This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2015R1A6A3A01020610 to HJP) and a grant from the Korean Health Technology R&amp;D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (HI15C0493 to SJH).

<ol>
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S.-J.H. has a patent and receives patent royalties related to the PD-1 pathway. The other authors have no financial conflicts of interest.

على الرغم من مجرد عدد قليل من الخلايا ريج T موجودة في الفئران والبشر، فمن المهم أن نفهم وظيفة لأنها تلعب دورا حاسما في تنظيم الاستجابة المناعية والحفاظ على التسامح المناعي. عدد والقمعية وظائف تي ريج خلايا زيادات خلال عدوى فيروس المزمن 15-20 وكذلك سرطان التقدم 13،14. هذا ربما يرجع إلى استمرار التحفيز مستضد. لتقييم خلايا تي ريج تعمل تحت استمرار مستضد وتطور المرض، يحتاج إلى قياس نشاطهم القمعية. هنا، نحن تصف بروتوكول لتحليل النمط الظاهري خارج الحي من خلايا تي ريج وقياس نشاطهم القمعية باستخدام نظام المشاركة في ثقافة في المختبر من CD8 + T والخلايا ريج T. الخطوات الحاسمة في البروتوكول الحالي هي التحليل وعزل الخلايا T ريج. حية وطازجة معزولة T <subتظهر&gt; خلايا ريج النشاط القمعية واضح في المختبر. ونحن أيضا فحص الظواهر تي التحويل وخلايا T ريج خلال عدوى فيروس المزمن. خلايا تي التحويل، أي Foxp3 - أظهرت CD4 + و CD8 + T خلايا انخفاض في النشاط الخلوي بعد عدوى فيروس المزمن (الشكل 1). وقد upregulated التعبير PD-1 في كل من السكان الخلايا التائية التحويل. زيادة في عدد الخلايا التائية ريج ويصل تنظيم PD-1 التعبير هي السمة المميزة لاستجابة جهاز المناعة خلال عدوى فيروس المزمن. بالإضافة إلى ذلك، المضادة للهيئات لالمستقبلات المثبطة الأخرى مثل TIM-3 و CTLA-4 يمكن استخدامها في تركيبة مع PD-1 لدراسة الخلية النمط الظاهري T من نظام مراقبة الأصول الميدانية بعد عدوى فيروس choric. من أجل التحقيق في نشاط القمعية، كانت خلايا تي التن وخلايا T ريج شارك في تربيتها في نسبة 1 إلى 1. التعاون مع ثقافة تي ريجالخلايا من الفئران المصابة بشكل مزمن إلى خفض كبير CD8 + T تكاثر الخلايا وإفراز الإنترفيرون جاما بالمقارنة مع خلايا ريج T من الفئران ساذجة. وهذا يعني أن إنتاج انتشار وخلوى الخلايا CD8 + T شارك في تربيتها مع خلايا تي ريج ترتبط عكسيا مع وظيفة القمعية الخلايا ريج T. على الرغم من أن هذا البروتوكول يوصي استخدام نظام فصل الخلية المغناطيسي للزنزانة انفرادية تي ريج، التلوث مع خلايا ريج غير T-لا يمكن استبعاده. خلايا تي ريج تم الحصول عليها من Foxp3-GFP الفئران مراسل 28-30 هي أفضل المرشحين للتحقيق بدقة وظيفة خلايا تي ريج من نظام فصل الخلية المغناطيسي، وغالبا ما هى overexpressed CD25 على CD4 + T التحويل وكذلك خلايا ريج T تفعيلها. تم تقييم وظيفة القمعية الخلايا ريج T من مختلف في المختبرالمقايسات قمع 16،28،31-34. ميزة في المختبر قمع الفحص هو أنه يقيم التأثير المباشر للخلايا T ريج على تثبيط استجابة الخلايا CD8 + T، فقط لأن الخلايا التركيب تي هي شارك في تربيتها مع خلايا تي ريج. بالإضافة إلى خلايا CD8 + T، وCD25 - خلايا CD4 + T يمكن استخدامها بدلا من خلايا تي التن 25. آثار تي ريج أو خلايا تي التحويل على الخلايا المناعية مثل الخلايا الجذعية أو خلايا القاتل الطبيعي يمكن تقييم من خلال تغيير الظروف التجريبية. جزيئات مثل CD103 35، LAG-3 36، بروتين سكري والتكرار تسود 37، CD43 38، CD11a 38 أو 1 38 استخدمت الخلايا القاتلة مثل كتين مستقبلات فصيلة عضوا G كعلامات الخلايا ريج T تفعيلها في أمراض معينة. في هذا البروتوكول، وكنا PD-1 كمؤشر لتحديد تنشيط خلايا T ريج خلال عدوى فيروس المزمن. هذا البروتوكول يمكن أن تستخدم لتحليلات متعددة الأوجه (المظهري وظيفية) من خلايا تي ريج في ظل ظروف محددة.

