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Evaluation of Cross-presentation in Bone Marrow-derived Dendritic Cells in vitro and Splenic Dendritic Cells ex vivo Using Antigen-coated Beads
使用抗原包被珠子评价体外骨髓源性树突状细胞和离体脾树突状细胞的交叉呈递   

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Abstract

Antigen presentation by MHC class I molecules, also referred to as cross-presentation, elicits cytotoxic immune responses. In particular, dendritic cells (DC) are the most proficient cross-presenting cells, since they have developed unique means to control phagocytic and degradative pathways.

This protocol allows the evaluation of antigen cross-presentation both in vitro (by using bone marrow-derived DC) and ex vivo (by purifying CD8+ DC from spleen after incorporation of particulate antigen) using ovalbumin (OVA)-coupled particles. Cross-presentation efficiency is measured by three different readouts: the B3Z hybridoma T cell line (Karttunen et al., 1992) and stimulation of antigen-specific CD8+ T cells (OT-I) (Kurts et al., 1996), either analyzing OT-I activation by CD69 expression or OT-I proliferation after labeling them with carboxyfluorescein succinimidyl ester (CFSE). By using this approach, we could show recently that DCs are able to increase cross-presentation efficiency transiently upon engagement of TLR4 (Alloatti et al., 2015).

Background

In mouse, antigen-presenting cells (APC) are able to take up exogenous antigens to process them and to load peptides derived from such exogenous antigens onto major histocompatibility complex (MHC) class I molecules. Peptide-MHC I complexes are subsequently transported to the plasma membrane, where they might be presented to CD8+ T cells thereby promoting T cell activation, a process referred to as cross-presentation (Joffre et al., 2012). Among the different APC, dendritic cells (DC) excel at cross-presentation and comprise of different subpopulations expressing the XCR1 marker, which have been shown to cross-present antigens very efficiently (i.e., CD8+ resident DC from spleen and CD103+ migratory DC from skin and lung) (Dorner et al., 2009; Crozat et al., 2011). While the purification of DC residing in spleen or migratory DC is feasible, it is laborious and expensive. In order to study the cell biology of DC, primary cultures of bone marrow-derived DC (BMDC) can be easily differentiated from myeloid progenitors by culturing them with GM-CSF. Even though BMDC cannot be associated with any particular DC subtype (perhaps inflammatory DC), they constitute a valuable tool to study the main characteristics of DC cell biology. Herein, we introduce a detailed protocol to analyze cross-presentation of particulate antigen by BMDC, but also by CD8+ splenic DC. Although previous protocols included different antigen forms and read-outs, the protocol described here aims to analyze cross-presentation in a comprehensive and concise way in different DC types.

Materials and Reagents

  1. 2 ml Eppendorf tubes
  2. 15 ml centrifuge tubes
  3. 50 ml centrifuge tubes
  4. 14 ml tubes
  5. FisherbrandTM cell strainer (Thermo Fisher Scientific, Fisher Scientific, catalog number: 22-363-548 )
  6. Pre-separation filters (30 μm) (Miltenyi Biotec, catalog number: 130-041-407 )
  7. 1 ml insulin syringes (Terumo Medical, catalog number: SS+01H1 )
  8. 2.5 ml syringes
  9. 25 G needles (Terumo, catalog number: AN*2516R1 )
  10. Non-treated 96-well plates (Coring, Falcon®, round bottom, catalog number: 351177 )
  11. Non-treated 6-well plates (Sigma-Aldrich, catalog number: M8562-100EA )
  12. Non-treated Petri dish, 145 x 20 mm (Greiner Bio One, catalog number: 639161 )
  13. Mice: C57BL/6 and C57BL/6 recombination activating gene 1-deficient OT-I TCR (Vα2, Vβ5) transgenic mice were obtained from Charles River Laboratories (CDTA, Orleans, France)
  14. B3Z T cell line (a Kb-restricted, OVA-specific CD8+ T cell hybridoma) (Kurts et al., 1996)
  15. Low endotoxin OVA (50 mg/ml stock) (Worthington Biochemical, catalog number: LS003062 )
  16. OVA peptide 257-264 (SIINFEKL) (Polypeptide, catalog number: SC1302 )
  17. Polybead® polystyrene 3.0 micron microspheres (Polysciences, catalog number: 17134 )
  18. Polybead® dyed blue 1.0 micron microspheres (Polysciences, catalog number: 15712 )
  19. PBS (1x, pH 7.4) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010-023 )
  20. Glycine (Sigma-Aldrich, catalog number: 50046 )
  21. Glutaraldehyde (25%) (Sigma-Aldrich, catalog number: G5882 )
  22. Iscove’s modified Dulbecco’s medium (IMDM) (Sigma-Aldrich, catalog number: I3390-500ML )
  23. Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  24. RPMI with GlutaMAXTM (Thermo Fisher Scientific, catalog number: 61870-010 )
  25. GlutaMAXTM supplement (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 35050061 )
  26. β-mercaptoethanol (Thermo Fisher Scientific, GibcoTM, catalog number: 21985-023 )
  27. MEM non-essential amino acids solution (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 11140050 )
  28. Sodium pyruvate (100 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11360070 )
  29. CPRG (Roche Diagnostics, catalog number: 10884308001 )
  30. Fixable viability dye eFluor 780 (dilution 1/10,000) (Affymetrix, eBioscience, catalog number: 65-0865-14 )
  31. Low endotoxin fetal bovine serum (FBS, heat-inactivated for 20 min at 56 °C) (Biowest, catalog number: S1860 )
  32. Low endotoxin BSA (fraction V) (Euromedex, catalog number: UA1315 )
  33. CFSE (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: C34554 )
  34. Liberase TM (Roche Diagnostics, catalog number: 05401119001 )
  35. DNase I (Roche Diagnostic, catalog number: 04536282001 )
  36. Red blood cell lysis buffer (Sigma-Aldrich, catalog number: R7757 )
  37. EasySepTM Mouse Pan-DC Enrichment Kit (STEMCELL Technologies, catalog number: 19763 )
  38. EasySepTM Mouse Naïve CD8+ T Cell Isolation Kit (STEMCELL Technologies, catalog number: 19858 )
  39. FACS antibodies (all anti-mouse):
    1. CD69-eFluor® 450 (clone H1.2F3, dilution 1/300) (Affymetrix, eBioscience, catalog number: 48-0691-82 )
    2. CD8a-PerCP-Cy5.5 (clone 53-6.7, dilution 1/300) (Affymetrix, eBioscience, catalog number: 45-0081-82 )
    3. TCR vβ 5.1-PE (clone MR9-4, dilution 1/500) (BD, PharmingenTM, catalog number: 553190 )
    4. CD25-FITC (clone 7D4, dilution 1/200) (BD, PharmingenTM, catalog number: 553072 )
    5. CD4-PE-Cy7 (clone RM4-5, dilution 1/300) (BD, PharmingenTM, catalog number: 552775 )
    6. CD25-APC (clone PC61.5, dilution 1/300) (Affymetrix, eBioscience, catalog number: 17-0251-81 )
    7. CD19 eFluor® 450 (clone 1D3, dilution 1/500) (Affymetrix, eBioscience, catalog number: 48-0193 )
    8. CD3 eFluor® 450 (clone 17A2, dilution 1/500) (Affymetrix, eBioscience, catalog number: 48-0032-80 )
    9. CD11c-FITC (clone HL3, dilution 1/500) (BD, PharmingenTM, catalog number: 553801 )
    10. CD11c-APC (clone N418, dilution 1/400) (Affymetrix, eBioscience, catalog number: 17-0114 )
    11. CD8-PE (clone 53-6.7, dilution 1/500) (BD, PharmingenTM, catalog number: 553032 )
    12. CD11b-PE (clone M1/70, dilution 1/300) (Affymetrix, eBioscience, catalog number: 12-0112 )
    13. CD40-PE (clone 3/23, dilution 1/150) (BD, PharmingenTM, catalog number: 553791 )
    14. CD86-PE (clone GL1, dilution 1/300) (Affymetrix, eBioscience, catalog number: 12-0862 )
    15. MHC Class II I-Ab-PE (clone AF6-120.1, dilution 1/300) (Affymetrix, eBioscience, catalog number: 12-5320 )
  40. BMDC culture medium (see Recipes)
  41. B3Z and T cell culture (see Recipes)
  42. Digestion medium for spleens (see Recipes)
  43. MACS buffer (see Recipes)

