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In vitro Ag Cross-presentation and in vivo Ag Cross-presentation by Dendritic Cells in the Mouse
小鼠树突细胞进行抗原交叉递呈的体外和体内实验   

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Abstract

Antigen cross presentation is important for effective immune responses to tumors and viral infections. Dendritic cells are professional antigen presenting cells and are unique in their ability to cross-present exogenous antigens on MHC class I molecules and activate antigen specific cytotoxic T cells. This protocol describes antigen cross presentation by dendritic cells (DCs) (bone marrow derived DCs and splenic DCs) in an in vitro and in an in vivo assay system using soluble ovalbumin protein.

Keywords: Antigen cross presentation(抗原交叉presentation), BMDC(BMDC), Splenic DC(splenic直流), B3Z(b3z), Soluble OVA antigen(抗原溶…), Dendritic cell isolation(dendritic细胞分离), In vivo cross-presentation(在体内跨解决方案), OVA(…), OT-1(阿尔卡特OT - 1), T cell proliferation(T细胞增殖)

Materials and Reagents

  1. Anti-Mouse CD45.1-APC (eBiosciences, catalog number: 17-0453-81 )
  2. Rat anti-mouse CD8-PerCpCy5.5 (Biolegend, catalog number: 100733 )
  3. Anti-mouse CD3-PE (Biolegend, catalog number: 100307 )
  4. Anti-mouse CD19-PE (Biolegend, catalog number: 115507 )
    Note: The above antibodies have been tested by the authors but may be substituted with antibodies conjugated with other fluorochromes obtained from other manufacturers.
  5. B3Z CD8+ T cell hybridoma is a kind gift from Dr. PK Srivastava (University of Connecticut Health Center)
  6. RPMI Medium 1640 + L-Glutamine (Life Technologies, catalog number: 11875-093 )
  7. Phosphate buffered saline (PBS) (Life Technologies, Gibco®, catalog number: 10010-023 )
  8. Fetal bovine serum (FBS) (Hyclone, catalog number: SH303397.03 )
  9. RBC lysis buffer (Sigma-Aldrich, catalog number: R7757 )
  10. β-mercaptoethanol (Sigma-Aldrich, catalog number: M7522 )
  11. Antibiotic-Pen-Strep (Life Technologies, Gibco®, catalog number: 15140-122 )
  12. Nonidet-P40 (NP40 (US Biological, catalog number: N3500 )
  13. CPRG Chlorophenol red-D-galactopyranoside (Roche Diagnostics, catalog number: 10884308001 )
  14. Endograde ovalbumin (Biovendor, catalog number: 321000 )
  15. CFSEcarboxyfluorescein diacetate, succinimidyl ester (Life Technologies, Molecular Probes®, catalog number: C34554 )
  16. CD8a (Ly-2) microbeads (Miltenyi Biotec, catalog number: 130-049-401 )
  17. Anti-PE MicroBeads (Miltenyi Biotec, catalog number: 130-048-801 )
  18. CD11c MicroBeads, mouse (Miltenyi Biotec, catalog number: 130-052-001 )
  19. Recombinant GM-CSF (Pierce Biotechnology, catalog number: RMGMCSF20 )
  20. 16% paraformaldehyde (Electron Microscopy Sciences, catalog number: 15710 )
  21. L-glutamine
  22. BMDC medium (see Recipes)
  23. Soluble ovalbumin protein solution (see Recipes)
  24. FACS buffer (see Recipes)
  25. MACS buffer (see Recipes)
  26. CPRG solution (see Recipes)
  27. CPRG lysis solution (see Recipes)
  28. CFSE dye solution (see Recipes)

Equipment

  1. LSR II flow cytometer (Becton-Dickinson)
  2. MACS Multi Stand (Mitenyi Biotec, catalog number: 005126 )
  3. Microplate reader (Bio-Rad Laboratories)
  4. FlowJo software (Tree Star)
  5. 96-well plates round bottom (Corning Incorporated, catalog number: 3799 )
  6. 96-well plates flat bottom (Cell Star, catalog number: 655180 )
  7. 100 mm TC-Treated culture dishes (Corning Incorporated, catalog number: 430293 )
  8. 40 μm cell strainer (BD Biosciences, Falcon®, catalog number: 352340 )
  9. FACS tube, polystyrene (BD Biosciences, Falcon®, catalog number: 352054 )
    Note: Perform the entire method with sterile pyrogen free dishes or plates, pipettes, tips, microfuge and conical tubes. Autoclave and filter all the buffers through 0.22 μm filter.
  10. Incubator
  11. 10 mm culture dish
  12. 5 ml conical tube