التنظيمية خلايا تي (T ريج) تعبر عن P3 مربع forkhead (Foxp3) باعتباره عامل النسخ لتنميتها وظيفة 1. بالإضافة إلى ذلك، خلايا تي ريج تعبر عن مختلف الجزيئات الأخرى مثل CD25 2، اللمفاويات التنشيط الجيني 3 (LAG-3) 3، الناجم عن جلايكورتيكود عامل نخر الورم مستقبلات 4، والسامة للخلايا اللمفاوية التائية-بروتين يرتبط 4 (CTLA-4) 5 على سطحها أو منطقة داخل الخلايا. خلال العدوى المزمنة مع أنواع مختلفة من مسببات الأمراض مثل الفيروسات 6،7، 8،9 البكتيريا، والطفيليات 10-12، أو في سياق تطور مرض السرطان 13،14، تصبح خلايا T ريج متباينة في خلايا تفعيلها، تظهر تعزيز وظيفة القمعية خلايا تستهدف المستجيب CD4 + و CD8 + T. وقد اقترح عدد من الأوراق أن الخلايا ريج T توسيع وتنشيط تساهم في ضعف في رد و الخلايا CD8 + Tالبريد خلال صديق الارتجاعي (اناث) عدوى 15-17. الناجم FV-خلايا تي ريج تمنع IFN-γ أو التعبير باء granzyme للتفاعل السامة للخلايا من خلايا CD8 + T 15-17. وعلاوة على ذلك، في الهربس البسيط نموذج عدوى فيروس، أفيد أن استنفاد الخلايا ريج CD4 + CD25 + T أدى إلى توسيع الخلايا CD8 + T الفيروس محددة وتلف الأنسجة الشديد نتيجة لتسلل immunopathogenic خلايا CD4 + T 18-20. الفئران المصابة بشكل مزمن مع سلالة استنساخ 13 من فيروس المشيمي اللمفاوي (LCMV CL13) 21-24 وقد استخدمت على نطاق واسع لوصف النمط الظاهري وظيفة الخلايا التائية المستجيب (T EFF) والخلايا ريج T خلال عدوى فيروس المزمن. خلال العدوى LCMV المستمر، الفيروس محددة الخلايا التائية ممثل المؤسسة يفقد تدريجيا وظيفة المستجيب وأصبحت منهكة خلايا تي (T EXH). من ناحية أخرى، Tخلايا ريج تعزز قدرتها على قمع الفيروس محددة تي استجابة الخلية 25. انخفاض في قدرة فاعلة من الخلايا ممثل المؤسسة T يمكن تفسيرها من خلال عدة عوامل مثل upregulation من المستقبلات المثبطة على خلايا ممثل المؤسسة T، وظيفة تغيير الخلايا العارضة للمستضد، وإنتاج السيتوكينات immunoregulatory، وزيادة تواتر أو تعزيز وظيفة تي ريج خلايا 26. ومن بين العوامل التي تدخل في تي قمع الخلايا، موت الخلية المبرمج البروتين 1 (PD-1) واعتبرت على نطاق واسع -expressing خلايا EXH تي وخلايا T ريج باعتبارها من أهم دعامات استمرار مستضد والبيئة القمعية. في الآونة الأخيرة، أفيد أن الحصار المفروض على مسار PD-1 والاجتثاث من خلايا ريج T يؤدي إلى تعزيز وظيفة الخلايا التائية وانخفض الحمل الفيروسي خلال LCMV عدوى مزمنة 27. وعلاوة على ذلك، يتم تنشيط خلايا T ريج خلال العدوى المزمنة من الفئران مع LCMV 23،25 </sتصل&gt; وتعزيز وظيفتها القمعية 25. وأعربت PD-1 كبير على خلايا T ريج وكذلك خلايا T EXH، ومستوى PD-1 أعرب من قبل خلايا ريج T يرتبط مع قوة من وظيفتها القمعية لمنع انتشار الخلايا التائية 25. هنا، نحن تصف طريقة لمقارنة خصائص الخلايا ريج T تنشيط معزولة من الفئران المصابة مع LCMV CL13 ويستريح خلايا تي ريج معزولة من الفئران ساذجة. وعلاوة على ذلك، نفسر سلسلة من العمليات لفصل تنشيط خلايا T ريج ودراسة على النمط الظاهري خارج الحي، وكذلك قياس النشاط القمعية في المختبر.

