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In vivo OVA-specific Cytotoxic CD8+ T Cell Killing Assay
测量卵清蛋白(OVA)特异性CD8+T淋巴细胞杀伤力的体内实验   

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Abstract

Cytotoxic CD8+ T cells are responsible for the lysis of cells expressing peptides associated with MHC class I molecules and derived from infection with a pathogen or from mutated antigens. In order to quantify in vivo this antigen-specific CD8+ T cell killing activity, we use the in vivo killing assay (IVK). Here we describe the protocol for the lysis of cells expressing a CD8+ T cell epitope of the OVA protein (SIINFEKL). Mice are previously immunized with the OVA protein and 7 days after immunization, they receive a mix of target cells, prepared from naive C57BL/6 spleen cells pulsed with the SIINFEKL peptide and labeled with high level of CFSE and of non-pulsed control cells labeled with low level of CFSE. One day later, the spleen cells of recipient mice are isolated and analyzed by FACS to measure the amount of CFSEhigh cells and CFSElow cells. The percentage of lysis is calculated by the difference between CFSE high versus low in immunized vs non-immunized mice.

Measuring the ability of antigen-specific CD8+ T cell to lyse their antigen in vivo is very important to evaluate the adaptive cytotoxic response induced against a pathogen or a tumor antigen.

Keywords: Cytotoxic T cells(细胞毒性T细胞), CD8 T cells(CD8 T细胞), Killing assay(杀法), In vivo assay(体内试验), Ovalbumin(卵清蛋白)

Materials and Reagents

  1. 0.5 ml insulin syringe (Terumo Corporation, catalog number: SS05M2713M )
  2. Falcon® 50 ml high clarity PP centrifuge tube (Corning, catalog number: 352070 )
  3. 40 μm nylon mesh cell strainer (Corning, catalog number: 352340 )
  4. 6-well plate (TPP Techno Plastic Products AG., catalog number: 92006 )

  5. Cell counting material 

  6. 5 ml round bottom polystyrene test tube (Corning, catalog number: 352235 )
  7. C57BL/6 mice
  8. CpG B-1826 (5’-TCCATGACGTTCCTGACGTT-3’) (Sigma-Aldrich, PrOligo, custom made) 

  9. N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoniumethyl sulfate (DOTAP) (Roche Diagnostics, catalog number: 11202375001 )
  10. Albumin from chicken egg white (OVA protein) (Sigma-Aldrich, catalog number: A5503 )
  11. Sterile DPBS, 1x (Life Technologies, Gibco, catalog number: 14190-094 )
    Note: Currently, it is “DPBS, no calcium, no magnesium (Thermo Fisher Scientific, GibcoTM, catalog number: 14190-094 )”.
  12. Carboxyfluorescein succinimidyl ester (CFSE) (Life Technologies, catalog number: C1157 )
    Note: Currently, it is “(5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester) (CFSE) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: C1157)”.
  13. ACK lysing buffer (1x) (Lonza Group Ltd., catalog number: 10548E ) 

  14. RPMI 1640 Medium, GlutaMAXTM (Thermo Fisher Scientific, GibcoTM, catalog number: 61870-010 )
  15. Penicillin/streptomycin (Life Technologies, GibcoTM, catalog number: 15140-12 )
    Note: Currently, it is “Penicillin/streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )”.
  16. Peptide SIINFEKL (PolyPeptide Group, custom made)
  17. Fetal calf serum (Thermo Fischer Scientific, HycloneTM, catalog number: SV30160.03 )
  18. Bovine serum albumin (Sigma-Aldrich, catalog number: A7906 )
  19. FACS buffer (see Recipes)

Equipment

  1. Dissection kit (containing scissors, curved forceps)
  2. Centrifuge (Eppendorf, model: 5810R )
  3. MyBathTM 4 mini water bath (Benchmark Scientific, model: B2000-4 )
  4. Flow cytometer: Cyan (Beckman Coulter) or Fortessa (Becton Dickinson)