Procedure

  1. In vitro Ag cross-presentation
    1. Isolate bone marrow derived dendritic cells (BMDCs) in RPMI Medium 1640 + L-Glutamine + 10% heat inactivated FBS + 50 μM β-mercaptoethanol +1% Pen-Strep + 20 ng/ml GM-CSF and grow for 5 days at 37 °C in a 5% (v/v) CO2 incubator. Briefly, sacrifice mouse, remove the skin and clean the tissue from the femurs and tibia of freshly prepared mouse hind legs using forceps and scissors. Flush the bone marrow from both ends of the bone with 25 g needle and a 10 cc syringe filled with 1x PBS (pH 7.4) into a 50 ml tube. Centrifuge cells at 300 x g for 5 min at room temperature (RT). Lyse RBCs with 3 ml of RBC lysis buffer for 5 min at RT. Wash cell suspension twice in RPMI Medium 1640 + L-glutamine + 10% heat inactivated FBS + 50 μM β-mercaptoethanol +1% Pen-Strep by centrifuging at 300 x g for 5 min at RT. Pass cell suspension through 40 μm cell strainer. Determine the number of cells/ml. Seed 2 x 106 cells in 10 ml BMDC medium (RPMI Medium 1640 + L-Glutamine + 10% heat inactivated FBS + 50 μM β-mercaptoethanol +1% Pen-Strep) containing 20 ng/ml GM-CSF in a 10 mm tissue culture treated culture dish. Grow the cells at 37 °C under a 5% (v/v) CO2 for 5 days. Feed the cells at day 3 by adding 10 ml BMDC medium. Extract the DCs by pipetting the non-adherent or loosely adherent cells and transferring to a 50ml tube and centrifuging at 300 x g for 5 min at RT.
    2. Isolate CD8+ splenic DCs as described (Ghosh et al., 2012). Briefly, isolate total splenocytes by mechanical shear using the frosted surface of glass slides in 10 ml 1x PBS (pH 7.4). Perform RBC lysis with 3 ml of RBC lysis buffer for 5 min at RT. Wash twice with 10 ml 1x PBS (pH 7.4) by centrifuging at 300 x g for 5 min at RT. Pass the cell suspension through 40 μm cell strainer. Incubate the cell suspension (1 x 107 cells in 0.1 ml MACS buffer) with anti-mouse CD3-PE and CD19–PE Abs (1:100) in MACS buffer (1x PBS, pH 7.2 + 0.5% FBS + 2 mM EDTA) for 30 min at 4 °C in the dark. After washing twice with 3 ml MACS buffer by centrifuging at 300 x g for 10 min at 4 °C, resuspend cells (1 x 107) in 90 μl MACS buffer. Magnetically label the R-Phycoerythrin (PE)-conjugated CD3+ and CD19+ cells with anti-PE microbeads (according to manufacturer’s instruction using a MACS Multi Stand. Add 10 μl of anti-PE microbeads and incubate for 15 min at 4 °C in the dark.
    3. Wash cell suspension twice with 3 ml MACS buffer by centrifuging at 300 x g for 10 min at 4 °C.
    4. Resuspend cells in 500 μl of MACS buffer.
    5. Load the magnetically labeled cells onto a MACS column in the magnetic field of a MACS separator. The magnetically labeled CD3-PE and CD19-PE cells will be retained within the column while the unlabeled cells that are depleted of CD3+ and CD19+ cells will be in the flow through fraction. Pass the cell suspension through a MACS column twice to completely deplete CD3+ T cells and CD19+ B cells.
    6. Next, magnetically label the flow-through fraction depleted of CD3+ and CD19+ cells with CD11c and CD8 microbeads according to manufacturer’s instruction, by magnetic separation using a MACS Multi Stand. Resuspend cells in 80 μl MACS buffer. Add 10 μl each of CD11c and CD8 microbeads and incubate for 15 min at 4 °C in the dark.
    7. Wash cell suspension twice with 3 ml MACS buffer by centrifuging at 300 x g for 10 min at 4 °C.
    8. Resuspend cells in 500 μl of MACS buffer.
    9. Load the magnetically labeled cells onto a MACS column in the magnetic field of a MACS separator. The magnetically labeled CD11c+ CD8+ cells will be retained within the column while the unlabeled cells will be in the flow through fraction. Remove the column from the magnetic field. Add 5 ml MACS buffer onto the column and place in a 5 ml conical tube. Elute the magnetically labeled CD8+ CD11c+ cells as positively selected fraction by flushing the column with a plunger. Increase the purity of the cell population by passing the cells over a second MACS column. Verify the purity of the cell population by flow cytometry.
    10. Seed 4 x 104 BMDCs or CD8+ CD11c+ splenic DCs in RPMI + 10% FBS on round-bottomed 96-well plates – 50 μl/well.
    11. Add 1 x 105 B3Z cells in RPMI + 10% FBS – 50 μl/well.
      B3Z is a CD8+ T-cell hybridoma that specifically recognizes OVA residues 257–264 (SIINFEKL) presented on the murine Kb MHC class I molecule (Karttunen et al., 1992) containing the LacZ reporter gene driven by transcriptional control of the nuclear factor of activated T cells (NFAT) element of the human IL2 enhancer. The presentation of the SIINFEKL epitope (SL8) to B3Z cells activates NFAT and results in the induction of β-gal synthesis by B3Z. The amount of β-gal produced can be measured by the hydrolysis of the chromogenic substrate CPRG and is an indication of the amount of SL8/Kb complexes presented on the surface of antigen presenting cells. The advantage of using the B3Z is that the LacZ assay is sensitive enough to detect antigen, expressing APCs at a low frequency and these cells are also easy to maintain in cultures. Culture these cells at 37 °C (5% CO2) in DMEM with 10% heat-inactivated fetal calf serum and supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin sulfate, 0.05 mM MEM nonessential amino acids, and 1 mM sodium pyruvate. Add varying concentrations of endofree ovalbumin protein (0.625-1 mg/ml) in 1x PBS (pH 7.4).
    12. Incubate at 37 °C for 18-24 h in a 5% (v/v) CO2 incubator.
    13. Next day, spin the plate at 500 x g for 2 min at RT, gently aspirate the medium and wash the cell cultures twice with 100 μl of 1x PBS. The cells are non-adherent or loosely adherent so centrifuging the plate is necessary to avoid loss of cells during the washing step.
    14. Add 100 μl of 0.15 mM of CPRG solution in CPRG lysis buffer (0.5% NP-40 in 1xPBS).
    15. Incubate at RT in the dark for 6-24 h.
    16. Transfer the contents to flat bottom 96-well plate by pipetting. In this step centrifuging the plate is not necessary as the cells are already lysed.
    17. Avoid the bubbles present in the wells while transferring the contents.
    18. Read the absorption at 570 nm, with 650 nm as the reference wavelength in a microplate reader. BMDCs incubated with B3Z but without the antigen serve as background control.