Equipment

  1. Incubator (37 °C and 5% CO2)
  2. Refrigerated centrifuge for tubes of 2 ml, 15 ml and 50 ml size as well as 96-well plates
  3. Stuart test tube rotator wheel tolerating 4 °C (Bibby Scientific, model: SB3 )
  4. Multicolor flow cytometer (MAQSquant, Miltenyi; FACSverse, Becton Dickinson or similar)
  5. Multicolor FACS sorter (for example, BD, model: FACSAria III )
  6. Fluorometric plate reader (OD: 590 nm)

Software

  1. FlowJo 10 software (FlowJo, LLC.)
  2. Prism 6 software (GraphPad Software, Inc.)

Procedure

Part I. Cross-presentation analysis of bead-bound OVA in BMDC in vitro

  1. Preparation of beads (Day: -1, sterile environment)
    The day before the cross-presentation experiment, particles are coupled to OVA (bead-bound OVA: bbOVA). Polystyrene beads of 3 μm diameter are coated with soluble OVA and soluble BSA ranging from 100% OVA (most concentrated) to 100% BSA (background). Since BSA is not cross-presented, the preparation of beads coated with 100% BSA is the accurate control to check whether beads without any antigen coating are able to induce any additional activation of DC.
    1. Estimate the amount of 3 μm beads that will be used during the cross-presentation experiment on day 0. Consider to prepare a minimum of at least four different conditions:
      1. 100% OVA and 0% BSA: 10 mg/ml OVA
      2. 50% OVA and 50% BSA: 5 mg/ml OVA + 5 mg/ml BSA
      3. 25% OVA and 75% BSA: 2.5 mg/ml OVA + 7.5 mg/ml BSA
      4. 0% OVA and 100% BSA: 10 mg/ml BSA
    2. Dilute the beads differentially depending on the readout. For B3Z T cell hybridomas, beads are diluted 1/10 (which corresponds to a bead/cell ratio of approximately 200), while for OT-I T cells the dilution is 1/25 (which corresponds to a bead/cell ratio of 600).
      The beads are added to BMDC in a final volume of 100 μl. In order to analyze cross-presentation in 10 samples with the 3 different read-outs of this protocol, you will need per condition:
      10 samples for B3Z 1 ml of culture medium 100 μl of beads (1/10 dilution)
      10 samples for OT-I CD69 expression 40 μl of beads (1/25 dilution)
      10 samples for OT-I proliferation 40 μl of beads (1/25 dilution)
      Therefore, a final bead volume of 200-250 μl is needed for 10 samples.
    3. Put a maximum of 250 μl of beads per 2 ml Eppendorf tube, and wash them with 1.5 ml of PBS. Centrifuge at 19,000 x g for 3 min at 4 °C. Repeat washing twice.
      Note: If you are assessing 10 samples with the 3 readouts, and doing 4 different conditions of bbOVA (100%, 50%, 25% and 0%) you should have at this point 4 tubes with 200-250 μl of beads (1 for each condition).
    4. Discard supernatant, and resuspend the beads in the different protein suspensions as shown in the following (250 μl of beads per 750 μl of soluble protein mix):
      Stock concentration of OVA: 50 mg/ml (Working concentration: 10 mg/ml)
      Stock concentration of BSA: 50 mg/ml (Working concentration: 10 mg/ml)
      Cond. 1) 100% OVA: 0% BSA → 150 μl of OVAstock + 600 μl PBS
      Cond. 2) 50% OVA: 50% BSA → 75 μl of OVAstock + 75 μl of BSAstock + 600 μl PBS
      Cond. 3) 25% OVA: 75% BSA → 37.5 μl of OVAstock + 112.5 μl of BSAstock + 600 μl PBS
      Cond. 4) 0% OVA: 100% BSA → 150 μl of BSAstock + 600 μl PBS
    5. Incubate overnight at 4 °C with permanent agitation on a rotator wheel.
  2. Washing the beads (Day: 0, sterile environment)
    1. Spin the bbOVA with 19,000 x g for 5 min at 4 °C. Subsequently, discard supernatant with a pipette trying not to touch the beads.
    2. Resuspend the bbOVA in 1 ml of cold PBS by pipetting. Centrifuge at 19,000 x g for 5 min at 4 °C.
    3. Repeat washing step twice.
    4. Resuspend the bbOVA in the original volume (200-250 μl in this example). The beads are ready to use. Keep bbOVA on ice.
  3. Loading BMDC with bbOVA, Co-culture with T cells (Day: 0, sterile environment)
    Plate in 96-well round bottom plates: 100,000 BMDC per well for B3Z readout and 10,000 BMDC per well for both OT-I readouts (Figure 1).
     

    Figure 1. Scheme of an average 96-well plate loaded with BMDCs. Plate 100,000 BMDCs/well in 50 μl of BMDC culture medium for B3Z readout, or 10,000 cells/well in 50 μl of BMDC culture medium for OT-I readouts. Add the antigen to the right concentration in 50 μl of BMDC culture medium for a final volume of 100 μl. In the scheme, sOVA and SIINFEKL peptide are also analyzed (see Notes 2 and 3). In the scheme, a minimum setup is shown: two different BMDC samples are analyzed, which are plated in duplicates. Additional replicates are suggested to increase robustness of the obtained data.

