이 연구는 중국 국립 자연 과학 재단 (No. 32300785에서 X.C.까지), 중국 국가 혁신 인재 박사 후 과정 (No. BX20230449 to X.C.), 국가 과학 기술 전공 프로젝트(No. 2021YFC2300602 to L.Y.).

급성 LCMV 감염에서 Early Follicular Helper T-Cell Commitment의 해독

<ol>
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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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저자는 상충되는 이해관계가 없음을 선언합니다.

TFH 세포 분야의 연구는 B 세포를 돕는 TFH 세포의 특화된 기능이 발견된 이후 주목을 받았습니다. 축적된 연구는 TFH 세포 분화가 다단계 및 다인자 과정(multistage and multifactorial process)이라는 것을 나타냈다(30), 여기서 TFH 세포 운명 헌신은 초기 단계(early stage5)에서 결정된다. 따라서 조기 분화된 TFH 세포의 기전을 더 잘 이?...

다양한 병원체나 위협에 직면했을 때, 미성숙 CD4+ T 세포는 특화된 기능을 가진 다양한 보조 T(TH) 세포 subset으로 분화하여 면역 반응을 조정합니다1. 급성 바이러스 감염 시나리오에서 naive CD4+ T 세포의 상당 부분은 B 세포 2,3에 도움을 제공하는 여포 도우미 T(TFH) 세포로 분화합니다. 다른 CD4+ TH 세포 subset(예를 들어, TH1, TH2, TH9 및 TH17 세포)와 구별되듯이, TFH 세포는 상당한 수준의 CXCR5를 발현하는데, 이는 B 세포 귀환 chemokine CXCL13에 대한 chemokine 수용체로서, TFH 세포가 B 세포 소낭선으로 이동할 수 있게 한다. B 세포 난포에서 TFH 세포는 생식 중심 반응을 시작하고 유지하는 데 있어 B 세포를 동족 형성을 지원하여 빠른 고친화성 항체 생산과 장기 체액성 기억을 가능하게 합니다 2,3.
급성 바이러스 감염 시, 바이러스 특이적 TFH 세포의 조기 운명 결정은 72 h 4,5 이내에 발생하며, TFH 세포 운명 결정을 관장하는 "마스터 조절자" 역할을 하는 전사 억제 인자 B 세포 림프종-6(Bcl-6)5,6,7,8에 의해 조절됩니다. Bcl-6의 결핍은 TFH 세포 분화를 심각하게 둔화시키는 반면, 이소성 Bcl-6 발현은 TFH 세포 운명 헌신을 실질적으로 촉진합니다. Bcl-6 외에도 여러 분자가 초기 TFH 세포 운명 헌신을 지시하는 데 관여합니다. 전사 인자 TCF-1 및 LEF-1은 Bcl-6 9,10,11의 유도를 통해 TFH 세포 분화를 시작합니다. Bcl-6 및 TCF-1 모두에 의한 Blimp1의 억제는 초기 TFH 세포 운명 헌신11,12에 필요합니다. STAT1 및 STAT3는 초기 TFH 세포 분화에도 필요합니다13. 게다가, 히스톤 메틸전이효소 EZH214,15 및 m6A 메틸전이효소 METTL316에 의한 후성유전학적 변형은 TFH 세포 전사 프로그램(특히 Bcl6 및 Tcf7)을 안정화하는 데 도움이 되므로 초기 TFH 세포 운명 헌신을 촉진합니다. 앞서 언급한 분자 및 다른 분자를 포함한 발전이 다른 곳에서 요약된3, 초기 TFH 세포 운명 헌신의 전사 및 후성유전학적 규칙을 이해하는 데 이루어졌지만, 이전에 알려지지 않은 분자는 학습되어야 할 과제로 남아 있습니다.
급성 림프구성 맥락막염 바이러스(LCMV) 감염의 마우스 모델에서, LCMV 당단백질 에피토프 I-AbGP66-77을 특이적으로 인식하는 입양 전달 동종성 TCR-형질전환 SMARTA(SM) CD4+ T 세포는 바이러스 감염 중에 TFH 또는 TH1 세포 분화를 겪습니다. 이 TFH/TH1 분기 분화 패턴은 바이러스 특이적 TFH 세포의 생물학을 연구하는 데 있어 SM/급성 LCMV 감염 모델의 발전을 지원합니다. 실제로, SM/급성 LCMV 감염 모델은 TFH 세포 연구 분야에서 널리 사용되어 왔으며 TFH 세포 생물학의 이정표적 발견에 중요한 역할을 했습니다. 여기에는 TFH 세포 5,6의 계통 정의 전사 인자로서의 전술한 Bcl-6의 식별뿐만 아니라 T FH 세포분화를 안내하는 다른 중요한 전사 인자(예: Blimp-16, TCF-1/LEF 9,10,11, STAT1/STAT313, STAT517, KLF218 및 Itch19)가 포함되며, TFH 세포 분화, 전사 후 규정( 예를 들어, TFH 세포 분화, TFH 세포 기억 및 가소성21,22, 및 TFH 세포를 표적으로 하는 합리적인 백신 접종 전략(예: 셀레늄23)의 METTL316 및 miR-17~9220)을 포함할 수 있다.
본 연구는 (1) 조기 분화된 TFH 세포에 접근하는 데 적합한 급성 LCMV 감염 SM 키메라 마우스 모델 확립, (2) 조기 분화 TFH 세포와 관련된 분자의 유세포 분석 염색 수행, (3) SM CD4+에서 레트로바이러스 벡터 기반 유전자 조작 수행을 포함하여 바이러스 특이적 TFH 세포의 조기 운명 헌신에 접근하기 위한 재현 가능한 방법을 설명합니다 T 세포. 이러한 방법은 바이러스 특이적 TFH 세포의 조기 운명 헌신을 조사하는 연구에 유용할 것입니다.