  2. In vivo Ag cross-presentation
    1. Harvest the spleen and lymph nodes (inguinal and mesenteric) from Rag-/- CD45.1 OT-1 mice and isolate single cell suspension by mechanical shear using the frosted surface of glass slides in MACS buffer (1x PBS + 0.5% FBS + 2 mM EDTA).
    2. Lyse RBCs (for spleen only as processing the lymph node does not require lysis of RBCs) with 3 ml of RBC lysis buffer for 5 min at RT.
    3. Wash cell suspension twice in 1x PBS by centrifuging at 300 x g for 5 min at RT.
    4. Pass cell suspension through 40 μm cell strainer and wash twice with 3 ml MACS buffer by centrifuging at 300 x g for 5 min at RT.
    5. Determine cell number.
    6. Resuspend 1 x 107 cells in 90 μl MACS buffer.
    7. Add 10 μl of CD8a microbeads per 1 x 107 cells in 90 μl MACS buffer and incubate for 15 min at 4 °C in the dark. Magnetically label the CD8+ cells with CD8 microbeads (the microbeads are not conjugated to any fluorophore).
    8. Wash cell suspension twice with 3 ml MACS buffer by centrifuging at 300 x g for 10 min at 4 °C.
    9. Resuspend cells in 500 μl of MACS buffer.
    10. Subject to magnetic separation using MACS column according to the manufacturer’s instruction using a MACS Multi Stand. Load the magnetically labeled CD8+ cells onto a MACS column in the magnetic field of a MACS separator. The magnetically labeled CD8+ cells will be retained within the column while the unlabeled cells will be in the flow through fraction. Remove the column from the magnetic field. Add 5 ml MACS buffer onto the column and place in a 5 ml conical tube. Elute the magnetically labeled CD8+ cells as positively selected fraction by flushing the column with a plunger. Increase the purity of the CD8+ cell population by passing the cells over a second MACS column.
    11. Resuspend 1 x 107 cells/ml of CD8+ T lymphocytes and label with 10 μM of CFSE proliferative dye for 10 min at room temperaturein the dark (add 2 μl of 5 mM CFSE dye to 107 cells in 1 ml 1x PBS).
    12. Wash cells once with 10 ml 1x PBS and resuspend in sterile 1x PBS to a concentration of 1 x 107 cells/ml.
    13. On day 0, adoptively transfer 1 x 106 CFSE labeled CD8+ T lymphocytes to mice (0.1 ml of 1 x 107cells/ml) intravenously.
    14. On day 1, after 24 h, challenge mice with 100 μg of endofree soluble ovalbumin protein/mice in 0.1 ml of 1x PBS intradermally by injecting into the right flank of the mouse.
    15. After 3 days, harvest spleen and lymph nodes (inguinal and mesenteric) by mechanical shear as in step 1. Briefly, isolate total splenocytes by mechanical shear using the frosted surface of glass slides in 10 ml 1x PBS (pH 7.4). Perform RBC lysis with 3 ml of RBC lysis buffer for 5 min at RT. Wash twice with 10 ml 1x PBS (pH 7.5) by centrifuging at 300 x g for 5 min at RT.
    16. Lyse RBCs (for spleen only as processing the lymph node does not require lysis of RBCs) as in step 2 and isolate single cell suspension in 5 ml FACS buffer (1x PBS+ 2% heat inactivated FBS).
    17. Pass cells suspension through 40 μm filter.
    18. Determine cell number.
    19. Immunostain 1 x 106 cells with anti-CD45.1-APC (1:100) and anti-CD8-PerCpCy5.5 (1:100) antibodies in 0.1 ml FACS buffer for 30 min at 4 °C in the dark in FACS tubes.
    20. Wash cells thrice with 3ml FACS buffer by centrifuging at 300 x g for 5 min at RT.
    21. Fix cells with 0.5 ml 2% Paraformaldehyde in 1x PBS (pH 7.4) for 30 min at 4 °C.
    22. Wash cells thrice with 3 ml FACS buffer by centrifuging at 300 x g for 5 min at RT.
    23. Resuspend cells in 0.5 ml of FACS buffer.
    24. Analyze cells by flow cytometry in LSRII for CFSE content (488 nm wavelength) in CD8+ CD45.1+ T-lymphocytes.
    25. Calculate the division index as the average number of divisions for all the cells in the original population using FlowJo software.
    26. Flow cytometric analysis.