    1. The correct amount of BMDC is plated in 50 μl of BMDC culture medium per well.
    2. The final dilution of bbOVA in BMDC culture medium is, as mentioned before: 1/10 for B3Z and 1/25 for OT-I. Please consider that 50 μl of bbOVA in BMDC culture medium will be added to 50 μl of BMDC for a final volume of 100 μl per well. Thereby, bbOVA concentration must be twice the final dilution (1/5 for B3Z, 1/12.5 for OT-I).
    3. For the OT-I T cell proliferation readout, incubate BMDC with bbOVA for 1 h at 37 °C. Consequently, cross-presenting BMDC are co-cultured with 60,000-100,000 CFSE-stained OT-I T cells and T cell proliferation is analyzed 72 h later (continues in step 5a).
    4. For B3Z and OT-I CD69 expression readouts, incubate BMDC with bbOVA for 4-5 h at 37 °C.
    5. Wash the cross-presenting BMDC with 100 μl/well of 0.1% PBS-BSA (always keep reagents and solutions on ice) and centrifuge at 800 x g for 2 min at 4 °C. Flick the plate and repeat washing twice.
    6. Fix cross-presenting BMDC: add 50 μl/well of freshly made PBS-glutaraldehyde (GTA) 0.008% (vol/vol) and incubate for 5 min on ice. Mix cells and PBS-GTA by pipetting.
    7. Add 50 μl of PBS-glycine 0.4 M to the PBS-GTA 0.008% solution, and centrifuge at 800 x g for 2 min at 4 °C. Flick the plate.
    8. Add 100 μl of PBS-glycine 0.2 M and centrifuge at 800 x g for 2 min at 4 °C. Flick the plate.
    9. Wash twice with 200 μl/well of B3Z and T cell culture medium (see Recipes). Centrifuge again and flick the plate.
    10. Resuspend fixed, cross-presenting BMDC in 100 μl/well of cell culture medium.
    11. Add 60,000-100,000 effector cells (B3Z or OT-I, see Notes 4 and 5) per well in 100 μl cell culture medium (to a final volume of 200 μl).
    12. Incubate for 16 h at 37 °C for B3Z T cells or for 18-20 h at 37 °C for OT-I T cells.
  4. Analysis of B3Z read-out and CD69 expression of OT-I cells (Day: 1)
    1. B3Z measurement:
      1. Centrifuge at 800 x g for 2 min at 4 °C. Supernatant can be kept for subsequent cytokine analysis.
      2. Wash once with 100 μl/well of 0.1% PBS-BSA, spin down and flick.
      3. Add 120 μl/well of CPRG (Chlorophenol red-β-D-galactopyranoside). Mix properly to resuspend the B3Z cells.
      4. Incubate at 37 °C and check every 30 min until the solution changes its color to orange/red (usually, after 2 h).
      5. Measure OD at 590 nm every 30-60 min until the reaction gets saturated (after 4-6 h).
    2. Measurement of CD69 expression of OT-I cells:
      1. Centrifuge at 800 x g for 2 min at 4 °C. Supernatant can be kept for subsequent cytokine analysis.
      2. Wash once with 100 μl/well of 0.1% PBS-BSA, spin down and flick the plate.
      3. Stain the cells for 15 min at 4 °C with 100 μl/well of fixable viability dye eFluor 780 + Fc block (1/100) in PBS. Centrifuge and flick the plate.
      4. Wash once with 0.1% PBS-BSA, centrifuge and flick the plate.
      5. Stain the cells for 40 min at 4 °C with 70 μl/well of a combination of CD69-eFluor450, CD8a-PerCP-Cy5.5, TCR vβ 5.1-PE, CD25-FITC and CD4-PE-Cy7 diluted in 1% PBS-BSA (dilution factors are provided in Materials and Reagents). Centrifuge and flick the plate.
      6. Wash 3 times with 0.1% PBS-BSA, centrifuge and flick the plate.
      7. Resuspend in 100 μl/well of 0.1% PBS-BSA and analyze by flow cytometry (see Figure 2a).
         

        Figure 2. Gating strategy for cross-presentation analysis. a. Activation of OT-I T cells by measuring CD69 expression at the cell surface. BMDC were incubated 5 h with bbOVA (upper panel) and sOVA (see Note 2) (lower panel), fixed and co-cultured with OT-I T cells. After 16 h, CD69 expression was measured. b. Proliferation of OT-I T cells. BMDC were incubated for 1 h in the presence of bbOVA (upper panel) and sOVA (lower panel) and co-cultured for 72 h with CFSE-labeled OT-I T cells. Proliferation was addressed by measuring loss of CFSE staining by flow cytometry and by calculating the proliferation index (right panel).

  5. Analysis of OT-I T cell proliferation (Day: 3)
    1. Centrifuge at 800 x g for 2 min at 4 °C. Supernatant can be kept for subsequent cytokine analysis.
    2. Wash once with 100 μl/well of 0.1% PBS-BSA, spin down and flick the plate.
    3. Stain the cells for 15 min at 4 °C with 100 μl/well of fixable viability dye eFluor 780 + Fc block (1/100) in PBS. Centrifuge and flick the plate.
    4. Wash once with 0.1% PBS-BSA, centrifuge and flick the plate.
    5. Stain the cells for 40 min at 4 °C with 70 μl/well of a combination of CD8a-PerCP-Cy5.5, TCR vβ 5.1-PE, CD25-APC and CD4-PE-Cy7 diluted in 1% PBS-BSA (dilution factors are provided in Materials and Reagents). Centrifuge and flick the plate.
    6. Wash 3 times with 0.1% PBS-BSA, centrifuge and flick the plate.
    7. Resuspend in 100 μl/well of 0.1% PBS-BSA and analyze by flow cytometry (see Figure 2b).

Part II. Cross-presentation of bbOVA by splenic CD8+ dendritic cells, analyzed ex vivo

In this protocol, blue dyed beads coated with OVA are injected intravenously into mice. Subsequently, after DC have internalized the particulate antigen and cross-presented it in vivo, spleens are harvested and digested. CD11c+ cells from whole digested spleens are then purified, and the enriched CD11c+ fraction is stained. CD8+ DC that have phagocytosed beads are sorted and co-cultured with OT-I T cells for further analysis (Figure 3).
 

Figure 3. Workflow of the antigen cross-presentation approach in vivo

  1. Preparation of beads (Day: -1)
    1. Calculate the number of blue dyed 1.0 μm beads that will be used. 4.5 x 109 beads are injected per mouse (bead stock solution is around 4.5 x 1010 particles/ml). Therefore, 100 μl per mouse are injected.
    2. Wash the beads with 1.5 ml cold PBS (again 250 μl of beads per 2 ml Eppendorf tube). Centrifuge at 19,000 x g for 5 min at 4 °C. Repeat.
    3. Resuspend each 250 μl of beads in 500 μl of OVA 50 mg/ml, and pool them (if needed) into a 15 ml centrifuge tube. Incubate overnight at 4 °C with permanent agitation on a rotator wheel.
  2. Washing the beads (Day: 0)
    1. For washing purposes, divide the beads in 2 ml Eppendorf tubes (250 μl per tube).
    2. Spin the bbOVA at 19,000 x g at 4 °C for 5 min. Subsequently, discard supernatant with a pipette, trying not to touch the beads.
    3. Resuspend the bbOVA in 1.5 ml of cold PBS. Centrifuge at 19,000 x g at 4 °C for 5 min.
    4. Repeat washing step twice.
    5. Resuspend the bbOVA in the original volume (250 μl in this example) and pool them. The beads are ready to use. Store them on ice.
  3. Injection of mice, purification of DC and co-culture with T cells (Day: 0) (see Figure 3)
    1. Inject 100 μl of beads per mouse intravenously (either in the eye vein or in the tail). Use Terumo 1 ml insulin syringes with 25 G needle. Wait 2 h (time for the bbOVA to be phagocytosed and cross-presented).
    2. Sacrifice the mice and harvest spleens.
    3. Flush the spleens in non-treated 6-well plates with 2 ml of digestion medium (see Recipes) per spleen by using 25 G needles and 2.5 ml syringes. Incubate for 15 min at 37 °C.
    4. Mince the spleens with scalpel blades in the same well and incubate for another 15 min. Stop Liberase/DNAse reaction by adding 2 ml of FBS.
    5. Smash and filter the digested spleens through the cell strainer with the flat part of a syringe plunger into 50 ml centrifuge tubes. Centrifuge and discard supernatant.
    6. Lyse red blood cells at RT (5 min of 2 ml of red blood cell lysis buffer per spleen is sufficient). Add 30 ml of PBS and centrifuge (350 x g, 5 min, 4 °C).
    7. Discard supernatant and resuspend each spleen in 600 μl of cold sorting medium (PBS + 0.5 % FBS). Pass the cells through the suggested Miltenyi pre-separation filters (or similar) and collect them in 14 ml tubes for magnetic purification.
    8. In order to isolate CD11c+ cells, we use the EasySep Mouse Pan-DC Enrichment Kit, but any other negative selection-based enrichment procedure might be used. Follow the manufacturer’s indications.
    9. Stain the CD11c+-enriched cell fraction for 40 min at 4 °C with 70 μl/well of a combination of CD19-eFluor 450, DAPI, CD3-eFluor 450, CD11c-FITC and CD8-PE (see Note 2).
    10. Sort the cells following the gating strategy shown in Figure 4.
    11. Plate phagocytic CD8+ DC at different amounts to obtain three different, final DC/T cell ratios:
      30,000 DC/100,000 T cells
      3,000 DC/100,000 T cells
      300 DC/100,000 T cells
    12. Fix CD8+ DC with 0.008% PBS-GTA as described before (Procedure of part I).
    13. Co-culture fixed DCs with 100,000 OT-I T cells to a final volume of 200 μl of B3Z and T cell culture medium.
    14. For T cell analysis, proceed as described before (T cell read-outs from Procedure of part I).


      Figure 4. Gating strategy to sort CD8+ DC from spleen. Total splenic cells were gated in order to exclude B, T and dead cells. Subsequently, gates were performed on CD11c+ and CD8+ cells. CD11c+ CD8+ cells that have phagocytosed at least one bead were sorted and co-cultured with OT-I T cells.

Data analysis

Each single experiment needs to be performed with a sufficient number of technical replicates (at least in triplicates). Conclusions can be drawn from the analysis of an appropriate number of biological replicates. The results shown here were confirmed by a minimum of 3 independent experiments. Analysis of flow cytometry data was performed using FlowJo 10 software (FlowJo, LLC.). Statistical analysis was performed using Prism 6 software (GraphPad Software, Inc.).

Notes

Part I. Cross-presentation analysis of bead-bound OVA in BMDC in vitro

  1. BMDC are generated by culture in GM-CSF-containing medium (see Recipes) for 10 days. Percentages of CD11c and CD11b are always higher than 85% in used cultures.
  2. Cross-presentation of soluble OVA (sOVA) can be measured in parallel. In our lab, we generally use sOVA ranging from 2 mg/ml to 0 mg/ml (4 different concentrations are usually sufficient).
  3. The best control for cross-presentation, to our knowledge, is the OVA peptide 257-264 (SIINFEKL). This peptide is incorporated into the cell and loaded onto MHC class I molecules without being processed. We strongly recommend using such a control in different dilutions (a range from 3 ng/ml to 0.001 ng/ml is sufficient) for all cross-presentation assays.
  4. Culturing B3Z T cells: Grow B3Z T cells in B3Z and T cell culture medium (see Recipes), split cells every 2-3 days; detach the cells manually by hitting smoothly against the flask. The day before the cross-presentation experiment, seed them at a concentration of 0.1 x 106 cells/ml. Co-culture them with cross-presenting BMDC. After 16 h, T cell activation is measured detecting β-galactosidase activity by optical density at 590 nm using CPRG as substrate for the reaction.
  5. In order to get OT-I T cells for co-culture with BMDC, we use kits for mouse naïve CD8+ T cell purification (obtained either from Stemcell or Miltenyi) of spleens and lymph nodes of OT-I mice. We use MACS buffer (recipes 4) for washing purposes.
  6. To obtain CFSE-labeled OT-I T cells, resuspend 1 x 107 cells/ml of CD8+ T cells and label them with 10 μM of CFSE proliferative dye for 10 min at room temperature in the dark. Wash cells once with 10 ml of PBS and resuspend in sterile PBS to a concentration of 1 x 107 cells/ml.

Part II. Cross-presentation of bbOVA by splenic CD8+ dendritic cells, analyzed ex vivo

  1. Cross-presentation of sOVA can be measured in parallel. Consider injecting 100 μl of OVA (i.v.) at a concentration of 20 mg/ml. Wait for 30 min instead of 2 h and harvest spleens to purify cross-presenting DC.
  2. With the labeling of CD19, CD3 and DAPI, it is possible to exclude B, T and dead cells, respectively, from the analysis. Be aware that blue dyed beads are visible in both, the APC and the APC-Cy7 channel. No fluorophore should be used in those channels.
  3. Consider that the proportion of phagocytic CD8+ DC in spleen is really low. You should get around 100,000-200,000 cells per spleen. While this amount is enough for OT-I T cells co-culture (to analyze both OT-I CD69 expression and CFSE OT-I proliferation), co-culture with B3Z T cells might require the sacrifice of many more mice as well as many hours of cell sorting. By the time you will get sufficient cells for B3Z analysis, they might be already dead. Therefore, we suggest to not address this readout for ex vivo analysis of cross-presentation.

Recipes

  1. BMDC culture medium
    IMDM containing 10% heat-inactivated FBS
    100 IU/ml penicillin
    100 μg/ml streptomycin
    2 mM glutamax
    50 μM β-mercaptoethanol
    1x MEM non-essential amino acids
    1x sodium pyruvate
    Supernatant from J558 plasmacytoma cells was used as GM-CSF source (Winzler et al., 1997).
  2. B3Z and T cell culture
    RPMI containing 10% heat-inactivated FBS
    100 IU/ml penicillin
    100 μg/ml streptomycin
    2 mM glutamax
    50 μM β-mercaptoethanol
    1x MEM non-essential amino acids
    1x sodium pyruvate
  3. Digestion medium for spleens
    Liberase 0.2 mg/ml (resuspend one bottle of 5 mg Liberase in 3 ml of RPMI-1640, which is sufficient for 15 spleens; use 0.2 ml of this solution per spleen) and DNase I 0.1 mg/ml in RPMI-1640 (final volume: 1.5 ml per spleen).
  4. MACS buffer
    1x PBS
    2% FBS (or 2% BSA)
    5 mM EDTA

Acknowledgments

This work was supported by the French National Research Agency through the ‘Investments for the Future’ program (France-BioImaging, ANR-10-INSB-04), ANR-11-LABX-0043 and by the CelTisPhyBio Labex (N- ANR-10-LBX-0038), part of the IDEX PSL (ANR-10-IDEX-0001-02 PSL). We are grateful to the financial support by the European Research Council (2013-AdG No.340046 DCBIOX), by La Ligue Nationale contre le Cancer (EL2014.LNCC/SA), by Fonds Wetenschappelijk Onderzoek (FWO; 1526615N), by an EMBO long-term fellowship (ALTF 883-2011) and by fellowships of Fondation Recherche Médicale (SPF20101221176) and the omics@VIB program (co-financed by the Marie Curie FP7 People Cofund).

References

  1. Alloatti, A., Kotsias, F., Pauwels, A. M., Carpier, J. M., Jouve, M., Timmerman, E., Pace, L., Vargas, P., Maurin, M., Gehrmann, U., Joannas, L., Vivar, O. I., Lennon-Dumenil, A. M., Savina, A., Gevaert, K., Beyaert, R., Hoffmann, E. and Amigorena, S. (2015). Toll-like receptor 4 engagement on dendritic cells restrains phago-lysosome fusion and promotes cross-presentation of antigens. Immunity 43(6): 1087-1100.
  2. Crozat, K., Tamoutounour, S., Vu Manh, T. P., Fossum, E., Luche, H., Ardouin, L., Guilliams, M., Azukizawa, H., Bogen, B., Malissen, B., Henri, S. and Dalod, M. (2011). Cutting edge: expression of XCR1 defines mouse lymphoid-tissue resident and migratory dendritic cells of the CD8α+ type. J Immunol 187(9): 4411-4415.
  3. Dorner, B. G., Dorner, M. B., Zhou, X., Opitz, C., Mora, A., Guttler, S., Hutloff, A., Mages, H. W., Ranke, K., Schaefer, M., Jack, R. S., Henn, V. and Kroczek, R. A. (2009). Selective expression of the chemokine receptor XCR1 on cross-presenting dendritic cells determines cooperation with CD8+ T cells. Immunity 31(5): 823-833.
  4. Joffre, O. P., Segura, E., Savina, A. and Amigorena, S. (2012). Cross-presentation by dendritic cells. Nat Rev Immunol 12(8): 557-569.
  5. 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.
  6. Kurts, C., Heath, W. R., Carbone, F. R., Allison, J., Miller, J. F. and Kosaka, H. (1996). Constitutive class I-restricted exogenous presentation of self antigens in vivo. J Exp Med 184(3): 923-930.
  7. Winzler, C., Rovere, P., Rescigno, M., Granucci, F., Penna, G., Adorini, L., Zimmermann, V. S., Davoust, J. and Ricciardi-Castagnoli, P. (1997). Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures. J Exp Med 185(2): 317-328.

简介

通过MHC I类分子的抗原呈递,也称为交叉呈递,引起细胞毒性免疫应答。特别地,树突细胞(DC)是最熟练的交叉呈递细胞,因为它们已经开发了控制吞噬和降解途径的独特手段。 <此协议允许在体外(通过使用骨髓衍生的DC)和离体(通过纯化CD8 + )评价抗原交叉呈递。 (OVA)偶联的颗粒后,来自脾脏的DC/DC结合颗粒抗原)。通过三种不同的读数测量交叉呈递效率:B3Z杂交瘤T细胞系(Karttunen等人,1992)和抗原特异性CD8 + T细胞的刺激OT-I)(Kurts等人,1996),在用羧基荧光素琥珀酰亚胺酯(CFSE)标记它们之后分析CD69表达或OT-I增殖的OT-I活化。通过使用这种方法,我们可以最近显示DCs能够在TLR4结合时瞬时增加交叉表达效率(Alloatti等人,2015)。

[背景] 在小鼠中,抗原呈递细胞(APC)能够吸收外源抗原以加工它们,并将源自这些外源抗原的肽加载到主要组织相容性复合体(MHC)I类分子上。肽-MHC I复合物随后被转运到质膜,在那里它们可以呈递到CD8 + T细胞,从而促进T细胞活化,这被称为交叉呈递(Joffre等人, al 。,2012)。在不同的APC中,树突状细胞(DC)在交叉呈递方面表现优异,并且包含表达XCR1标记的不同亚群,其已经显示非常有效地交叉呈递抗原(即CD8 + 来自脾的DC和来自皮肤和肺的CD103 + 迁移DC)(Dorner等人,2009; Crozat等人, ,2011)。虽然存在于脾或迁移性DC中的DC的纯化是可行的,但是费力和昂贵。为了研究DC的细胞生物学,骨髓衍生的DC(BMDC)的原代培养物可以通过用GM-CSF培养而容易地与骨髓祖细胞分化。即使BMDC不能与任何特定的DC亚型(可能是炎症性DC)相关联,它们构成了研究DC细胞生物学的主要特征的有价值的工具。在这里,我们介绍一个详细的协议,以分析颗粒抗原的交叉表示BMDC,但也由CD8支持+脾DC。虽然以前的协议包括不同的抗原形式和读数,这里描述的协议旨在以一种全面和简洁的方式在不同的直流类型分析交叉呈现。

材料和试剂

  1. 2 ml Eppendorf管
  2. 15ml离心管
  3. 50ml离心管
  4. 14 ml管
  5. Fisherbrand 细胞滤网(Thermo Fisher Scientific,Fisher Scientific,目录号:22-363-548)
  6. 预分离过滤器(30μm)(Miltenyi Biotec,目录号:130-041-407)
  7. 1ml胰岛素注射器(Terumo Medical,目录号:SS + 01H1)
  8. 2.5 ml注射器
  9. 25G针(Terumo,目录号:AN * 2516R1)
  10. 未处理的96孔板(Coring,Falcon ,圆底,目录号:351177)
  11. 未处理的6孔板(Sigma-Aldrich,目录号:M8562-100EA)
  12. 未处理的培养皿,145×20mm(Greiner Bio One,目录号:639161)
  13. 小鼠:从Charles River Laboratories(CDTA)获得C57BL/6和C57BL/6重组激活基因1-缺陷型OT-I TCR(Vα2,Vβ5) ,Orleans,France)
  14. B3Z T细胞系(Kb限制性,OVA特异性CD8 + T细胞杂交瘤)(Kurts等人,1996)
  15. 低内毒素OVA(50mg/ml母液)(Worthington Biochemical,目录号:LS003062)
  16. OVA肽257-264(SIINFEKL)(多肽,目录号:SC1302)
  17. Polybead聚苯乙烯3.0微米微球(Polysciences,目录号:17134)
  18. Polybead染色的蓝色1.0微米微球(Polysciences,目录号:15712)
  19. PBS(1x,pH 7.4)(Thermo Fisher Scientific,Gibco TM ,目录号:10010-023)
  20. 甘氨酸(Sigma-Aldrich,目录号:50046)
  21. 戊二醛(25%)(Sigma-Aldrich,目录号:G5882)
  22. Iscove's改良的Dulbecco培养基(IMDM)(Sigma-Aldrich,目录号:I3390-500ML)
  23. 青霉素 - 链霉素(10,000U/ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  24. RPMI与GlutaMAX TM (Thermo Fisher Scientific,目录号:61870-010)
  25. GlutaMAX TM补充(100x)(Thermo Fisher Scientific,Gibco TM ,目录号:35050061)
  26. β-巯基乙醇(Thermo Fisher Scientific,Gibco TM ,目录号:21985-023)
  27. MEM非必需氨基酸溶液(100x)(Thermo Fisher Scientific,Gibco TM ,目录号:11140050)
  28. 丙酮酸钠(100mM)(Thermo Fisher Scientific,Gibco< sup> TM,目录号:11360070)
  29. CPRG(Roche Diagnostics,目录号:10884308001)
  30. 可固定活力染料eFluor 780(稀释1/10,000)(Affymetrix,eBioscience,目录号:65-0865-14)
  31. 低内毒素胎牛血清(FBS,在56℃热灭活20分钟)(Biowest,目录号:S1860)
  32. 低内毒素BSA(级分V)(Euromedex,目录号:UA1315)
  33. CFSE(Thermo Fisher Scientific,Molecular Probes TM ,目录号:C34554)
  34. Liberase TM(Roche Diagnostics,目录号:05401119001)
  35. DNase I(Roche Diagnostic,目录号:04536282001)
  36. 红细胞裂解缓冲液(Sigma-Aldrich,目录号:R7757)
  37. EasySep TM 鼠标Pan-DC富集试剂盒(STEMCELL Technologies,目录号:19763)
  38. EasySep TM Mouse Naive CD8 + T细胞分离试剂盒(STEMCELL Technologies,目录号:19858)
  39. FACS抗体(所有抗小鼠):
    1. CD69-eFluor 450(克隆H1.2F3,稀释度1/300)(Affymetrix,eBioscience,目录号:48-0691-82)
    2. CD8a-PerCP-Cy5.5(克隆53-6.7,稀释1/300)(Affymetrix,eBioscience,目录号:45-0081-82)
    3. TCRvβ5.1-PE(克隆MR9-4,稀释度1/500)(BD,Pharmingen TM ,目录号:553190)
    4. CD25-FITC(克隆7D4,稀释1/200)(BD,Pharmingen TM ,目录号:553072)
    5. CD4-PE-Cy7(克隆RM4-5,稀释1/300)(BD,Pharmingen ,目录号:552775)
    6. CD25-APC(克隆PC61.5,稀释度1/300)(Affymetrix,eBioscience,目录号:17-0251-81)
    7. CD19 eFluor 450(克隆1D3,稀释1/500)(Affymetrix,eBioscience,目录号:48-0193)
    8. CD3克隆(克隆17A2,稀释1/500)(Affymetrix,eBioscience,目录号:48-0032-80)
    9. CD11c-FITC(克隆HL3,稀释1/500)(BD,Pharmingen TM ,目录号:553801)
    10. CD11c-APC(克隆N418,稀释1/400)(Affymetrix,eBioscience,目录号:17-0114)
    11. CD8-PE(克隆53-6.7,稀释1/500)(BD,Pharmingen TM ,目录号:553032)
    12. CD11b-PE(克隆M1/70,稀释度1/300)(Affymetrix,eBioscience,目录号:12-0112)
    13. CD40-PE(克隆3/23,稀释度1/150)(BD,Pharmingen TM ,目录号:553791)
    14. CD86-PE(克隆GL1,稀释度1/300)(Affymetrix,eBioscience,目录号:12-0862)
    15. MHC II类I-Ab-PE(克隆AF6-120.1,稀释1/300)(Affymetrix,eBioscience,目录号:12-5320)
  40. BMDC培养基(见配方)
  41. B3Z和T细胞培养(见配方)
  42. 脾消化介质(见配方)
  43. MACS缓冲区(参见配方)

设备

  1. 培养箱(37℃和5%CO 2)
  2. 用于2ml,15ml和50ml大小的管以及96孔板的冷冻离心机
  3. Stuart试管旋转轮耐受4°C(Bibby Scientific,型号:SB3)
  4. 多色流式细胞仪(MAQSquant,Miltenyi; FACSverse,Becton Dickinson或类似物)
  5. 多色FACS分选机(例如,BD,型号:FACSAria III)
  6. 荧光读板仪(OD:590nm)

软件

  1. FlowJo 10软件(FlowJo,LLC。)
  2. Prism 6软件(GraphPad Software,Inc。)

程序

第一部分。在体外BMDC中珠结合的OVA的交叉呈递分析

  1. 珠子的制备(天:-1,无菌环境)
    交叉呈递实验前一天,将颗粒偶联到OVA(珠粒结合的OVA:bbOVA)。直径为3μm的聚苯乙烯珠粒用范围从100%OVA(最浓)至100%BSA(背景)的可溶性OVA和可溶性BSA包被。因为BSA不是交叉呈递的,所以用100%BSA包被的珠的制备是检查没有任何抗原涂层的珠是否能够诱导任何额外的DC活化的精确对照。
    1. 估计在第0天在交叉呈递实验期间使用的3μm珠的量。考虑至少准备至少四种不同条件:
      1. 100%OVA和0%BSA:10mg/ml OVA
      2. 50%OVA和50%BSA:5mg/ml OVA + 5mg/ml BSA
      3. 25%OVA和75%BSA:2.5mg/ml OVA + 7.5mg/ml BSA
      4. 0%OVA和100%BSA:10mg/ml BSA
    2. 根据读数差异稀释珠子。对于B3Z T细胞杂交瘤,将珠稀释1/10(其对应于约200的珠/细胞比),而对于OT-I T细胞,稀释为1/25(其对应于珠/细胞比率600 )。
      将珠子加入到BMDC中,终体积为100μl。为了分析10个样品中的交叉呈现,使用此协议的3种不同的读数,您将需要每个条件:
      10个样本用于B3Z span> 1ml培养基100μl珠子(1/10稀释)
      OT-I CD69表达的10个样品40μl珠子(1/25稀释)
      因此,10个样品需要200-250μl的最终珠体积
    3. 每2毫升Eppendorf管最多250微升珠,并用1.5毫升PBS洗涤。在4℃下以19,000×g离心3分钟。重复清洗两次。
      注意:如果你用3个读数评估10个样品,并做4个不同的bbOVA条件(100%,50%,25%和0%),你应该在这一点有4个管,珠子(每种条件1个)。
    4. 弃去上清液,并将珠子悬浮在如下所示的不同蛋白质悬浮液中(250μl珠子/750μl可溶性蛋白质混合物):
      OVA原液浓度:50mg/ml(工作浓度:10mg/ml)
      BSA原液浓度:50mg/ml(工作浓度:10mg/ml)
      条件。 1)100%OVA:0%BSA→150μlOVA sub-stock +600μlPBS
      条件。 2)50%OVA:50%BSA→75μlOVA sub-stock +75μlBSA sub-stock +600μlPBS
      条件。 3)25%OVA:75%BSA→37.5μlOVA原液+112.5μlBSA缓冲液+600μlPBS
      条件。 4)0%OVA:100%BSA→150μlBSA缓冲液+600μlPBS
    5. 在4℃下在旋转轮上永久搅拌孵育过夜。
  2. 洗涤珠子(日:0,无菌环境)
    1. 在4℃下用19,000×g的bbOVA旋转5分钟。随后,用移液管丢弃上清液,试图不接触珠子
    2. 通过吸移重悬在1ml冷PBS中的bbOVA。在4℃下以19,000xg离心5分钟。
    3. 重复洗涤步骤两次。
    4. 将bbOVA重悬于原始体积(本例中为200-250μl)。珠子准备使用。保持bbOVA在冰上。
  3. 用bbOVA装载BMDC,与T细胞共培养(日:0,无菌环境)
    96孔圆底板中的板:对于B3Z读出为100,000个BMDC /孔,对于OT-I读出为10,000个BMDC /孔(图1)。  

    图1.装载有BMDCs的平均96孔板的方案。在50μl用于B3Z读出的BMDC培养基中铺板100,000 BMDCs /孔,或在50μlBMDC培养基中以10,000细胞/孔用于OT-I读数。添加抗原到正确的浓度在50微升的BMDC培养基,最终体积为100微升。在该方案中,还分析了sOVA和SIINFEKL肽(参见注释2和3)。在该方案中,显示了最小设置:分析两个不同的BMDC样品,其被重复铺板。建议使用其他重复,以增加获得的数据的鲁棒性
    1. 将正确量的BMDC接种在每孔50μlBMDC培养基中
    2. 如前所述,BMDC培养基中bbOVA的最终稀释:B3Z为1/10,OT-I为1/25。请考虑在BMDC培养基中的50微升bbOVA将添加到50微升的BMDC中,每孔最终体积为100微升。因此,bbOVA浓度必须是最终稀释度的两倍(B3Z为1/5,OT-I为1/12.5)。
    3. 对于OT-I T细胞增殖读出,将BMDC与bbOVA在37℃温育1小时。因此,交叉呈递BMDC与60,000-100,000个CFSE染色的OT-I T细胞共培养,72小时后分析T细胞增殖(在步骤5a中继续)。
    4. 对于B3Z和OT-I CD69表达读数,将BMDC与bbOVA在37℃孵育4-5小时。
    5. 用100μl/孔的 0.1% PBS-BSA(总是将试剂和溶液保持在冰上)并在4℃下以800×g离心2分钟。轻拂板,重复洗涤两次。
    6. 固定交叉呈递BMDC:添加50μl/孔新鲜制备的0.008%(体积/体积)的PBS-戊二醛(GTA),并在冰上孵育5分钟。通过移液混合细胞和PBS-GTA
    7. 向PBS-GTA 0.008%溶液中加入50μl的PBS-甘氨酸0.4M,并在4℃下以800×g离心2分钟。轻推板。
    8. 加入100μlPBS-甘氨酸0.2M,并在4℃下以800×g离心2分钟。轻推板。
    9. 用200μl/孔的B3Z和T细胞培养基洗涤两次(见Recipes)。再次离心并轻弹板。
    10. 重悬在100微升/孔的细胞培养基中固定,交叉呈递BMDC
    11. 在100μl细胞培养基(至终体积200μl)中每孔加入60,000-100,000个效应细胞(B3Z或OT-I,参见注释4和5)。
    12. 在37℃下对于B3Z T细胞孵育16小时或在37℃下对OT-I T细胞孵育18-20小时。
  4. 分析OT-I细胞的B3Z读出和CD69表达(天:1)
    1. B3Z测量:
      1. 在4℃下以800×g离心2分钟。可以保留上清液用于随后的细胞因子分析
      2. 用100μl/孔的
      3. 在4℃下以800×g离心2分钟。可以保留上清液用于随后的细胞因子分析
      4. 用100μl/孔的 0.1% PBS-BSA,离心并轻弹板
      5. 将细胞在4℃下用70μl/孔的在 1%  PBS -BSA(稀释因子在材料和试剂中提供)。离心并轻弹板。
      6. 0.1% ; PBS-BSA,离心并轻弹板
      7. 重悬于100μl/孔的
        图2.交叉表示分析的门控策略。通过测量细胞表面的CD69表达来激活OT-I T细胞。 BMDC与固定并与OT-I T细胞共培养的bbOVA(上图)和sOVA(参见注释2)(下图)孵育5小时。 16小时后,测量CD69表达。 b。 OT-I T细胞的增殖。 BMDC在bbOVA(上图)和sOVA(下图)存在下孵育1小时,并与CFSE标记的OT-I T细胞共培养72小时。通过用流式细胞术测量CFSE染色的损失和通过计算增殖指数来解决增殖(右图)。
  5. OT-I T细胞增殖分析(天:3)
    1. 在4℃下以800×g离心2分钟。可以保留上清液用于随后的细胞因子分析
    2. 用100μl/孔的 0.1% PBS-BSA,离心并轻弹板
    3. 染色细胞在4℃下40分钟,用70微升/孔的CD8a-PerCP-Cy5.5,TCR v6.1-PE,CD25-APC和CD4-PE-Cy7的组合以 1%  PBS-BSA因素在材料和试剂中提供)。离心并轻弹板。
    4. 0.1% ; PBS-BSA,离心并轻弹板
    5. 重悬于100μl/孔的 第二部分。 bbOVA由脾CD8 + 树突状细胞的交叉呈递,离体分析

      在该方案中,用OVA包被的蓝色染色珠被静脉内注射到小鼠中。随后,在DC已经内化颗粒抗原并在体内交叉呈递之后,收获脾并消化。然后纯化来自完整消化的脾的CD11c +细胞,并将富集的CD11c +级分染色。将具有吞噬珠粒的CD8 + sup/+ DC分选并与OT-I T细胞共培养用于进一步分析(图3)。
       

      图3.抗原交叉表现方法在体内的工作流程


      1. 珠的制备(天:-1)
        1. 计算将使用的蓝色染色的1.0μm珠的数量。每只小鼠注射4.5×10 9个珠(珠储备溶液为约4.5×10 10个颗粒/ml)。因此,每只小鼠注射100μl
        2. 用1.5ml冷PBS(再次每250ml珠子每2ml Eppendorf管)洗涤珠。在4℃下以19,000×g离心5分钟。重复。
        3. 重悬每个250微升的珠子在500微升的OVA 50毫克/毫升,并将他们(如果需要)将其合并到15毫升离心管。在4℃下在旋转轮上永久搅拌孵育过夜。
      2. 洗涤珠子(天:0)
        1. 为了洗涤目的,将珠子在2ml Eppendorf管(每管250μl)中分开
        2. 在4℃下将bboOVA以19,000×g离心5分钟。随后,用移液管丢弃上清液,试图不接触珠子
        3. 将bbOVA重悬于1.5ml冷PBS中。在4℃下以19,000×g离心5分钟
        4. 重复洗涤步骤两次。
        5. 将bbOVA重悬在原始体积(在本实施例中为250μl)中并混合。珠子准备使用。将它们储存在冰上。
      3. 注射小鼠,纯化DC并与T细胞共培养(Day:0)(参见图3)
        1. 每只小鼠静脉注射(在眼静脉或尾部)100微升珠。使用Terumo 1毫升胰岛素注射器与25 G针。等待2小时(bbOVA被吞噬和交叉呈递的时间)
        2. 牺牲老鼠和收获脾脏。
        3. 通过使用25G针头和2.5ml注射器,在每个脾脏中用2ml消化培养基(参见Recipes)冲洗未处理的6孔板中的脾脏。在37℃下孵育15分钟。
        4. 用同一个孔中的解剖刀刀片剁脾脏,再孵育15分钟。通过加入2ml FBS停止Liberase/DNAse反应
        5. 粉碎和过滤消化的脾通过细胞过滤器与注射器柱塞的平坦部分到50ml离心管中。离心并弃去上清液。
        6. 在室温溶解红细胞(每脾脏2ml红细胞裂解缓冲液5分钟就足够了)。加入30ml PBS并离心(350×g,5分钟,4℃)。
        7. 弃去上清液并将每个脾脏重悬于600μl冷分选培养基(PBS + 0.5%FBS)中。让细胞通过建议的Miltenyi预分离过滤器(或类似的),并收集在14毫升磁力净化管中。
        8. 为了分离CD11c + 细胞,我们使用EasySep Mouse Pan-DC富集试剂盒,但可以使用任何其他基于负选择的富集程序。按照制造商的说明。
        9. 将CD11c +富含的细胞级分在4℃下用70μl/孔的CD19-eFluor 450,DAPI,CD3-eFluor 450,CD11c-FITC和CD8-PE的组合染色40分钟(见注2)。
        10. 按照图4所示的门控策略对单元格进行排序。
        11. 以不同的量平板吞噬CD8 + DC,以获得三种不同的最终DC/T细胞比率:
          30,000 DC/100,000 T细胞
          3,000 DC/100,000 T细胞
          300 DC/100,000 T细胞
        12. 如前所述(第I部分的程序),用0.008%PBS-GTA固定CD8 + DC。
        13. 共培养将具有100,000个OT-I T细胞的DC固定至最终体积为200μl的B3Z和T细胞培养基。
        14. 对于T细胞分析,如前所述进行(来自第I部分的程序的T细胞读数)

          图4.对来自脾脏的CD8 + DC进行分选的门控策略。对脾脏细胞进行门控以排除B,T和死细胞。随后,在CD11c +和CD8 +细胞上进行门控。将已经吞噬了至少一个珠的CD11c +/CD8 +细胞分选并与OT-I T细胞共培养。

      数据分析

      每个单个实验需要以足够数量的技术重复(至少一式三份)进行。结论可以从适当数量的生物学重复的分析中得出。这里显示的结果通过最少3次独立实验确认。使用FlowJo 10软件(FlowJo,LLC。)进行流式细胞术数据的分析。使用Prism 6软件(GraphPad Software,Inc。)进行统计分析。

      笔记

      第一部分。在体外BMDC中珠结合的OVA的交叉呈递分析

      1. BMDC通过在含有GM-CSF的培养基(参见Recipes)中培养10天而产生。在使用的培养物中,CD11c和CD11b的百分比总是高于85%
      2. 可以并行测量可溶性OVA(sOVA)的交叉呈递。在我们的实验室中,我们通常使用2mg/ml至0mg/ml的sOVA(4种不同的浓度通常就足够了)。
      3. 据我们所知,交叉呈递的最佳对照是OVA肽257-264(SIINFEKL)。将该肽掺入细胞中并加载到MHC I类分子上而不进行处理。我们强烈建议在所有交叉表达试验中使用不同稀释度的这种对照(范围从3 ng/ml到0.001 ng/ml就足够了)。
      4. 培养B3Z T细胞:在B3Z和T细胞培养基(参见Recipes)中培养B3Z T细胞,每2-3天分裂细胞;通过平稳地撞击烧瓶手动分离细胞。在交叉呈递实验前一天,以0.1×10 6个细胞/ml的浓度接种它们。与交叉呈递BMDC共培养他们。 16小时后,使用CPRG作为反应的底物,通过590nm的光密度检测β-半乳糖苷酶活性来测量T细胞活化。
      5. 为了获得用于与BMDC共培养的OT-I T细胞,我们使用用于小鼠幼稚CD8 + T细胞纯化的试剂盒(从Stemcell或Miltenyi获得)在OT-I T细胞的脾和淋巴结上, I小鼠。我们使用MACS缓冲液(配方4)进行洗涤
      6. 为了获得CFSE标记的OT-I T细胞,重悬悬浮1×10 7个细胞/ml的CD8 + T细胞,并用10μM的CFSE增殖染料标记10在室温下在黑暗中。用10ml PBS洗涤细胞一次,并在无菌PBS中重悬至浓度为1×10 7个细胞/ml。

      第二部分。通过脾CD8 +树突状细胞交叉呈递bbOVA,离体分析

      1. sOVA的交叉呈递可以并行测量。考虑注射100μl浓度为20mg/ml的OVA(i.v.)。等待30分钟而不是2小时,收获脾脏净化交叉呈递DC
      2. 使用CD19,CD3和DAPI的标记,可以分别从分析中排除B,T和死细胞。请注意,蓝色染色的珠子在APC和APC-Cy7通道都是可见的。在这些通道中不应使用荧光团。
      3. 考虑到脾脏中吞噬CD8 + sup + DC的比例确实很低。你应该每脾脏约100,000-200,000个细胞。尽管该量足以用于OT-I T细胞共培养(分析OT-I CD69表达和CFSE OT-I增殖),但与B3Z T细胞的共培养可能需要更多的小鼠的牺牲以及许多小时的细胞分选。当你得到足够的细胞进行B3Z分析时,他们可能已经死了。因此,我们建议不要针对交叉表示的离体分析解决此读出

      食谱

      1. BMDC培养基
        含有10%热灭活FBS的IMDM
        100 IU/ml青霉素
        100μg/ml链霉素 2mM glutamax
        50μMβ-巯基乙醇 1×MEM非必需氨基酸
        1×丙酮酸钠
        来自J558浆细胞瘤细胞的上清液用作GM-CSF来源(Winzler等人,1997)。
      2. B3Z和T细胞培养物
        含有10%热灭活的FBS的RPMI 100 IU/ml青霉素
        100μg/ml链霉素 2mM glutamax
        50μMβ-巯基乙醇 1×MEM非必需氨基酸
        1×丙酮酸钠
      3. 脾消化介质
        缓冲液0.2mg/ml(在每个脾脏中再悬浮一瓶5mg Liberase在3ml RPMI-1640中,对于15个脾脏是足够的;在每个脾脏中使用0.2ml该溶液)和DNase I 0.1mg/ml在RPMI-1640体积:每脾1.5ml)
      4. MACS缓冲区
        1x PBS
        2%FBS(或2%BSA) 5 mM EDTA

      致谢

      这项工作由法国国家研究机构通过"未来投资"计划(法国生物成像,ANR-10-INSB-04),ANR-11-LABX-0043和CelTisPhyBio Labex(N-ANR- 10-LBX-0038),IDEX PSL的一部分(ANR-10-IDEX-0001-02 PSL)。我们感谢欧洲研究委员会(2013-AdG No.340046 DCBIOX),La Ligue Nationale Conte le Cancer(EL2014.LNCC/SA),Fonds Wetenschappelijk Onderzoek(FWO; 1526615N)的资助,EMBO长期研究金(ALTF 883-2011)以及Fondation RechercheMédicale(SPF20101221176)和omics @ VIB计划(由Marie Curie FP7 People Cofund共同资助)的奖学金。

      参考文献

      1. Alloatti,A.,Kotsias,F.,Pauwels,AM,Carpier,JM,Jouve,M.,Timmerman,E.,Pace,L.,Vargas,P.,Maurin,M.,Gehrmann,U.,Joannas, L.,Vivar,OI,Lennon-Dumenil,AM,Savina,A.,Gevaert,K.,Beyaert,R.,Hoffmann,E.and Amigorena,S。(2015)。< a class ="ke-插入文件"href ="http://www.ncbi.nlm.nih.gov/pubmed/26682983"target ="_ blank">树突状细胞上的Toll样受体4接合抑制结肠溶酶体融合并促进抗原的交叉呈递。 免疫力 43(6):1087-1100。
      2. Croizat,K.,Tamoutounour,S.,Vu Manh,TP,Fossum,E.,Luche,H.,Ardouin,L.,Guilliams,M.,Azukizawa,H.,Bogen,B.,Malissen, Henri,S.和Dalod,M。(2011)。  切割边缘:XCR1的表达定义了CD8α + 型的小鼠淋巴样组织驻留和迁移树突状细胞。 J Immunol 187(9):4411-
      3. Dorner,BG,Dorner,MB,Zhou,X.,Opitz,C.,Mora,A.,Guttler,S.,Hutloff,A.,Mages,HW,Ranke,K.,Schaefer, ,Henn,V。和Kroczek,RA(2009)。  趋化因子受体XCR1在交叉呈递树突细胞上的选择性表达决定了与CD8 + T细胞的合作。免疫 31(5):823-833。
      4. Joffre,OP,Segura,E.,Savina,A.和Amigorena,S。(2012)。  通过树突细胞的交叉呈递。 Nat Rev Immunol 12(8):557-569。
      5. Karttunen,J.,Sanderson,S.和Shastri,N。(1992)。 
      6. Kurts,C.,Heath,WR,Carbone,FR,Allison,J.,Miller,JF和Kosaka,H。(1996)。  自体抗原的组成型I型限制性外源性表达在体内。 em> 184(3):923-930。
      7. Winzler,C。,Rovere,P.,Rescigno,M.,Granucci,F.,Penna,G.,Adorini,L.,Zimmermann,VS,Davoust,J.and Ricciardi-Castagnoli, ; 成熟阶段的小鼠树突状细胞生长因子依赖的长期, 185(2):317-328。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Alloatti, A., Kotsias, F., Hoffmann, E. and Amigorena, S. (2016). Evaluation of Cross-presentation in Bone Marrow-derived Dendritic Cells in vitro and Splenic Dendritic Cells ex vivo Using Antigen-coated Beads. Bio-protocol 6(22): e2015. DOI: 10.21769/BioProtoc.2015.
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