<table border="0" cellpadding="0" cellspacing="0" width="976">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">FITC Rat Anti-Mouse CD4</td>
			<td style="width:67px;">RM4-5</td>
			<td style="width:173px;">BD Biosciences</td>
			<td style="width:104px;">553047</td>
			<td style="width:249px;">Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytofix/Cytoperm</td>
			<td></td>
			<td>BD Biosciences</td>
			<td>554714</td>
			<td>Use this reagent for cell surface staining.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">U-Bottom Tissue Culture Plates</td>
			<td></td>
			<td>BD Biosciences</td>
			<td>353077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fixation buffer</td>
			<td></td>
			<td>BD Biosciences</td>
			<td align="right">554655</td>
			<td>Use this reagent for cell surface staining.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC Rat Anti-Mouse CD25</td>
			<td>7D4</td>
			<td>BD Biosciences</td>
			<td>553072</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell strainer, 70mm</td>
			<td></td>
			<td>BD Biosciences</td>
			<td>352350</td>
			<td>Use this strainer for grinding the whole spleen.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell strainer, 40mm</td>
			<td></td>
			<td>BD Biosciences</td>
			<td>352340</td>
			<td>Use this strainer for filtering the cells before column enrichment.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brilliant Violet 421 Anti-mouse CD279 (PD-1)</td>
			<td>29F.1A12</td>
			<td>BioLegend</td>
			<td>135217</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brilliant Violet 605 Anti-Mouse CD4</td>
			<td>RM4-5</td>
			<td>Biolegend</td>
			<td align="right">100547</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">APC Anti-Mouse/Rat Foxp3&#160;</td>
			<td>FJK-16s</td>
			<td>eBioscience</td>
			<td>17-5773</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Foxp3 / Transcription Factor Staining Buffer Set</td>
			<td></td>
			<td>eBioscience</td>
			<td>00-5223</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PerCP-Cyanine5.5 Anti-Mouse CD8a</td>
			<td>53-6.7</td>
			<td>eBiosicence</td>
			<td>45-0081</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse IFN-gamma Platinum ELISA</td>
			<td></td>
			<td>eBiosicence</td>
			<td>BMS606</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td></td>
			<td>GE Life Sciences</td>
			<td>SH30027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS (1X)</td>
			<td></td>
			<td>GE Life Sciences</td>
			<td>SH30256</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ACK Lysing Buffer</td>
			<td></td>
			<td>Gibco</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine, 200mM solution</td>
			<td></td>
			<td>Gibco&#160;</td>
			<td>25030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin, 10,000U/mL</td>
			<td></td>
			<td>Gibco&#160;</td>
			<td>10378-016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit</td>
			<td></td>
			<td>Life technologies</td>
			<td>L-34975</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 500X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD8a+ T Cell Isolation Kit, mouse</td>
			<td></td>
			<td>Miltenyibiotec</td>
			<td>130-104-075</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD4+CD25+ Regulatory T Cell Isolation Kit, mouse</td>
			<td></td>
			<td>Miltenyibiotec</td>
			<td>130-091-041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACS Separation Columns, LD columns</td>
			<td></td>
			<td>Miltenyibiotec</td>
			<td>130-042-901</td>
			<td>Use this column for Treg cell isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACS Separation Columns, LS columns</td>
			<td></td>
			<td>Miltenyibiotec</td>
			<td>130-042-401</td>
			<td>Use this column for CD8+ T cell and Treg cell isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA, 0.5M (pH 8.0)</td>
			<td></td>
			<td>Promega</td>
			<td>V4231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercaptoethanol</td>
			<td></td>
			<td>Sigma Life Science</td>
			<td>M7522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td></td>
			<td>Thermo Fisher Scientific</td>
			<td>SH30919.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTrace Violet Cell Proliferation Kit</td>
			<td></td>
			<td>Thermo Fisher Scientific</td>
			<td>C34557</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Canto II flowcytometer</td>
			<td></td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometer*</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flowjo</td>
			<td></td>
			<td>TreeStar</td>
			<td></td>
			<td>Flow cytometry software&#8224;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hematocytomer</td>
			<td></td>
			<td>Marienfeld superior</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