<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

Follicular Helper T (TFH) cell은 동족 B 세포가 친화성이 높은 항체를 생성하여 장기적인 체액성 면역을 형성하는 데 도움을 주는 독립적인 CD4+ T 세포 계통으로 인식됩니다. 급성 바이러스 감염 시 바이러스 특이적 TFH 세포의 운명 결정은 초기 감염 단계에서 결정되며, 조기 분화된 TFH 세포에 대한 연구는 T 세포 의존성 체액성 면역을 이해하고 백신 설계를 최적화하는 데 중요합니다. 이 연구에서는 급성 림프구성 맥락수막염 바이러스(LCMV) 감염 마우스 모델과 LCMV 당단백질 에피토프 I-AbGP66-77을 특이적으로 인식하는 CD4+ T 세포를 가진 TCR-transgenic SMARTA(SM) 마우스를 사용하여 유세포 염색을 기반으로 바이러스 특이적 TFH 세포의 조기 운명 헌신에 접근하는 절차를 설명했습니다. 또한, SM CD4+ T 세포의 레트로바이러스 transduction을 이용하여 조기 분화된 바이러스 특이적 TFH 세포에서 유전자 발현을 조작하는 방법도 제공합니다. 따라서 이러한 방법은 바이러스 특이적 TFH 세포의 조기 투여의 기저에 있는 메커니즘을 탐색하는 연구에 도움이 될 것입니다.

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.

저자 스포트라이트: 급성 LCMV 문제에서 TFH 세포 분화의 경로 규명

급성 LCMV에 감염된 마우스에서 바이러스 특이적 여포 도우미 CD4+ T 세포의 조기 분화에 접근

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.

급성 LCMV 감염 중 조기 분화 바이러스 특이적 TFH 세포의 특성 바이러스 특이적 TFH 세포의 초기 운명 헌신을 조사하기 위해, LCMV GP 에피토프 I-AbGP66-77을 특이적으로 인식하는 naive congenic SM CD4+ T 세포를 CD45.2+ C57BL/6 수용자에게 입양 전달했습니다. 다음 날, 이 수혜자들은 급성 분해된 LCMV Armstrong의 고용량으로 정맥 주사로 감염되었습니...

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

현재 연구는 바이러스 특이적 TFH 세포의 조기 운명 헌신을 평가하고 이러한 세포에서 유전자 발현을 조작하기 위한 프로토콜을 보여줍니다.

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

436 조회수.  Shenzhen University. 당사의 연구는 급성 LCMV에 감염된 단클론 마우스 모델 구축, 유세포 분석 염색 수행 및 레트로바이러스 벡터 기반 유전자 조작 수행을 포함하여 바이러스 특이적 TFH 세포의 조기 운명 헌신을 평가하기 위한 재현 가능한 방법을 설명합니다. 체외 T세포 자극과 비교했을 때, 체내 자극은 T세포를 12시간 내에 더 빠르게 활성화할 수 있으며, 다음 레트로?...

비디오: 저자 스포트라이트: 급성 LCMV 문제에서 TFH 세포 분화의 경로 규명

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, thus establishing long-term humoral immunity. During acute viral infection, the fate commitment of virus-specific TFH cells is determined in the early infection phase, and investigations of the early-differentiated TFH cells are crucial in understanding T cell-dependent humoral immunity and optimizing vaccine design. In the study, using a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection and the TCR-transgenic SMARTA (SM) mouse with CD4+ T cells specifically recognizing LCMV glycoprotein epitope I-AbGP66-77, we described procedures to access the early fate commitment of virus-specific TFH cells based on flow cytometry stainings. Furthermore, by exploiting retroviral transduction of SM CD4+ T cells, methods to manipulate gene expression in early-differentiated virus-specific TFH cells are also provided. Hence, these methods will help in studies exploring the mechanism(s) underlying the early commitment of virus-specific TFH cells.

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

연구

면역학 및 감염병학

Mapping the Early Development of LCMV-Specific Follicular Helper T-Cells in Mice

To begin, obtain a six to eight week old CD 45.1 positive SMARTA mouse and inject it with 200 micrograms of LCMV GP 61-77 peptide in 100 microliters of RPMI medium intravenously. After acquiring the splenocytes from the mouse, suspend them in 2%RPMI to achieve a cell density of one times 10 to the eighT-cells per milliliter and place the tube with cells on ice. Dispense 50 microliters of cell suspension along with 150 microliters of staining buffer to each well of a round-bottom 96-well plate.Centrifuge the 96-well plate at 800 G for one minute at four degrees Celsius and carefully decant the supernatant. Vortex the plate and add 200 microliters of staining buffer to each well. After pelleting the cells, resuspend and incubate them with surface antibody cocktail on ice for 30 minutes in the dark.After washing two times, resuspend the cells in 200 microliters of staining buffer and transfer them into a flow cytometry tube. Perform flow cytometry analysis to check the activation status of SMARTA CD4 positive T-cells. Then seed one times 10 the six SMARTA CD4 positive T-cells per well into a 24 well plate.Spin down the cells at 800 G for three minutes at four degrees Celsius and carefully remove the supernatant medium. Next, add one milliliter of retrovirus medium and eight micrograms of polybrene to each well. Spin transduce the cells at 800 G for two hours at 37 degrees Celsius.After discarding the retrovirus medium, incubate the cells with one milliliter of prewarmed 10%RPMI supplemented with interleukin 2. Transfer the cell suspension into a 15 milliliter conical tube and centrifuge it. After discarding the supernatant, resuspend the cells with 2%RPMI to achieve a cell density of one times 10 to the eighth cells per milliliter.For assessing the transduction efficiency, aliquot 50 microliters of cell suspension into a round bottom 96-well plate. Incubate them on ice with the surface antibody cocktail including CD4, V alpha two, and vector tag-associated antibody. Wash the cells and perform flow cytometry as demonstrated earlier.Then inject one times 10 of the six CD 45.1 positive SMARTA CD4 positive T-cells to a C57BL/6 recipient mouse intravenously followed by one times 10 to the six plaque forming units of LCMV Armstrong the next day. After acquiring the splenocytes from the mouse, stain them to check the desired markers. To detect transcriptional factors, incubate the cells with 200 microliters of 2%paraformaldehyde at room temperature for 20 minutes in the dark.Finally, perform flow cytometry to detect factors at the early acute LCMV infection stage. On day three after infection, a balanced bifurcation of follicular helper T-cells and T-helper 1 cells Among MIGR1 SMARTA CD4 positive T-cells was found. A predominant follicular helper T-cell directed differentiation and enhanced BCL6 expression levels were observed in MIGR1 BCL6 SMARTA CD4 positive T-cells.