Procedure

  1. Day 0. Immunize mice with:
    1. CpG-B 1826 (30 μg/mouse)
    2. DOTAP (60 μg/mouse)
    3. Ovalbumin (100 μg/mouse)
      All reagents are diluted in sterile 1x DPBS (to a final total volume of 200 μl/mouse) and the final solution is inoculated by intravenous (i.v.) retro-orbital injection in the eye of each mouse. As control for immunization, some mice receive retro-orbital i.v. injections of sterile 1x DPBS: these naive/non-immunized mice are used to calculate the % of specific lysis, as compared to immunized mice.
      Naive non-injected mice will also be used for isolating splenocytes and pulsing them with peptides, and will also be used as control mice (see step B14).

  2. Day 7. Injection of splenocytes pulsed with the peptide and labeled with a high concentration of CFSE + splenocytes not pulsed with the peptide but labeled with a low concentration of CFSE
    1. Isolate the spleen of naive non-injected mice (1 naive non-injected mouse for every 3 OVA-immunized or DPBS-injected mice + control mouse) and place each spleen in a well (6-well plate).
    2. Mechanically disrupt the spleen to obtain single-cell suspensions in a small volume of DPBS (1-2 ml), and then wash cells with DPBS and centrifuge (652 x g, 5 min, + 4 °C).
    3. Resuspend the pellet and treat with ACK lysing buffer to lyse red blood cells: Add 2 ml of ACK per spleen and incubate for 2 min (at room temperature, RT).
    4. Wash each spleen suspension with 2-3 ml of RPMI + Penicillin/Streptomycin and filter them on cell strainers (40 μm). Pool the suspensions all together in one Falcon 50 tube (or more if many spleens are used for the experiments), add RPMI + Penicillin/Streptomycin until you reach 50 ml and centrifuge (625 x g, 5 min, + 4 °C).
    5. Discard the supernatant and resuspend the pellet in RPMI + Penicillin/Streptomycin; divide splenocytes into two halves. Add the peptide to one half (peptide SIINFEKL 10 μg/ml in the final volume of 2 ml per half spleen). For example: If 10 spleens are used for the experiment, 10 half-spleens will be pulsed with SIINFEKL (meaning 10 x 2 ml = 20 ml final volume) while the other half (equal volume as the final volume of spleen+ SIINFEKL; in our example: 20 ml) will remain without peptide.
    6. Incubate both suspensions for 30 min at 37 °C in a water bath. Gently mix every 10 min.
    7. Wash in RT DPBS 1x and centrifuge (625 x g, 5 min, + 4 °C) twice.
    8. In the meantime, prepare two solutions of CFSE (dilutions in 1x DPBS) in the dark:
      1. CFSEhigh solution: 2.5 μM (diluted from the stock solution kept at -20 °C)
      2. CFSElow solution: 0.25 μM (dilution 1/10 from the CFSEhigh solution)
    9. Discard the supernatants and resuspend each pellet in 1 ml of DPBS and count the cells.
    10. Then, add the necessary volume of the CFSE solutions to their respective cell suspension to obtain a final concentration of 107 cells/ml as follows:
      1. CFSEhigh solution to the splenocytes pulsed with peptide (2.5 μM of CSFE) 

      2. CFSElow solution to the splenocytes without peptide (0.25 μM of CSFE)
      Incubate 15 min at RT in the dark.
    11. Wash each suspension with 40 ml of RPMI + 10% of FCS and centrifuge (625 x g, 5 min, 4 °C).
    12. Discard the supernatant and wash again with RPMI + 10% of FCS and centrifuge (625 x g, 5 min, 4 °C).
    13. Discard the supernatant, resuspend the pellet in 1x DPBS and count cells:
      1. Splenocytes pulsed with peptide (and CFSEhigh)
      2. Splenocytes without peptide (and CFSElow)
      Resuspend each suspension in DPBS to obtain a final concentration of 5 x 106/100 μl.
    14. Pool suspensions (both CFSEhigh andlow) together (ratio 1:1) and inject i.v. 200 μl of the mix to OVA-immunized and DPBS-injected mice. Do not forget to inject a naive (previously non-injected, non-immunized) mouse as control.
    15. Wait 20-24 h.