      Figure 1. Activation of CD8+ T lymphocyte activation by in vivo cross presentation of soluble OVA antigen. CFSE-labeled CD45.1+ OT-I cells were intravenously injected into mice, followed by intradermal administration of soluble OVA after 24 h. 3 days later splenic lymphocytes were isolated and stained with anti-CD8-PerCpCy5.5 and anti-CD45.1-APC antibodies and OT-I cell proliferation was analyzed by flow cytometry. 1. Total splenocytes were gated on live cell population. 2. Total live cell population was gated on CD8+ T- cells. 3. Total CD8+ T-cell population was gated on CD8+ CD45.1+ T-cells.  4. CFSE content of CD8+ CD45.1+ OT-1 cells was determined as a measure of T-cell proliferation. 5. Number of cell division in the CD8+ CD45.1+ T-cell population indicated by the division index was determined by FlowJo software.

Recipes

  1. BMDC medium (500 ml)
    445 ml RPMI Medium 1640 + L-Glutamine
    50 ml heat inactivated FBS
    2 μl of 14 M β-mercaptoethanol
    5 ml of 100% Pen-Strep
    20 μl of 10 μg/ml GM-CSF [Reconstitute 10 μg of GM-CSF in 1 ml 1x PBS (pH7.4) Store at -80 °C in aliquots]
  2. Soluble ovalbumin protein solution (1 ml)
    10 mg of ovalbumin
    1 ml 1x PBS (pH 7.4)
  3. FACS buffer (500 ml)
    490 ml 1x PBS (pH 7.4)
    10 ml 100% Heat-inactivated FBS
  4. MACS buffer (500 ml)
    495.5 ml 1x PBS (pH 7.2)
    2.5 ml 100% Heat-inactivated FBS
    2 ml 500 mM EDTA (pH 8.0)
    (FBS can be replaced with 0.5% BSA. Addition of EDTA prevents cell clumping.)
  5. CPRG solution (500 ml)
    45.5 mg CPRG powder
    500 ml CPRG lysis solution
  6. CPRG lysis solution (500 ml)
    2.5 ml 100% NP-40
    498 ml 1x PBS
  7. CFSE dye solution (18 μl of 5 mM stock solution) (prepare according to manufacturer’s instruction)
    50 μg of CFSE
    18 μl DMSO (supplied by the manufacturer)

Acknowledgments

We thank Drs. Sreyashi Basu and Pramod K Srivastava, UConn Health for advice and help. The B3Z CD8+ T cell hybridoma was a gift from Dr. Pramod K Srivastava, UConn Health. In addition, we thank the UConn Health Flow Cytometry Core. This work was supported by Public Health Service grants, the National Cancer Institute (CA-106345), and the National Heart, Lung, and Blood Institute (HL-70694).

References

  1. Ghosh, M., McAuliffe, B., Subramani, J., Basu, S. and Shapiro, L. H. (2012). CD13 regulates dendritic cell cross-presentation and T cell responses by inhibiting receptor-mediated antigen uptake. J Immunol 188(11): 5489-5499.
  2. Karttunen, J., Sanderson, S. and Shastri, N. (1992). Detection of rare antigen-presenting cells by the lacZ T-cell activation assay suggests an expression cloning strategy for T-cell antigens. Proc Natl Acad Sci U S A 89(13): 6020-6024.

