この作品は、オーストラリア国立保健医療研究評議会、肝炎やHIVのウイルス学EOIのためのオーストラリアのセンターから＃1010395（BQとCP）と＃525431（CR）、プログラムグラント＃455395（CP）を付与し、プロジェクトによってサポートされていました2012助成金（CRとRJJ）とゴードンとグレーテルうしかい座財団（BQとCR）からの助成金。我々はJCSMRのFACS研究室のその優れたメンテナンスのためHarpreet Vohra著、マイケルデヴォイに感謝したい、ペプチド合成のためのオーストラリアのがん研究財団生体分子資源施設、JCSMR、ANU、およびデビッド·ボイル、CSIROのアニマルヘルス研究所、ジーロン、オーストラリア親HIVワクチン株を提供するため。

<ol>
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著者は、彼らが競合する経済的利益を持っていないことを宣言します。

FTAに基づくアッセイの利点は、フローサイトメトリーによる単一の宿主動物から&gt; 250生存および完全に機能する標的細胞集団の識別を可能にすることである。これは以前には不可能であったin vivoでのフローサイトメトリーベースのアッセイに複雑さのレベルを提供します。これは、6の濃度で7の異なるウイルスエピトープに対する応答は、AUCおよびEC 50などのパラメータは<em...

T細胞は、適応免疫応答において中心的な役割を果たし、多くの場合、免疫療法における操作のために標的化される。 CD4 +エフェクターT細胞が免疫の多くの局面を調節し、直接抗体を製造するためにB細胞を助けることができるサイトカインを分泌することによって外来抗原に応答する。 CD8 +細胞傷害性T細胞（CTL）は、サイトカインを分泌すること、ならびに直接に外来抗原を発現する細胞を死滅させる中心的な役割を再生することにより、外来抗原に応答することができる。これらのT細胞エフェクター機能を開始する基本的な相互作用は、細胞表面上のMHC分子上に表示された外来ペプチドとT細胞受容体（TCR）との相互作用を伴う。 CD4 + T細胞は、抗原提示細胞上のMHCクラスII分子上に提示されるペプチドを認識し、CD8 + T細胞は、典型的には、微生物に感染した細胞上に表示されるMHCクラス-I分子上に提示されるペプチドを認識する。 順番にT細胞は免疫応答において果たす役割を評価するためには、それらのエフェクター機能が確実かつ敏感な技術によって測定することが必須である。 T細胞応答の評価のための一般的な方法としては、 MHC class-I/II/peptide四量体の反応性; ELISPOTおよび細胞内サイトカイン染色によるサイトカイン産生; 51 Cr放出アッセイによって容量を殺す。これらのアッセイは、しかしながら、典型的には、 インビトロ刺激によりエクスビボで行うか、またはT細胞機能に制限された洞察を提供している。 T細胞応答を測定する際に、発生したin vitro刺激によって起こり得る機能的パラメータの変化を回避するように理想的には、T細胞の無操作に、in vivoで、in situでそれらを評価することが有益であろう。最も一般的にインビボで T細胞機能アッセイにおいて使用されるのいくつかは、インビボで列挙されるMHCクラスI結合ペプチドをパルスした標的細胞のCTL媒介死滅を測定することに基づいているこのようCFSEなどの重要な染料で蛍光標識を通じて検出による。これらのタイプのアッセイは、それらがインビボで起こる場合、それらは以前に定性的なパラメータは、例えば可能にするために必要とされる、異なる濃度のペプチドエピトープの異なるタイプを提示する複数の標的の死滅を評価するための比較的限られた能力を持っていた標的のCTL媒介死滅を監視することができるがなどの官能アビディティおよびエピトープ変異体の交差反応性を評価することができる。これらのアッセイはまた、CD4 + T細胞媒介応答に関するあらゆる情報を提供しない。 T細胞応答を評価するために使用される現在の方法で制限の多くを克服するために、我々は最近ずつの動物に同時に&gt; 250標的細胞に対するT細胞応答のモニタリングを可能にする蛍光性標的アレイ（のFTA）に基づいて、多重アッセイを開発した1、2フローサイトメトリー。自由貿易協定は、SEVで標識されたリンパ球から構成されているERAL濃度およびCFSE、CTV及びCPDのような生体染色色素の組み合わせは、ユニークな蛍光の&gt; 250細胞クラスターが生成されることを可能にする。これらの細胞は生存可能で、完全に機能的なままであるので、それらは、インビボ3におけるエフェクターT細胞との相互作用のモニタリングを可能にするために動物に注射することができる。例えば、FTA細胞クラスターは、標的細胞1の抗原特異的CTL媒介死滅の評価を可能にするためにMHCクラス-I結合性ペプチドでパルスすることができる。また、FTA細胞クラスタはまた、CD69、CD44および/ ​​または活性化マーカーを評価することによって（活性化を評価することによって、抗原特異的Tヘルパー細胞（T H）活性の評価を可能にする、MHCクラス-II結合性ペプチドでパルスすることができる同族ペプチド2を有するFTA内のB細胞のCD62L）。 250以上の標的を同時に検出することができるので、n個でパルス多くの標的細胞クラスタに対するCTLおよびT H応答を測定することが可能である異なる濃度や多くの反復を含めることでumerousペプチド。 FTAのアッセイは、したがって、in vivoでのT細胞エフェクター応答評価の前例のないレベルを提供します。 ここでは、詳細にFTAの構成を説明し、それらはin vivoで T細胞応答の評価に適用することができる方法を示す。手順は、MHCクラスIおよびII結合ペプチドをパルスした42細胞クラスターの6反復から成る3の重要な染料を使用することで252識別可能な細胞クラスターで構成される自由貿易協定（FTA）の構築を記載する。標識の量を減少させることによって、必要に応じて42細胞クラスターの標識は、チューブ底の10mlの円錐形で発生し、それを表1に示すように管ラックにこれらを配置することが有用である。この方法は、識別可能なクラスタの少ない数のために調整することができる各色素の1を行った。 我々は、それが的応答を測定することができますどのように示すことによって、アッセイの有用性を強調マウスの小さなコホートにおける複数のエピトープに対する組換えポックスウイルスワクチン接種によって生成されたES。これは、FTAアッセイは、それぞれを通る曲線（AUC）の評価および半最大応答（EC 50）を生成するために必要な効果的なペプチド濃度の被測定領域の使用を累積応答および機能的結合活性を測定するために使用することができる方法を示している。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">RPMI</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">R8758</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Fetal calf serum</td>