  3. Day 8
    1. Kill recipient mice, remove their spleen and put it on a cell strainer (40 μm) on a Falcon 50 tube. Mechanically disrupt the spleens (individually) to obtain single-cell suspensions, treat splenocytes with ACK buffer (2 ml/spleen) as described in steps B1-3.
    2. Wash with DPBS 1x and centrifuge (625 x g, 5 min, RT).
    3. Resuspend pellets in 1 ml of FACS buffer.
    4. Count cells and resuspend them to get 5 x 107 cell/ml.
    5. Filter using a 40 μm cell strainer (to avoid FACS blocking).
    6. FACS acquisition: On the Forward Scatter (FSC) vs. Side scatter (SSC) plot, gate cells on total splenocytes, then when plotting CFSE (in the FL-1, GFP or FITC channel) vs. SSC, gate on total CFSE+ cells. Within this gate, using another CFSE vs. SSC plot, distinguish between the CFSEhigh and CFSElow populations. Acquire 10,000 total CFSE+ events (i.e. of both populations: CFSEhigh and low). The number of total CFSE+ events should be the same for all the samples analyzed in order to better compare between OVA-immunized and non-immunized mice. In case 10,000 events cannot be reached, 5,000 events can be acquired, as long as the final number is the same for all samples.

  4. Data analysis
    1. For the analysis of the FACS data after acquisition, 2 populations (the CFSEhigh and the CFSEhigh) are distinguished by using the following gating strategy: Define the splenocyte gate on FSC and SSC, then after excluding the doublets, gate on the total CFSE+ cells. Within the CFSE+ cells, gate on the CFSEhigh and the CFSElow and determine (i) the percentage of CFSEhigh within total CFSE+ cells and (ii) the percentage of CFSElow within total CFSE+ cells.
    2. To calculate the percentage of specific lysis, use the following equation for each mouse (OVA or DPBS) or sample, compared to one control “naive” un-injected mouse:
      % specific lysis = 100 - [100 x (% CFSEhigh immunized mouse/% CFSElow immunized mouse)/(% CFSEhigh naive mouse/% CFSElow naive mouse).

Representative data


Figure 1. Representative dot plot of the FACS analysis of an in vivo killing assay. The dot plots in Figure 1 represent the CFSE staining (vs. side scatter, SSC) in a non-immunized control mouse (A), a non-immunized naive mouse (B) and an OVA-immunized mouse (C) after gating on total splenocytes and then on total CFSE+ cells, as detailed in steps C21 and D22. The CFSEhigh and CFSElow gates are indicated for each mouse, and the respective percentages are shown in pink above each gate. The CFSEhigh population represents target cells pulsed with the SIINFEKL peptide, while CFSElow populations represent target cells not pulsed with any peptide.

C57BL/6 mice are immunized with the OVA protein (or with DPBS; non-immunized mice) and 7 days later, they receive a mix of spleen cells from C57BL/6 mice, pulsed with the SIINFEKL peptide and stained with a high concentration of CFSE and cells not pulsed with the peptide, stained with a low concentration of CFSE, at a 1:1 ration. One day later, the spleen cells from recipient mice are isolated and analyzed by FACS to determine the % of CFSEhigh and CFSElow cells in each mouse (OVA-immunized mouse in Figure 1C and non-immunized mice in Figure 1B).
One naive non-injected mouse is used as the “control naive mouse” (Figure 1A) in the formula detailed above (this mouse is mentioned in step B14). All OVA-immunized (ex: Figure 1C) but also the naive DPBS-injected mice (a.k.a. non-immunized mice, ex: Figure 1B) will be compared to this control mouse in order to quantify and compare the specific lysis of non-immunized mice and OVA-immunized mice.
In this example, using the formula detailed above, the % of specific lysis for the non-immunized mouse in B is calculated as follows:
% specific lysis = 100 - [100 x (42.6/57.1)/(45/54.6)]
% specific lysis = 100 - [100 x 0.746/0.824]
% specific lysis = 100 - [100 x 0.905]
% specific lysis = 100 - 90.5
% specific lysis = 9.5
The % of specific lysis for the OVA-immunized mouse in C is calculated as follows:
% specific lysis = 100 - [100 x (3.2/96.5)/(45/54.6)]
% specific lysis = 100 - [100 x 0.033/0.824]
% specific lysis = 100 - [100 x 0.040]
% specific lysis = 100 - 4.0
% specific lysis = 96
As shown in the example, the % of specific lysis of each mouse is calculated as compared to the naive non-injected mouse set at control mouse.