phenotypic and functional analysis of activated regulatory t cells isolated from chronic lymphocytic choriomeningitis virus-infected mice

التنظيمية تي (T ريج) الخلايا، التي تعبر عن Foxp3 كعامل النسخ، هي مجموعات فرعية من خلايا CD4 + T. خلايا تي ريج تلعب أدوارا حاسمة في التسامح المناعة، وصيانة التوازن من خلال تنظيم الاستجابة المناعية. الدور الأساسي للخلايا ريج T هو قمع انتشار المستجيب تي (T EFF) الخلايا وإنتاج السيتوكينات مثل IFN-γ، TNF-α، و IL-2. وقد ثبت أن قدرة خلايا T ريج &quot;لمنع وظيفة الخلايا ممثل المؤسسة تي تتعزز خلال العدوى الممرض استمرار وتطور مرض السرطان. لتوضيح وظيفة الخلايا ريج T تحت يستريح أو الملتهبة الظروف، ومجموعة متنوعة من فحوصات في المختبر قمع باستخدام الماوس أو تي الخلايا البشرية ريج وقد وضعت. والهدف الرئيسي من هذه الدراسة هو تطوير طريقة لمقارنة الاختلافات في النمط الظاهري وظيفة القمعية بين الراحة والمفعلين تي ريجالخلايا. لعزل تنشيط خلايا T ريج، والفئران المصابة مع الليمفاوي فيروس التهاب السحايا المشيمي (LCMV) استنساخ 13 (CL13)، سلالة مزمن في LCMV. خلايا تي ريج معزولة عن الطحال من الفئران المصابة CL13-LCMV أظهرت كل من النمط الظاهري تنشيط وتعزيز النشاط القمعية مقارنة مع يستريح خلايا ريج تي المعزولة من الفئران ساذجة. هنا، نحن تصف البروتوكول الأساسي للخارج الحي تحليل النمط الظاهري للتمييز تنشيط خلايا T ريج من يستريح خلايا تي ريج. وعلاوة على ذلك، نحن تصف بروتوكول لقياس النشاط القمعية من خلايا ريج T تشغيلها بالكامل.

ولدت لنا الفئران مع عدوى فيروس المستمر من قبل حقنها بمادة 2 × 10 6 PFU من LCMV CL13 عن طريق الوريد. ولبحث التغيرات المظهرية في الخلايا ريج T و T التحويل الخلايا خلال عدوى فيروس المزمن، كانت ملطخة الخلايا الليمفاوية الطحال تم الحصول عليها من الفئران ساذجة والمصابة مع مختلف الأجسام المضادة وتحليلها بواسطة التدفق الخلوي. في 16 د بي، وupregulated PD-1 في كل من Foxp3 - CD4 + T التحويل (الشكل 1A، اللوحة العليا) وFoxp3 - خلايا CD8 + T التحويل (1B الشكل، لوحة العليا). وكان تردد من خلايا ريج Foxp3 + CD4 + T العالي مرتين في الفئران المصابة CL13-LCMV مما كانت عليه في الفئران ساذجة (الشكل 1A). على وجه الخصوص، أكثر من Foxp3 + الخلايا ريج CD4 + T عرض النمط الظاهري تنشيط، معربا عن مستويات عالية منPD-1 في هذه المرحلة الزمنية (الشكل 1A). لفي المختبر قمع الفحص، ويتطلب عددا كبيرا من خلايا التحويل CD8 + T والخلايا ريج CD4 + CD25 + T. للحصول على 1 × 10 7 خلية تتبع انتشار البنفسجي خلايا CD8 + T المسمى صبغ، وكان يتم تجميع splenocytes من اثنين من الطحال من الفئران ساذجة. للحصول على 2 × 10 6 أو 3 × 10 6 خلايا ريج CD4 + CD25 + T، لا يقل عن ثلاثة الطحال من الفئران ساذجة والمصابين CL13-LCMV، على التوالي، وكان يتم تجميع. تم فصل خلايا CD8 + T والخلايا التركيب تي بنجاح دون تلوث كبير مع الخلايا المناعية الأخرى (الشكل 2A). ويمكن أيضا أن تكون معزولة خلايا تي ريج، ويبلغ عدد سكانها السائد للخلايا CD25 + CD4 + T مع أكثر من 80٪ النقاء (الشكل 2B). لمقارنة وظيفة القمعية من خلايا تي ريج ساذجة والمزمنة، وحضنت يموزين تي معزولة والخلايا التركيب تي معا تحت التحفيز مع حبات مكافحة CD3 / المغلفة CD28 عند 37 درجة مئوية لمدة 3 أيام. وزادت٪ تثبيط CD8 + T تكاثر الخلايا التركيب في تي ريج خلية تعتمد DESE-الطريقة (الشكل 3A). عندما كانت CD8 + T الخلايا التركيب شارك في تربيتها مع خلايا تي ريج المزمنة في نسبة 1: 1، وتثبيط تكاثر الخلايا التركيب CD8 + T بشكل ملحوظ (الشكل 3B). كان إنتاج الإنترفيرون γ الخلايا التركيب CD8 + T أيضا تحول دون بشكل ملحوظ عندما كانت CD8 + الخلايا التركيب T شارك في تربيتها مع تنشيط خلايا T ريج من الفئران المصابة بشكل مزمن وليس مع يستريح خلايا ريج T من الفئران السذاجة في ريج T </فرعية&gt; خلية تعتمد على الجرعة بطريقة (الشكل 3C). <img alt="شكل 1" src="/files/ftp_upload/54138/54138fig1.jpg" /> الشكل 1: الظواهر من خلايا CD4 + و CD8 + T في الطحال من الفئران ساذجة أو الفئران المصابة CL13-LCMV في 16 د بي (A) التعبير عن PD-1 أو CD25 على Foxp3 - CD4 + T التحويل وFoxp3 + CD4 + خلايا تي ريج. (ب) التعبير عن PD-1 أو CD25 على Foxp3 - CD8 + T خلايا التحويل. كانت ملطخة الخلايا الليمفاوية الطحال مع الأجسام المضادة ضد CD4، CD8، CD25، PD-1، وFoxp3. البيانات هي ممثل لثلاث تجارب مستقلة. لوحة وتم تعديلها من 25 كمرجع. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54138/54138fig1large.jpg" target="_blank">يرجى النقر</a><html> هنا لعرض نسخة أكبر من هذا الرقم. <img alt="الشكل 2" src="/files/ftp_upload/54138/54138fig2.jpg" /> الشكل 2: النقاوات الخلايا التركيب تي المعزولة من الفئران ساذجة والخلايا ريج تي المعزولة من الفئران ساذجة أو المصابين CL13-LCMV (أ) النسبة المئوية للخلايا CD8 + T بعد العزلة. (ب) النسبة المئوية للخلايا CD4 + T ريج، معربا عن CD25 بعد العزلة. تم تحديد كل رباعي في الفقرة (ب) من خلال مستويات التعبير عن CD4 وCD25. البيانات هي ممثل لثلاث تجارب مستقلة. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54138/54138fig2large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a> ليه / ftp_upload / 54138 / 54138fig3.jpg &quot;/&gt; الشكل (3): تأثير خلايا T ريج تفعيلها على استجابة الخلايا CD8 + T (A)٪ تثبيط خلايا CD8 + T شارك في تربيتها مع خلايا تي ريج بطريقة تعتمد على الجرعة الخلايا التائية ريج. (ب) انتشار خلايا CD8 + T شارك في تربيتها مع الخلايا ريج تي في نسبة 1: 1. (C) IFN-γ إنتاج خلايا CD8 + T شارك في تربيتها مع خلايا تي ريج بطريقة تعتمد على الجرعة الخلايا التائية ريج. وقد تم قياس انتشار الخلايا CD8 + T من التخفيف من انتشار الخلايا تتبع البنفسجي الصبغة في تكاثرت الخلايا CD8 + T وإفراز الإنترفيرون γ تم تقييمها من قبل ELISA. وحفز تكاثر الخلايا تتبع البنفسجي خلايا CD8 + T المسمى صبغ مع anti-CD3 / حبات المغلفة CD28 لمدة 3 أيام في حالة غياب أو وجود T <sخلايا يو بي&gt; ريج معزولة عن إما الفئران ساذجة أو إصابة مزمنة. في الرسم البياني، والأرقام دون ومع قوسين يشير إلى نسبة انتشار الخلايا CD8 + T شارك في تربيتها مع وبدون خلايا تي ريج، على التوالي. شغل قمم الرمادية في الرسم البياني تشير إلى انتشار خلايا CD8 + T شارك في تربيتها دون خلايا تي ريج. وقد تم تصميم كل مجموعة في يثلث. تمثل القضبان يعني + ووزارة شؤون المرأة. تم تعديل هذا الرقم من 25 كمرجع. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54138/54138fig3large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a>

