |
| Purified Rat Anti-Mouse CXCR5 (clone 2G8) | BD Biosciences | 551961 | 1:50 dilution |
| Rat monoclonal PerCP anti-mouse CD4 (clone RM4-5) | Biolegend | 100538 | 1:200 dilution |
| recombinant murine IL-2 | Gibco | 212-12-1MG | |
| Red Blood Cell Lysis Buffer | Beyotime | C3702-500mL | |
| RPMI 1640 medium | Sigma | R8758 | |
| RPMI 2% | RPMI 1640 medium containing 2% FBS | ||
| SMARTA (SM) TCR transgenic mouse | 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). | ||
| Staining buffer | PBS containing 2% FBS and 0.01% NaN3 | ||
| Streptavidin PE-Cyanine7 | eBioscience | 25-4317-82 | 1:200 dilution |
| TCF1/TCF7 (C63D9) Rabbit mAb (Alexa Fluor 488 Conjugate) | Cell signaling technology | 6444S | 1:400 dilution |
| TFH cell staining buffer | FACS buffer containing 1% BSA and 2% mouse serum | ||
| TransIT-293 reagent | Mirus Bio | MIRUMIR2700 |
References
- O'shea, J. J., Paul, W. E. Mechanisms underlying lineage commitment and plasticity of helper CD4+ T cells. Science. 327 (5969), 1098-1102 (2010).
- Crotty, S. T follicular helper cell biology: A decade of di....
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