Notes

This protocol was optimized for the vaccination with the Ova protein and for the IVK test with the corresponding H-2b restricted OVA peptide that activates the CD8+ cytotoxic T cells. The protocol can be adapted to other vaccine candidates and their corresponding peptide with a CD8+ T cell epitope. The respective concentrations should be optimized.

Recipes

  1. FACS buffer
    1x DPBS
    5% fetal calf serum or bovine serum albumin

Acknowledgments

The protocol was adapted from a work supported by grants from the Ligue Nationale Contre le Cancer (Equipe Labellisée, 2014). N. Chaoul was supported by Conseil Regional Île-de-France/Canceropole Île-de-France and Fondation de France.

References

  1. Berraondo, P., Nouze, C., Preville, X., Ladant, D. and Leclerc, C. (2007). Eradication of large tumors in mice by a tritherapy targeting the innate, adaptive, and regulatory components of the immune system. Cancer Res 67(18): 8847-8855.
  2. Chaoul, N., Fayolle, C., Desrues, B., Oberkampf, M., Tang, A., Ladant, D. and Leclerc, C. (2015). Rapamycin impairs antitumor CD8+ T-cell responses and vaccine-induced yumor eradication. Cancer Res 75(16): 3279-3291.
  3. Dadaglio, G., Fayolle, C., Zhang, X., Ryffel, B., Oberkampf, M., Felix, T., Hervas-Stubbs, S., Osicka, R., Sebo, P., Ladant, D. and Leclerc, C. (2014). Antigen targeting to CD11b+ dendritic cells in association with TLR4/TRIF signaling promotes strong CD8+ T cell responses. J Immunol 193(4): 1787-1798.

简介

细胞毒性CD8 + T细胞负责裂解表达与MHC I类分子相关并且源自病原体感染或突变抗原的肽的细胞。为了在体内定量该抗原特异性CD8 + T细胞杀伤活性,我们使用体内杀伤试验(IVK)。在这里,我们描述了用于裂解表达OVA蛋白(SIINFEKL)的CD8 + T细胞表位的细胞的方案。小鼠先前用OVA蛋白免疫,并且在免疫后7天,它们接受从用SIINFEKL肽脉冲并用高水平的CFSE标记的天然C57BL/6脾细胞制备的靶细胞和标记的非脉冲对照细胞的混合物具有低水平的CFSE。一天后,分离受体小鼠的脾细胞并通过FACS分析以测量CFSE高度细胞和CFSE低度细胞的量。通过在免疫的和未免疫的小鼠中CFSE高与低之间的差计算裂解的百分比。
 测量抗原特异性CD8 + T细胞在体内裂解其抗原的能力对评价诱导针对病原体或肿瘤抗原的适应性细胞毒性应答是非常重要的。

关键字:细胞毒性T细胞, CD8 T细胞, 杀法, 体内试验, 卵清蛋白

材料和试剂

  1. 0.5ml胰岛素注射器(Terumo Corporation,目录号:SS05M2713M)
  2. (Corning,目录号:352070)
  3. 40μm尼龙网孔过滤器(Corning,目录号:352340)
  4. 6孔板(TPP Techno Plastic Products AG。,目录号:92006)
  5. 细胞计数物质
  6. 5ml圆底聚苯乙烯试管(Corning,目录号:352235)
  7. C57BL/6小鼠
  8. CpG B-1826(5'-TCCATGACGTTCCTGACGTT-3')(Sigma-Aldrich,PrOligo,custom made)
  9. N- [1-(2,3-二油酰氧基)丙基] -NNN三甲基铵甲基硫酸盐(DOTAP)(Roche Diagnostics,目录号:11202375001)
  10. 来自鸡蛋白(OVA蛋白)的白蛋白(Sigma-Aldrich,目录号:A5503)
  11. 无菌DPBS,1x(Life Technologies,Gibco,目录号:14190-094)
    注意:目前,"DPBS,无钙,无镁(Thermo Fisher Scientific,Gibco TM ",目录号:14190 -094)"。
  12. 羧基荧光素琥珀酰亚胺酯(CFSE)(Life Technologies,目录号:C1157)
    注意:目前,它是"(5-(和-6) - 羧基荧光素二乙酸酯,琥珀酰亚胺酯)(CFSE)(Thermo Fisher Scientific,Molecular Probes ,目录号:C1157)"。
  13. ACK裂解缓冲液(1x)(Lonza Group Ltd.,目录号:10548E)
  14. RPMI 1640 Medium,GlutaMAX (Thermo Fisher Scientific,Gibco TM ,目录号:61870-010)
  15. 青霉素/链霉素(Life Technologies,Gibco TM ,目录号:15140-12)
    注意:目前,它是"青霉素/链霉素(Thermo Fisher Scientific,Gibco ,目录号:15140122)/em>
  16. 肽SIINFEKL(多肽组,定制)
  17. 胎牛血清(Thermo Fischer Scientific,Hyclone ,目录号:SV30160.03)
  18. 牛血清白蛋白(Sigma-Aldrich,目录号:A7906)
  19. FACS缓冲区(参见配方)

设备

  1. 解剖套件(含剪刀,弯钳)
  2. 离心机(Eppendorf,型号:5810R)
  3. MyBath TM 4迷你水浴(Benchmark Scientific,型号:B2000-4)
  4. 流式细胞仪:青色(Beckman Coulter)或Fortessa(Becton Dickinson)

程序

  1. 第0天。
    1. CpG-B 1826(30μg/小鼠)
    2. DOTAP(60μg/小鼠)
    3. 卵白蛋白(100μg/小鼠)
      将所有试剂在无菌1×DPBS中稀释(至最终总体积为200μl/小鼠),并通过静脉内(i.v.)眼后眼注射在每只小鼠的眼中接种最终溶液。作为免疫的对照,一些小鼠接受无菌的1×DPBS的后眼窝注射:这些幼稚/未免疫的小鼠用于计算与免疫的小鼠相比的特异性裂解的%。
      原始非注射小鼠也将用于分离脾细胞并用肽脉冲它们,并且还将用作对照小鼠(参见步骤B14)。