简介

抗原交叉呈递对于对肿瘤和病毒感染的有效免疫应答是重要的。 树突状细胞是专业抗原呈递细胞,并且在其将MHC I类分子上的外源抗原交叉呈递并激活抗原特异性细胞毒性T细胞的能力方面是独特的。 该方案描述了在体外和在体内使用可溶性卵白蛋白蛋白质的体系中通过树突细胞(DC)(骨髓来源的DC和脾DC)的抗原交叉呈递 。

关键字:抗原交叉presentation, BMDC, splenic直流, b3z, 抗原溶…, dendritic细胞分离, 在体内跨解决方案, …, 阿尔卡特OT - 1, T细胞增殖

材料和试剂

  1. 抗小鼠CD45.1-APC(eBiosciences,目录号:17-0453-81)
  2. 大鼠抗小鼠CD8-PerCpCy5.5(Biolegend,目录号:100733)
  3. 抗小鼠CD3-PE(Biolegend,目录号:100307)
  4. 抗小鼠CD19-PE(Biolegend,目录号:115507)
    注意:上述抗体已经被作者测试,但可以用与从其他制造商获得的其他荧光染料结合的抗体替代。
  5. B3Z CD8 + T细胞杂交瘤是来自PK Srivastava博士(康涅狄格大学健康中心大学)的礼物。
  6. RPMI Medium 1640 + L-谷氨酰胺(Life Technologies,目录号:11875-093)
  7. 磷酸盐缓冲盐水(PBS)(Life Technologies,Gibco ,目录号:10010-023)
  8. 胎牛血清(FBS)(Hyclone,目录号:SH303397.03)
  9. RBC裂解缓冲液(Sigma-Aldrich,目录号:R7757)
  10. β-巯基乙醇(Sigma-Aldrich,目录号:M7522)
  11. 抗生素 - Pen-Strep(Life Technologies,Gibco ,目录号:15140-122)
  12. Nonidet-P40(NP40(US Biological,目录号:N3500))
  13. CPRG氯酚红-D-吡喃半乳糖苷(Roche Diagnostics,目录号:10884308001)
  14. 前列腺卵清蛋白(Biovendor,目录号:321000)
  15. CFSE羧基荧光素二乙酸酯,琥珀酰亚胺酯(Life Technologies,Molecular Probes ,目录号:C34554)
  16. CD8a(Ly-2)微珠(Miltenyi Biotec,目录号:130-049-401)
  17. 抗PE微珠(Miltenyi Biotec,目录号:130-048-801)
  18. CD11c MicroBeads,小鼠(Miltenyi Biotec,目录号:130-052-001)
  19. 重组GM-CSF(Pierce Biotechnology,目录号:RMGMCSF20)
  20. 16%多聚甲醛(Electron Microscopy Sciences,目录号:15710)
  21. L-谷氨酰胺
  22. BMDC介质(参见配方)
  23. 可溶性卵白蛋白溶液(参见配方)
  24. FACS缓冲区(请参阅配方)
  25. MACS缓冲区(参见配方)
  26. CPRG解决方案(参见配方)
  27. CPRG裂解液(见配方)
  28. CFSE染料溶液(参见配方)

设备

  1. LSR II流式细胞仪(Becton-Dickinson)
  2. MACS Multi Stand(Mitenyi Biotec,目录号:005126)
  3. 酶标仪(Bio-Rad Laboratories)
  4. FlowJo软件(Tree Star)
  5. 96孔板圆底(Corning Incorporated,目录号:3799)
  6. 96孔板平底(Cell Star,目录号:655180)
  7. 100mm TC处理的培养皿(Corning Incorporated,目录号:430293)
  8. 40μm细胞滤器(BD Biosciences,Falcon ,目录号:352340)
  9. FACS管,聚苯乙烯(BD Biosciences,Falcon ,目录号:352054)
    注意:使用无菌无热原培养皿或板,移液管,吸头,微量离心管和锥形管进行整个方法。 高压灭菌并通过0.22μm过滤器过滤所有缓冲液。
  10. 孵化器
  11. 10mm培养皿
  12. 5ml锥形管