Watch this Scientific Journal Video about Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice at JoVE.com

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

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.

المظهرية والتحليل الوظيفي من المنشط خلايا تنظيم تي المعزولة من الفئران المصابة بفيروس المزمنة الليمفاوي المشيمي

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 ...

phenotypic-functional-analysis-activated-regulatory-t-cells-isolated

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

9.7K Views.  Yonsei University. Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from naïve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

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

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

جيل من الخلايا المستحثة تي التنظيمية من المرحلة الابتدائية الإنسان خلايا T ساذجة والذاكرة

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

أدوات جديدة لتوسيع الخلايا التائية التنظيمية من الأفراد HIV-1-المصابة

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.
Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

حقن النظامية من الخلايا الجذعية العصبية / السلف في الفئران مع EAE المزمن

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

اشتقاق خلايا T<em&gt; في المختبر</em&gt; من الفأر الجنينية الخلايا الجذعية

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

توليد خلايا T الإنسان مستضد خيفي محددة من الدم المحيطي

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

عزل الخلايا اللمفاوية من الجلد البشري لتحليل المظهري و<em&gt; فيفو السابقين</em&gt; زراعة الخلايا

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

T خلايا التقاط البكتيريا التي كتبها Transinfection من الخلايا الجذعية

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSV&#39;s effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.

A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

تحديد بيوفيلم بدء في الخلايا المصابة بالفيروسات من قبل البكتيريا والفطريات

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the allograft early after transplantation, mount a graft reactive response against the transplanted organ, and initiate organ rejection2. Recent data suggest that suppressive macrophages facilitate successful long-term transplantation3 and are required for the induction of transplantation tolerance4. This suggests a multidimensional concept of macrophage ontogeny, activation, and function, which demands a new roadmap for the isolation and analysis of macrophage function5. Due to the plasticity of macrophages, it is necessary to provide a methodology to isolate and characterize macrophages, depending on the tissue environment, and to define their functions according to different scenarios. Here, we describe a protocol for immune characterization of graft-infiltrating macrophages and the methods we used to functionally evaluate their capacity to inhibit CD8+ T proliferation and to promote CD4+Foxp3+ Treg expansion in vitro.

Macrophages are plastic cells of the hematopoietic system that have a crucial role in protective immunity and homeostasis. In this report, we describe optimized in vitro techniques to phenotypically and functionally characterize graft-infiltrating regulatory macrophages that accumulate in the transplanted organ under tolerogenic conditions.

توصيف وظيفي من الضامة التنظيمية التي تمنع مناعة الكسب غير المشروع

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.