  2. 第7天注射用该肽脉冲并用高浓度的未用该肽脉冲但用低浓度CFSE标记的CFSE +脾细胞标记的脾细胞
    1. 分离幼稚的未注射小鼠的脾脏(每3只OVA免疫的或DPBS注射的小鼠+对照小鼠1只未注射的小鼠),并将每个脾脏置于孔(6孔板)中。
    2. 机械地破坏脾脏以在小体积的DPBS(1-2ml)中获得单细胞悬浮液,然后用DPBS洗涤细胞并离心(652×g,5分钟,+ 4℃) 。
    3. 重悬细胞沉淀,用ACK裂解缓冲液处理以裂解红细胞:每脾脏加入2ml ACK,孵育2分钟(室温,室温)。
    4. 用2-3ml RPMI +青霉素/链霉素洗涤每个脾悬浮液,并在细胞过滤器(40μm)上过滤。将悬浮液一起集中在一个Falcon 50管中(或更多,如果许多脾用于实验),加入RPMI +青霉素/链霉素,直至达到50ml并离心(625×g,5分钟, + 4°C)。
    5. 弃去上清液,并在RPMI +青霉素/链霉素中重悬沉淀;将脾细胞分成两半。将肽加入一半(肽SIINFEKL10μg/ml,终体积为每个半脾2ml)。例如:如果10个脾用于实验,10个半脾用SIINFEKL(意指10×2ml = 20ml最终体积)脉冲,而另一半(等于体积作为脾的最终体积+ SIINFEKL;在我们的示例:20 ml)将保持无肽
    6. 在37℃下在水浴中孵育两种悬浮液30分钟。每10分钟轻轻混匀。
    7. 在RT DPBS 1x中洗涤并离心(625x g,5分钟,+ 4℃)两次。
    8. 同时,在黑暗中制备两种CFSE溶液(1×DPBS中的稀释液):
      1. CFSE 高溶液:2.5μM(从保持在-20℃的储备溶液稀释)
      2. CFSE 低溶液:0.25μM(来自CFSE 高溶液的稀释1/10)
    9. 弃去上清液并将每个沉淀重悬于1ml DPBS中并计数细胞
    10. 然后,将必要体积的CFSE溶液加入到它们各自的细胞悬浮液中,以获得10 7个细胞/ml的最终浓度,如下:
      1. (2.5μM的CSFE)刺激的脾细胞的CFSE高度溶液。
      2. CFSE 低溶液施用于无肽的脾细胞(0.25μM的CSFE)
      在室温下在黑暗中孵育15分钟。
    11. 用40ml RPMI + 10%FCS洗涤每种悬浮液并离心(625×g,5分钟,4℃)。
    12. 弃去上清液并再次用RPMI + 10%FCS洗涤并离心(625×g,5分钟,4℃)。
    13. 弃去上清液,将沉淀重悬在1×DPBS中,计数细胞:
      1. 用肽(和CFSE )脉冲的脾细胞
      2. 无肽的脾细胞(和CFSE
      将每种悬浮液重悬于DPBS中,以获得5×10 6 /100μl的终浓度。
    14. 将细胞悬浮液(CFSE )(比例为1:1),并注射200μl混合物至OVA免疫, DPBS注射的小鼠。 不要忘记注射一个幼稚的(以前没有注射,非免疫的)小鼠作为控制
    15. 等待20-24小时。

  3. 第8天
    1. 杀死受体小鼠,取出其脾脏,并将其放在Falcon 50管上的细胞滤器(40μm)上。 机械地破坏脾脏(单独)以获得单细胞悬浮液,如步骤B1-3中所述用ACK缓冲液(2ml /脾)处理脾细胞。
    2. 用DPBS 1x洗涤并离心(625×g,5分钟,室温)
    3. 将沉淀重悬于1ml FACS缓冲液中
    4. 计数细胞并重悬以获得5×10 7细胞/ml
    5. 使用40μm细胞过滤器过滤(以避免FACS阻断)
    6. FACS采集:在前向散射(FSC)对侧向散射(SSC)图上,在总脾细胞上的门细胞,然后当绘制CFSE(在FL-1,GFP或FITC通道) sup> + 细胞。在该门内,使用另一个CFSE对SSC图,区分CFSE 和CFSE 群体。获取10,000个CFSE + 事件(,即两个群体:CFSE )。对于所有分析的样品,总CFSE + 事件的数目应相同,以便更好地比较OVA免疫和未免疫的小鼠。如果无法达到10,000个事件,则可以获取5,000个事件,只要所有样本的最终数量相同即可。

  4. 数据分析
    1. 为了分析获得后的FACS数据,通过使用以下门控策略来区分2个群体(CFSE 和CFSE ):定义FSC上的脾细胞门和SSC,然后在排除双联体之后,选择总CFSE + 细胞。在CFSE + 单元格中,选通CFSE 和CFSE ,并确定(i)CFSE /sup>在总CFSE + 细胞内的百分比和(ii)CFSE 在总CFSE
    2. 为了计算特异性裂解的百分比,与一个对照的"幼稚"未注射的小鼠相比,对每只小鼠(OVA或DPBS)或样品使用以下等式:
      %特异性裂解= 100- [100×(%CFSE高/高免疫小鼠/%CFSE低/低免疫小鼠)/(%CFSE高//%CFSE 幼稚鼠标)。