程序

  1. 体外 Ag交叉呈递
    1. 在RPMI培养基1640 + L-谷氨酰胺+ 10%热灭活的FBS +50μMβ-巯基乙醇+ 1%Pen-Strep + 20ng/ml GM-CSF中分离骨髓衍生的树突细胞(BMDC),并在37℃下生长5天在5%(v/v)CO 2培养箱中培养。简而言之,处死小鼠,取出皮肤,使用镊子和剪刀从新鲜制备的小鼠后腿的股骨和胫骨清洁组织。用25g针头和填充1×PBS(pH 7.4)的10cc注射器将骨头两端的骨髓冲洗到50ml管中。在室温(RT)下以300xg离心细胞5分钟。用3ml RBC裂解缓冲液在室温下裂解RBC 5分钟。通过在室温下以300×g离心5分钟,在RPMI培养基1640 + L-谷氨酰胺+ 10%热灭活的FBS +50μMβ-巯基乙醇+ 1%Pen-Strep中洗涤细胞悬浮液两次。将细胞悬液通过40μm细胞过滤器。确定细胞数/ml。在含有20ng/ml的10mM BMDC培养基(RPMI培养基1640 + L-谷氨酰胺+ 10%热灭活的FBS +50μMβ-巯基乙醇+ 1%Pen-Strep)的10ml BMDC培养基中接种2×10 6个细胞GM-CSF在10mm组织培养处理的培养皿中。在37℃下在5%(v/v)CO 2下生长细胞5天。在第3天通过加入10ml BMDC培养基饲养细胞。通过吸取非粘附或松散粘附的细胞并转移到50ml试管中并在室温下以300xg离心5分钟来提取DCs。
    2. 如所述分离CD8 +脾DC(Ghosh等人,2012)。简言之,通过机械剪切使用玻璃载玻片的磨砂表面在10ml 1x PBS(pH 7.4)中分离总脾细胞。在室温下用3ml RBC裂解缓冲液进行RBC裂解5分钟。用10ml 1x PBS(pH 7.4)通过在室温下以300×g离心5分钟洗涤两次。将细胞悬液通过40μm细胞过滤器。将细胞悬浮液(在0.1ml MACS缓冲液中的1×10 7个细胞)与在MACS缓冲液(1×PBS,pH7.2)中的抗小鼠CD3-PE和CD19-PE Ab(1:100) 0.5%FBS + 2mM EDTA)在4℃避光孵育30分钟。用3ml MACS缓冲液通过离心洗涤两次后 在300×g下在4℃温育10分钟,将细胞(1×10 7个)悬浮在90μlMACS缓冲液中。用抗PE微珠磁珠标记R-藻红蛋白(PE)结合的CD3 +和CD19 +细胞(根据制造商的说明使用MACS Multi Stand。加入10μl抗PE微珠,并在4℃下在暗。
    3. 通过在4℃下以300×g离心10分钟,用3ml MACS缓冲液洗涤细胞悬浮液两次。
    4. 将细胞重悬于500μlMACS缓冲液中。
    5. 将磁性标记的细胞加载到MACS分离器的磁场中的MACS柱上。磁性标记的CD3-PE和CD19-PE细胞将保留在柱内,而耗尽CD3 +和CD19 +/+细胞的未标记细胞将处于流动通过分数。将细胞悬液通过MACS柱两次,以完全耗尽CD3 + T细胞和CD19 + B细胞。
    6. 接下来,根据制造商的说明,通过使用MACS Multi Stand的磁性分离,用CD11c和CD8微珠磁性标记消耗CD3 +和CD19 +细胞的流过级分。重悬细胞在80微升MACS缓冲液。加入10μl每个CD11c和CD8微珠,并在4℃下在黑暗中孵育15分钟。
    7. 通过在4℃下以300×g离心10分钟,用3ml MACS缓冲液洗涤细胞悬浮液两次。
    8. 将细胞重悬于500μlMACS缓冲液中。
    9. 将磁性标记的细胞加载到MACS分离器的磁场中的MACS柱上。磁性标记的CD11c + CD8 + 细胞将保留在柱内,而未标记的细胞将处于流过级分中。从磁场中取出色谱柱。加入5毫升MACS缓冲液到柱子上,并放置在5毫升锥形管。通过用柱塞冲洗柱,洗脱磁性标记的CD8 + CD11c +细胞作为阳性选择的部分。通过将细胞通过第二个MACS柱提高细胞群体的纯度。通过流式细胞术验证细胞群的纯度
    10. 在圆底96孔板上在RPMI + 10%FBS中种子4×10 4个BMDCs或CD8 + CD11c +脾脾DC - 50 μl/孔
    11. 在RPMI + 10%FBS中加入1×10 5个B3Z细胞 - 50μl/孔。
      B3Z是特异性识别存在于鼠Kb MHC I类分子(Karttunen等,1992)上的OVA残基257-264(SIINFEKL)的CD8 + T细胞杂交瘤,其含有由以下驱动的LacZ报告基因:人IL2增强子的活化T细胞(NFAT)元件的核因子的转录控制。 SIINFEKL表位(SL8)对B3Z细胞的呈递激活NFAT并导致B3Z诱导β-gal合成。产生的β-gal的量可以通过显色底物CPRG的水解来测量,并且是存在于抗原呈递细胞表面上的SL8/Kb复合物的量的指示。使用B3Z的优点是LacZ测定足够灵敏以检测抗原,以低频率表达APC,并且这些细胞也容易在培养物中维持。将这些细胞在37℃(5%CO 2)下在含有10%热灭活的胎牛血清并补充有2mM L-谷氨酰胺,100U/ml青霉素,100μg/ml的DMEM中培养链霉素硫酸盐,0.05mM MEM非必需氨基酸和1mM丙酮酸钠。加入不同浓度的endofree卵白蛋白(0.625-1 mg/ml)在1x PBS(pH 7.4)中
    12. 在37℃,在5%(v/v)CO 2培养箱中孵育18-24小时
    13. 第二天,在室温下以500×g离心平板2分钟,轻轻地吸出培养基并用100μl的1×PBS洗涤细胞培养物两次。 细胞是非粘附的或松散粘附的,因此需要离心平板以避免在洗涤步骤期间细胞的损失。
    14. 在CPRG裂解缓冲液(0.5%NP-40,在1xPBS中)中加入100μl的0.15mM的CPRG溶液。
    15. 在室温下在黑暗中孵育6-24小时。
    16. 通过吸移将内容物转移到平底96孔板。 在该步骤中,不需要离心板,因为细胞已经裂解
    17. 避免在转移内容物时在孔中存在气泡。
    18. 读取570 nm的吸光度,在酶标仪中用650 nm作为参考波长。 与B3Z孵育但没有抗原的BMDC作为背景对照

  2. 体内 Ag交叉呈现
    1. 从Rag CD45.1 OT-1小鼠收获脾脏和淋巴结(腹股沟和肠系膜),并通过机械剪切使用载玻片的磨砂表面在MACS缓冲液(1x PBS + 0.5%FBS + 2mM EDTA)
    2. 用3ml RBC裂解缓冲液在室温下裂解RBC(仅用于脾脏,因为加工淋巴结不需要裂解RBC)5分钟。
    3. 在室温下,通过在300×g离心5分钟,在1x PBS中洗涤细胞悬浮液两次。
    4. 使细胞悬液通过40μm细胞过滤器,并通过在RT下以300×g离心5分钟用3ml MACS缓冲液洗涤两次。
    5. 确定单元格号。
    6. 在90μlMACS缓冲液中重悬1×10 7个细胞
    7. 在90μlMACS缓冲液中加入10μlCD8a微珠/1×10 7个细胞,并在4℃在黑暗中孵育15分钟。 用CD8微珠磁标记CD8 +细胞(微珠不与任何荧光团缀合)。
    8. 通过在4℃下以300×g离心10分钟,用3ml MACS缓冲液洗涤细胞悬浮液两次。
    9. 将细胞重悬于500μlMACS缓冲液中。
    10. 根据制造商的说明使用MACS多支架进行磁分离。将磁性标记的CD8 +细胞加载到MACS分离器的磁场中的MACS柱上。磁性标记的CD8 +细胞将保留在柱内,而未标记的细胞将处于流过级分中。从磁场中取出色谱柱。加入5毫升MACS缓冲液到柱子上,并放置在5毫升锥形管。通过用柱塞冲洗柱,将磁性标记的CD8 +细胞洗脱为阳性选择的级分。通过将细胞通过第二个MACS柱提高CD8 +细胞群的纯度
    11. 重悬悬浮1×10 7个细胞/ml的CD8 + T淋巴细胞,并在室温下在暗处用10μMCFSE增殖染料标记10分钟(加入2μl5 mM CFSE染料至1×1×PBS中的10μL细胞)
    12. 用10ml 1x PBS洗涤细胞一次,并在无菌1×PBS中重悬至浓度为1×10 7个细胞/ml。
    13. 在第0天,静脉内过继转移1×10 6 CFSE标记的CD8 + T淋巴细胞至小鼠(0.1ml的1×10 7个细胞/ml)。
    14. 在第1天,24小时后,通过注射入小鼠的右胁腹,在0.1ml的1×PBS中皮内攻击100μg无毒可溶性卵白蛋白/小鼠的小鼠。
    15. 3天后,通过机械剪切如步骤1收获脾脏和淋巴结(腹股沟和肠系膜)。简而言之,通过机械剪切使用玻璃载玻片的磨砂表面在10ml 1x PBS(pH 7.4)中分离总脾细胞。在室温下用3ml RBC裂解缓冲液进行RBC裂解5分钟。在室温下以300×g离心5分钟,用10ml 1×PBS(pH7.5)洗涤两次。
    16. 如步骤2中那样裂解RBC(仅用于脾,因为加工淋巴结不需要裂解RBC),并在5ml FACS缓冲液(1×PBS + 2%热灭活的FBS)中分离单细胞悬浮液。
    17. 将细胞悬浮液通过40μm过滤器
    18. 确定单元格号。
    19. 在4℃下用0.1ml FACS缓冲液中的抗CD45.1-APC(1:100)和抗CD8-PerCpCy5.5(1:100)抗体对抗体进行免疫染色1×10 6个细胞30分钟°F在黑暗中在FACS管中
    20. 通过在室温下以300×g离心5分钟,用3ml FACS缓冲液洗涤细胞三次。
    21. 用0.5ml 2%多聚甲醛在1×PBS(pH 7.4)中在4℃固定细胞30分钟
    22. 通过在室温下以300×g离心5分钟,用3ml FACS缓冲液洗涤细胞三次。
    23. 将细胞重悬于0.5ml FACS缓冲液中
    24. 通过流式细胞术在LSRII中分析细胞,用于CD8 + CD45.1 + T淋巴细胞中的CFSE含量(488nm波长)。
    25. 使用FlowJo软件计算分割指数,作为原始群体中所有单元格的平均分割数。
    26. 流式细胞分析。