			<td style="width:71px;">Serana</td>
			<td style="width:119px;">FCS-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">CTV</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">C34557</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">CFDA, SE</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">C1157</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">CPD</td>
			<td style="width:71px;">eBioscience</td>
			<td style="width:119px;">65-0840-90</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">anti-B220 PerCp-Cy5.5</td>
			<td style="width:71px;">eBioscience</td>
			<td style="width:119px;">110730</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">anti-CD69 brilliant violet 605</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">104529</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">PKH-26</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">PKH26GL-1KT</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">EQUIPMENT:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Material Name</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalogue Number</td>
			<td style="width:167px;">Comments (optional)</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Vortex</td>
			<td style="width:71px;">Scientific Industries Inc&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Centrifuge</td>
			<td style="width:71px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Flow cytometer (Fortessa or equivalent with blue, red and violet laser source)</td>
			<td style="width:71px;">BD Bioscience</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

the use of fluorescent target arrays for assessment of t cell responses in vivo

in vivoで T細胞応答をモニターする能力は、免疫応答および免疫療法の設計の我々の理解の開発のために重要である。赤色レーザ励起可能染料（細胞増殖色素eFluor 670：CPD：ここでは、そのようなカルボキシフルオレセインスクシンイミジルエステル（CFSE）などの重要な染料を利用した蛍光ターゲット配列（FTA）技術、紫色レーザー励起可能染料（CTV CellTraceバイオレット）の使用を記載）&gt; 250識別可能な蛍光細胞のクラスタにラベルをマウスリンパ球を、組み合わせた。これらのFTA内の細胞クラスタは、主要組織適合性複合体（MHC）クラスIおよびMHCクラスII結合ペプチドでパルスし、それによって、それぞれCD8 +およびCD4 + T細胞のための標的細胞として作用することができる。これらのFTA細胞は生存可能で、完全に機能的なままであり、したがって、FTA標的細胞およびCD4 + T細胞瞑想HELのCD8 + T細胞媒介死滅の評価を可能にするためにマウスに投与することができるフローサイトメトリーによるインビボでリアルタイムにFTA B細胞標的細胞におけるp。 &gt; 250標的細胞を一度に評価することができるので、この技術は、いくつかの濃度では、複数の反復のいくつかの抗原エピトープに対するT細胞応答のモニタリングを可能にする。このように、この技術は、定量的（ 例えば、累積的な応答の大きさ）および定性的（ 例えば、機能性アビディティおよび応答のエピトープの交差反応性）レベルの両方でT細胞応答を測定することができる。ここで、我々はこれらのFTAを構築する方法を記述し、それらは、組換えポックスウイルスワクチンによって誘導されるT細胞応答を評価するために適用することができる方法の例を与える。