代表数据

图1.体内杀伤试验的FACS分析的代表性点图。 图1中的点图代表未免疫的对照小鼠(A),未免疫的幼稚小鼠(B)和OVA免疫的小鼠(C)中的CFSE染色(与侧散射,SSC) ),然后对总CFSE细胞,如步骤C21和D22中详述的。对于每只小鼠指示CFSE 和CFSE 门,并且相应的百分比在每个门上方以粉红色显示。 CFSE 群代表用SIINFEKL肽脉冲的靶细胞,而CFSE 群代表不用任何肽脉冲的靶细胞。

用OVA蛋白(或用DPBS;未免疫的小鼠)免疫C57BL/6小鼠,7天后,它们接受来自C57BL/6小鼠的脾细胞的混合物,用SIINFEKL肽脉冲并用高浓度CFSE和未用肽脉冲的细胞,用低浓度的CFSE以1:1的比例染色。一天后,分离来自受体小鼠的脾细胞,并通过FACS分析以确定每只小鼠中CFSE高和高/低细胞的百分比(OVA免疫的小鼠图1C和图1B中的未免疫的小鼠) 一个未注射的未注射小鼠用作上文详述的式中的"对照未处理小鼠"(图1A)(该小鼠在步骤B14中提及)。所有OVA免疫的(例如:图1C),但是也将初始DPBS注射的小鼠(aka未免疫的小鼠,例如:图1B)与该对照小鼠进行比较,以便定量和比较未免疫的小鼠和OVA免疫的小鼠 在本实施例中,使用上面详述的公式,B中未免疫的小鼠的特异性裂解百分比计算如下:
%特异性裂解= 100- [100×(42.6/57.1)/(45/54.6)] %特异性裂解= 100- [100×0.746/0.824] %特异性裂解= 100- [100×0.905]
%特异性裂解= 100-90.5
%特异性裂解= 9.5
C中OVA免疫的小鼠的特异性裂解百分比计算如下:
%特异性裂解= 100- [100×(3.2/96.5)/(45/54.6)] %特异性裂解= 100- [100×0.033/0.824] %特异性裂解= 100- [100×0.040]
%特异性裂解= 100-4.0
%特异性裂解= 96
如实施例中所示,与设定在对照小鼠的幼稚非注射小鼠相比,计算每只小鼠的特异性裂解%。

笔记

该方案针对用Ova蛋白的接种和用激活CD8 +细胞毒性T细胞的相应的H-2限制性OVA肽的IVK测试进行了优化。该方案可适用于其他疫苗候选物及其具有CD8 + T细胞表位的相应肽。应优化各自的浓度。

食谱

  1. FACS缓冲区
    1x DPBS
    5%胎牛血清或牛血清白蛋白

致谢

该协议改编自由Ligue Nationale Contrele Cancer(EquipeLabellisée,2014)资助的工作。 N. Chaoul由法国法兰西岛地区法兰西岛法兰西岛和法兰西基金会支持。

参考文献

  1. Berraondo,P.,Nouze,C.,Preville,X.,Ladant,D.and Leclerc,C。(2007)。  通过靶向免疫系统的先天性,适应性和调节性组分的tritherapy消除小鼠中的大肿瘤。 (18):8847-8855。
  2. Chaoul,N.,Fayolle,C.,Desrues,B.,Oberkampf,M.,Tang,A.,Ladant,D。和Leclerc,C.(2015)。  雷帕霉素损害抗肿瘤CD8 + sup/+ T细胞应答和疫苗诱导的肿瘤根除。/a> Cancer Res 75(16):3279-3291
  3. Dadaglio,G.,Fayolle,C.,Zhang,X.,Ryffel,B.,Oberkampf,M.,Felix,T.,Hervas-Stubbs,S.,Osicka,R.,Sebo,P.,Ladant,D 。和Leclerc,C。(2014)。  抗原靶向对于与TLR4/TRIF信号传导相关的CD11b + 树突细胞,促进强的CD8 + T细胞应答。 ):1787-1798。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chaoul, N., Fayolle, C. and Leclerc, C. (2016). In vivo OVA-specific Cytotoxic CD8+ T Cell Killing Assay. Bio-protocol 6(12): e1838. DOI: 10.21769/BioProtoc.1838.
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