      图1.通过体内可溶性OVA抗原的交叉呈递激活CD8 + T淋巴细胞激活。 将CFSE标记的CD45.1 + OT-I细胞静脉内注射到小鼠中,随后在24小时后皮内施用可溶性OVA。 3天后,分离脾淋巴细胞并用抗CD8-PerCpCy5.5和抗CD45.1-APC抗体染色,并通过流式细胞术分析OT-I细胞增殖。总脾细胞在活细胞群体上门控。 2.在CD8 + T细胞上门控总活细胞群体。 3.在CD8 + CD45.1 + T细胞上门控总CD8 + T细胞群体。 4.测定CD8 + CD45.1 + OT-1细胞的CFSE含量作为T细胞增殖的量度。通过FlowJo软件测定由分裂指数指示的CD8 + CD45.1 + T细胞群中的细胞分裂数。

食谱

  1. BMDC培养基(500ml) 445ml RPMI培养基1640 + L-谷氨酰胺 50ml热灭活的FBS
    2μl14μMβ-巯基乙醇 5ml的100%Pen-Strep
    20μl10μg/ml GM-CSF [在1ml 1×PBS(pH7.4)中重组10μgGM-CSF)-80℃等分试样]
  2. 可溶性卵白蛋白溶液(1ml) 10mg卵清蛋白
    1ml 1×PBS(pH7.4)
  3. FACS缓冲液(500ml) 490ml 1×PBS(pH7.4) 10ml 100%热灭活的FBS
  4. MACS缓冲液(500ml)
    495.5ml 1×PBS(pH 7.2) 2.5ml 100%热灭活的FBS
    2ml 500mM EDTA(pH8.0) (FBS可以用0.5%BSA代替,加入EDTA可以防止细胞聚集。)
  5. CPRG溶液(500ml)
    45.5 mg CPRG粉
    500 ml CPRG裂解液
  6. CPRG裂解溶液(500ml) 2.5ml 100%NP-40
    498 ml 1x PBS
  7. CFSE染料溶液(18μl5mM储备溶液)(根据制造商的说明制备) 50μgCFSE
    18μlDMSO(由制造商提供)

致谢

我们感谢博士。 Sreyashi Basu和Pramod K Srivastava,UConn Health提供咨询和帮助。 B3Z CD8 + T细胞杂交瘤是来自UConn Health博士Pramod K Srivastava的礼物。此外,我们感谢UConn健康流式细胞仪核心。这项工作得到公共卫生服务补助金,国家癌症研究所(CA-106345)和国家心肺血液研究所(HL-70694)的支持。

参考文献

  1. Ghosh,M.,McAuliffe,B.,Subramani,J.,Basu,S.and Shapiro,L.H。(2012)。 CD13通过抑制受体介导的抗原摄取来调节树突状细胞交叉呈递和T细胞应答。 a> J Immunol 188(11):5489-5499
  2. Karttunen,J.,Sanderson,S。和Shastri,N。(1992)。 通过lacZ T细胞活化测定检测罕见的抗原呈递细胞提示了表达克隆策略T-cell antigens。 Proc Natl Acad Sci USA 89(13):6020-6024。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ghosh, M. and Shapiro, L. H. (2012). In vitro Ag Cross-presentation and in vivo Ag Cross-presentation by Dendritic Cells in the Mouse. Bio-protocol 2(24): e305. DOI: 10.21769/BioProtoc.305.
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Ting Wang
Anhui Medical University
It a very useful protocol to me and my group. Tanks a lot for the authors!
10/8/2015 8:02:25 PM Reply
Anupinder kaur
UCHC
Very well written protocol
4/11/2014 10:37:22 AM Reply
Linda H Shapiro
Center for Vascular Biology, University of Connecticut Health Center, USA

Thank you for your kind comment.

Regards

Mallika Ghosh

4/11/2014 12:45:09 PM


Paula Nunes Paula
University of Geneva
Hello,
Are the B3Z cells commercially available? If not how might I obtain them?
Many thanks for the nice and detailed protocol,
Best,
P. Nunes
4/3/2014 5:15:11 AM Reply
Mallika Ghosh
Center for Vascular Biology, University of Connecticut Health Center, USA

Dear Dr. Nunes,

Thank you for your kind comment. The B3Z cells used in the study was obtained from Dr. Sreyashi Basu and Dr. Pramod Srivastava in the Department of Immunology, University of Connecticut Health Center, Farmington, CT 06030.

Best Regards

Mallika Ghosh

4/3/2014 7:20:22 AM