FTAアッセイの使用の一例として、BALB / cマウスを、HIV-I CTLエピトープにHIV-Iエピトープ（VV-HIV）と応答を発現する組換えワクシニアウイルス（VV）で免疫化したGag、ギャグたmut、のEnvおよびPol、VV CTLエピトープF2LとのF2Lたmut、およびHIV-I T H細胞エピトープ 、Thをギャグ（2に記載のように）252パラメータFTAアッセイ（ 図1B）を使用して評価した。ギャグ（ギ...

Watch this Scientific Journal Video about The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo at JoVE.com

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

インビボで詳細に T細胞応答をモニターする能力は、免疫応答の我々の理解の開発のために重要である。ここでは、フローサイトメトリーで同時に&gt; 250のパラメータを評価するin vivoでの T細胞アッセイにおける蛍光の標的配列（のFTA）の使用を記載している。

T細胞応答の評価のための蛍光ターゲット配列の使用<em&gt;インビボ</em

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

the-use-fluorescent-target-arrays-for-assessment-t-cell-responses

Research

JoVE Journal

Immunology and Infection

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

14.3K ビュー.  Australian National University.  インビボで詳細に T細胞応答をモニターする能力は、免疫応答の我々の理解の開発のために重要である。ここでは、フローサイトメトリーで同時に> 250のパラメータを評価するin vivoでの T細胞アッセイにおける蛍光の標的配列（のFTA）の使用を記載している。 

ビデオ: The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

細菌付着の研究ではせん断応力を導入する

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

免疫・感染学

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.

臍帯血から細胞傷害性Tリンパ球の拡大をそのターゲットサイトメガロウイルス、エプスタイン - バーウイルス、およびアデノウイルス

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

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植片対宿主病の誘導および<emインビボで&gt;</emMHCマッチしたマウスモデルを使用する&gt; T細胞のモニタリング

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

マウスにおける同種のCD4 T細胞応答を研究する尾皮膚の移植

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

体外と体内の T, B, 骨髄細胞の抑制活性と移植から体液性応答の評価

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

生体内で予防接種モデルを用いた抗原特異的 T 細胞細胞傷害性関数の検出のための試金

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

サルモネラの分離-大食細胞のファゴゾームを含む

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

勉強する人間の T 細胞のリコール反応体内吸引水疱プロトコル

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

ジカ ウイルス特異 Immunoprivileged 臓器感染 Ifnar1-/-マウスの T 細胞応答の評価

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist pMHC, through a process termed co-agonism. This protocol describes an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptides as single polypeptides (single chain MHC) in a xenogeneic cell line. We expressed single chain MHCs under conditions where low levels of agonist single chain p-MHC complexes and high levels of non-stimulatory single chain p-MHC complexes were expressed. Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of non-stimulatory pMHC. The protocol describes cell line transfection with single chain MHC constructs, generation of stable cell lines, culture of hepatitis B virus-specific human CD8+ T cells and T cell activation experiments simultaneously quantifying cytokine production and degranulation. The presented methods can be used for research on different aspects of CD8+ T cell activation in human T cell systems with known peptide MHC specificity.

This protocol describes the use of single chain MHC class I complexes to investigate molecular interactions in human CD8+ T cell activation: generation of engineered antigen presenting cells expressing single chain constructs, culture of human CD8+ T cell clone and T cell activation experiments.

人間 cd8 T 細胞活性化における Co 作動を調査する単一のチェーン MHC 技術の使用

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist ...