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Differentiation of Myeloid-derived Suppressor Cells from Murine Bone Marrow and Their Co-culture with Splenic Dendritic Cells
源自小鼠骨髓的骨髓源性抑制细胞的分化及其与脾树突状细胞的共培养   

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Immunity
Feb 2017

Abstract

Myeloid-derived suppressor cells (MDSCs) possess the ability to suppress the immune response, and to amplify the regulatory properties of other immune cells, i.e., dendritic cells. Here we describe a protocol in which MDSCs were differentiated from murine bone marrow cells, and CD11c+ dendritic cells were purified from murine spleens. MDSCs and CD11c dendritic cells can be co-cultured and the immunoregulatory phenotype of the MDSCs-conditioned dendritic cells could be assessed by means of a specific functional in vivo experiment, i.e., a skin test as a measure of the delayed-type hypersensitivity reaction toward a poorly immunogenic antigen.

Keywords: Myeloid-derived suppressor cells (骨髓源性抑制细胞), Dendritic cells (树突状细胞), Co-culture (共培养)

Background

The myeloid-derived suppressor cells (MDSCs) are a group of myeloid cells comprised of precursor of macrophages, granulocytes, dendritic cells and myeloid cells at earlier stages of differentiation (Youn et al., 2008) accumulating in large numbers in lymphoid tissues of tumor-bearing mice as well as in mice with infectious diseases, sepsis and trauma. The main feature of these cells is their ability to suppress T cell responses in Ag-specific and/or nonspecific fashion. These cells are now considered as one of the major cell type responsible for tumor-associated immune defects; main factors implicated in MDSC-mediated immune suppression include high expression of Arg1 (Marvel and Gabrilovich, 2015). Arginase 1 (Arg1) and indoleamine 2,3-dioxygenase 1 (IDO1) are immunoregulatory enzymes catalyzing the degradation of L-arginine (L-Arg) and L-tryptophan (L-Trp), respectively, resulting in local amino acid deprivation. In addition, unlike Arg1, IDO1 is also endowed with non-enzymatic signaling activity in dendritic cells (DCs) (Mondanelli et al., 2017). In addition to their inherent immunosuppressive activity, MDSCs might amplify regulatory properties of other immune cells, particularly in tumor microenvironments. Although some mechanisms underlying MDSC-macrophage interaction have been established (Ugel et al., 2015), the cross-talk between MDSCs and DCs is still unclear (Ostrand-Rosenberg et al., 2012); to fill this gap, we have developed this protocol and we demonstrated that Arg1+ MDSCs confer to DCs an IDO1-dependent, immunosuppressive phenotype via Arg1 metabolites (i.e., polyamines such as putrescine and spermidine) (Mondanelli et al., 2017). The Arg and Trp immunoregulatory pathways are functionally integrated, this integration occurring both intra- (i.e., DCs) and inter-cellularly (MDSCs and DCs) (Mondanelli et al., 2017).

Materials and Reagents

  1. Petri dishes (Corning, Falcon®, catalog number: 351029 )
  2. 15 ml Falcon tubes (Corning, Falcon®, catalog number: 352096 )
  3. Cell strainers (Corning, Falcon®, catalog number: 352340 )
  4. 5 ml sterile pipettes (Corning, Falcon®, catalog number: 357543 )
  5. 10 ml sterile pipettes (Corning, Falcon®, catalog number: 357551 )
  6. 200 µl sterile tips (Biotix, Neptune®, catalog number: 2102 NS )
  7. 1,000 µl sterile tips (Biotix, Neptune®, catalog number: 2372 S )
  8. 24-well plates (Corning, Falcon®, catalog number: 353047 )
  9. 50 ml Falcon tubes (Corning, Falcon®, catalog number: 352070 )
  10. 10 ml syringe (Terumo Medical, catalog number: SS+10S21381 )
  11. 1 ml syringe with 26 G ½ needle (Terumo Medical, catalog number: SS+01H26131 )
  12. 2 ml syringe plunger (Terumo Medical, catalog number: SS-02S2238 )
  13. 6-well plates (Corning, Falcon®, catalog number: 353046 )
  14. Permeable support for 24-well plate with 0.4 μm translucent high density PET membrane (Corning, Falcon®, catalog number: 353495 )
  15. LS columns (Miltenyi Biotec, catalog number: 130-042-401 )
  16. MS columns (Miltenyi Biotec, catalog number: 130-042-201 )
  17. Pasteur pipette (Sigma-Aldrich, catalog number: Z627992-1000EA )
  18. C57BL/6 female mice, 6 weeks old (C57BL/6NCrl) (Charles River Laboratories, catalog number: 027 )
  19. RPMI 1640 medium (Thermo Fisher Scientific, catalog number: 11875093 )
  20. FCS (Thermo Fisher Scientific, catalog number: A3160801 )
  21. L-Glutamine (Thermo Fisher Scientific, catalog number: 25030024 )
  22. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  23. HEPES (Thermo Fisher Scientific, catalog number: 15630056 )
  24. 2-Mercaptoethanol (Thermo Fisher Scientific, GibcoTM, catalog number: 31350010 )
  25. Trypan blue (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
  26. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  27. Bovine serum albumin (BSA) (Rockland Immunochemicals, catalog number: BSA-50 )
  28. Ethylenediaminetetraacetate acid (EDTA) (AppliChem, catalog number: 131669.1211 )
  29. CD11b MicroBeads, human and mouse (Miltenyi Biotec, catalog number: 130-049-601 )
  30. CD11c MicroBeads UltraPure, mouse (Miltenyi Biotec, catalog number: 130-108-338 )
  31. HBSS, no calcium, no magnesium (Thermo Fisher Scientific, catalog number: 14170088 )
  32. Sodium chloride (NaCl) (CARLO ERBA Reagents, catalog number: 479687 )
  33. Tris (Bio-Rad Laboratories, catalog number: 1610719 )
  34. Potassium chloride (KCl) (CARLO ERBA Reagents, catalog number: 471177 )
  35. Recombinant murine GMCSF (PeproTech, catalog number: 315-03 )
  36. Recombinant murine IL-4 (PeproTech, catalog number: 214-14 )
  37. Collagenase from Clostridium histolyticum (Sigma-Aldrich, catalog number: C5138-1G )
  38. Histodenz (Sigma-Aldrich, catalog number: D2158-100G )
  39. Nor-NOHA (Cayman Chemicals, catalog number: 10006861 )
  40. MACS buffer (see Recipes)
  41. RPMI medium (see Recipes)
  42. TCCM (see Recipes)
  43. Collagenase 100 U/ml and 400 U/ml (see Recipes)
  44. Nycodenz (see Recipes)
  45. Nycodenz buffer (see Recipes)

Equipment

  1. Scissor (Isolab Laborgeräte, catalog number: 048.25.130 )
  2. Sterile biosafety cabinet
  3. P20L pipette (Pipetman L) (Gilson, catalog number: FA10003M )
  4. P200L pipette (Pipetman L) (Gilson, catalog number: FA10005M )
  5. P1000L pipette (Pipetman L) (Gilson, catalog number: FA10006M )
  6. Pipet controller (Corning, Falcon®, catalog number: 357471 )
  7. Centrifuge (Eppendorf, model: 5810 R )
  8. Incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: BB15 )
  9. -80 °C freeze (Thermo Fisher Scientific, Thermo ScientificTM, model: FormaTM 88000 Series )
  10. Light microscope (ZEISS Primostar)
  11. MiniMACS separator (Miltenyi Biotec, catalog number: 130-042-102 )
  12. MidiMACS separator (Miltenyi Biotec, catalog number: 130-042-302 )

Procedure

  1. MDSCs isolation from bone marrow
    1. Take the hind limbs from two C57BL/6 mice, cutting the bone extremities.
    2. Under a sterile biosafety cabinet, completely remove flesh from the bones. Put the bones in a Petri dish and inject 10 ml of complete RPMI medium (see Recipes) inside each thigh bone using a 10 ml syringe with a 26 G ½ needle, in order to extract the bone marrow.
    3. Aspirate and discharge the medium until the bone marrow cells are completely resuspended, using the same initial 10 ml of RPMI.
    4. Transfer the bone marrow cells suspension in a 15 ml Falcon tube supplied with a 40 µm cell strainer, then remove the cell strainer and centrifuge (582 x g, 10 min, RT).
    5. Discard the supernatant and resuspend cells in 10 ml of fresh complete RPMI.
    6. Count cells, properly diluting them in trypan blue (from 4 legs, approximately 50 x 106 cells can be obtained).
    7. Centrifuge the cell suspension (582 x g, 10 min, RT).
    8. Sort CD11b+ cells:
      1. Resuspend cell pellet in 80 µl of ice-cold MACS buffer (see Recipes) per 107 total cells.
      2. Add 20 µl of CD11b Microbeads per 107 total cells.
      3. Mix well and incubate for 15 min at 4 °C.
      4. Wash cells by adding 1-2 ml of ice-cold MACS buffer per 107 cells and centrifuge (582 x g, 10 min, 4 °C).
      5. Resuspend up to 108 cells in 500 µl of MACS buffer. Proceed with magnetic separation.
      6. Place the column in the magnetic field and rinse with the appropriate amount of buffer: MS column 500 µl (up to 108 cells), LS column 3 ml (from 108 cells to 109 cells).
      7. Apply the cell suspension onto the column. Collect unlabeled cells which pass through and wash column with the appropriate amount of buffer. Perform washing steps by adding ice-cold MACS buffer three times, each time once the column reservoir is empty (MS column: 3 x 500 µl, LS column: 3 x 3 ml). Collect the total effluent (the unlabeled cell fraction).
      8. Remove column from the separator and place it on a suitable collection tube.
      9. Pipette appropriate amount of buffer onto the column. Immediately flush out fraction with the magnetically labeled cells by firmly applying the plunger supplied with the column (MS: 1 ml, LS: 5 ml).
      10. Centrifuge the labeled cells (CD11b+ cells) (582 x g, 10 min, RT). Usually, from 50 x 106 BM cells, the recovery of CD11b+ cells is about 15 x 106 cells.
    9. Resuspend the sorted CD11b+ cells (1 x 106 cells/ml) in complete RPMI containing 30% TCCM (see Recipes), 20 ng/ml of GMCSF and 20 ng/ml of IL-4. Both GMCSF and IL-4 are resuspended in RPMI and aliquoted and stored at -80 °C.
    10. Plate the cells 1 ml/well in a 24-well plate. Incubate cells for 4 days at 37 °C.
    11. After 4 days change the medium: recover cells by pipetting them in a 50 ml Falcon tube and centrifuge (805 x g, 10 min, RT). Discard the supernatant and add new RPMI containing 30% TCCM, 20 ng/ml of GMCSF and 20 ng/ml of IL-4. Plate again the cells 1 ml/well in a 24-well plate.
    12. After 7 days, isolate CD11c+ DCs from spleens.

  2. CD11c+ DCs isolation from spleen
    1. Before starting, prepare 2 solutions of collagenase; for each spleen prepare 1 ml of each collagenase solution (15 ml of 100 and 15 ml of 400 U/ml) (see Recipes) as described in the Recipes section.
    2. Take spleens from 15 C57BL/6 mice.
    3. Put the spleens in a Petri dish and inject in each spleen 1 ml of the 100 U/ml collagenase solution (i.e., for 15 spleens, use 15 ml of 100 U/ml collagenase solution) using a 10 ml syringe with a 26 G ½ needle, until the spleen change colour (spleens colour should fade from red to white). Repeat this step twice.
    4. Recover the cell suspension and filter it in a 50 ml Falcon tube supplied with a 40 µm cell strainer, already containing 5 ml of complete RPMI. Keep this supernatant at RT.
    5. Incubate the spleens with 1 ml/spleen of 400 U/ml collagenase solution for 30 min at 37 °C.
    6. Recover the collagenase solution in a 10 ml syringe and homogenize the spleens using a 2 ml syringe plunger.
    7. Resuspend the spleens completely until all splenic cells are in solution using the collagenase solution recovered before. Be sure that cells are homogeneously resuspended, then filter the cell suspension through the cell strainer onto the same 50 ml Falcon tube already containing the first cell suspension recovered from spleens treated with 100 U/ml collagenase.
    8. Remove the cell strainer and centrifuge (582 x g, 10 min, RT).
    9. Discard the supernatant and resuspend the cellular pellet in 15 ml (1 ml/spleen) of a solution composed by 54% of Nycodenz (see Recipes) and 46% of Nycodenz buffer (see Recipes). Divide the obtained cellular suspension in three 15 ml Falcon tubes (5 ml/tube).
    10. Carefully overlay 3 ml of RPMI medium in each tube containing 5 ml of the cell suspension (Figure 1A).
    11. Centrifuge at 1,811 x g, 15 min, 4 °C without brake.
    12. Recover the ring at the interface of the two phases using a Pasteur pipette and transfer the cells into a new 50 ml Falcon tube (Figure 1B).


      Figure 1. Isolation of dendritic cells from splenocytes by density gradient. A. Density gradient of splenic cells resuspended in Nycodenz (lower phase) and medium RPMI (upper phase) before the centrifugation. B. Density gradient of splenic cells after the centrifugation: the ring at the interface contains CD11c+ dendritic cells.

    13. Add 10 ml of RPMI medium and centrifuge at 582 x g, 10 min, RT. Discard the supernatant and resuspend the cellular pellet in 10 ml of complete RPMI.
    14. Count cells and centrifuge again at 582 x g, 10 min, RT. Usually, the recovery is about 100 x 106 cells from one spleen.
    15. Resuspend cell pellet in 8.5 µl of ice-cold MACS buffer per 107 total cells.
    16. Add 1.5 µl of CD11c Microbeads per 107 total cells.
    17. Mix well and incubate for 15 min at 4 °C.
    18. Wash cells by adding 1-2 ml of ice-cold MACS buffer per 107 cells and centrifuge at 582 x g, 10 min, RT.
    19. Resuspend up to 108 cells in 500 µl of ice-cold MACS buffer. Proceed with magnetic separation.
    20. Place column in the magnetic field and rinse with the appropriate amount of buffer: MS column 500 µl (up to 108 cells), LS column 3 ml (from 108 cells to 109 cells).
    21. Apply cell suspension onto the column. Discard unlabeled cells which pass through and wash column with the appropriate amount of buffer. Perform washing steps by adding ice-cold MACS buffer three times, each time once the column reservoir is empty (MS column: 3 x 500 µl, LS column: 3 x 3 ml). Collect the total effluent (the unlabeled cell fraction).
    22. Remove column from the separator and place it on a suitable collection tube.
    23. Pipette appropriate amount of buffer onto the column. Immediately flush out fraction with the magnetically labeled cells by firmly applying the plunger supplied with the column (MS: 1 ml, LS: 5 ml).
    24. Centrifuge the labeled cells (CD11c+ cells) (582 x g, 10 min, RT), discard the supernatant, resuspend cells in 10 ml of complete RPMI and count them. Usually, the recovery of CD11c+ cells is about 1 x 106 cells from one spleen.
    25. Resuspend cells (1.5 x 106 cells/ml) in complete RPMI and plate 4 ml/well in a 6-well plate. Incubate CD11c+ cells overnight at 37 °C, 5% CO2.

  3. Co-culture of MDSCs and CD11c+ DCs
    For a co-culture of MDSCs and CD11c+ DCs at a 1:2 ratio:
    1. Recover and count CD11c+ cells. Resuspend 1 x 106 cells in 700 µl of complete RPMI. Plate cells in a 24-well plate. Consider 1 x 106 CD11c+ cells for each sample of the co-culture (i.e., CD11c+ alone, CD11c+ co-cultured with untreated MDSCs and CD11c+ co-cultured with MDSCs treated with Nor-NOHA to inhibit Arg1 enzymatic activity).
    2. Put onto each well containing CD11c+ cells a transwell filled with 100 µl of complete RPMI (without MDSCs) and incubate for 1 h at 37 °C.
    3. In the meanwhile, incubate MDSCs 1 x 106 cells/ml with the appropriate stimulus (i.e., with and without 150 µM Nor-NOHA to inhibit Arg1 enzymatic activity) for 1 h at 37 °C, then recover and centrifuge MDSCs (582 x g, 10 min, RT).
    4. Resuspend MDSCs 0.5 x 106 cells in 100 µl of complete RPMI.
    5. Add 100 µl of MDSCs suspension into the appropriated transwell and incubate overnight at 37 °C.
    6. Recover MDSCs and CD11c+ cells and supernatants and perform the desired analysis (i.e., a delayed-type hypersensitivity assay using a skin test (Mondanelli et al., 2017) to assess the functional phenotype of CD11c DCs after the co-culture with MDSCs).

Data analysis

Usually, co-culture of MDSCs with DCs must be repeated at least three times to obtain statistically relevant data. Unpaired Student’s t-test was used for in vitro analyses, using at least three values from 2-3 experiments per group (Mondanelli et al., 2017).

Notes

  1. All steps should be performed under sterile conditions. Even for the isolation of legs and spleens from euthanized mice, use of autoclaved dissection tools and spray ethanol is highly recommended.
  2. All media should be warmed at 37 °C before use.
  3. Usually, purity of MDSC is about 60% and purity of CD11c+ cells is about 95%. More in detail, more than 60% of the MDSCs after the TCCM and cytokines stimulation were CD11b and Gr1 positive cells. CD11c+ cells were 90-95% CD11c+, > 95% MHC I-A+, > 95% B7-2+, < 0.1% CD3+, and appeared to consist of 90-95% CD8-, 5-10% CD8+, and 1-5% B220+ PDCA+ (i.e., plasmacytoid DCs or pDCs) cells.
  4. As reported in literature, there are several methods of MDSCs differentiation, including the use of GCSF, GMCSF and IL-13, or GMCSF and IL-6, or by means of PGE2. We used TCCM, GMCSF and IL-4 because in these specific MDSCs we assessed the highest expression and functional activity of Arg1.
  5. BMDCs can also be used in this protocol; in order to use the same DCs we used to determine Arg1 expression and function (Mondanelli et al., 2017) we used splenic DCs for the co-culture with MDSCs.

Recipes

Note: All buffers and media should be sterile.

  1. RPMI medium
    Add to RPMI 1640 medium 10% FCS (Thermo Fisher Scientific)
    2 mM L-glutamine (stock solution 200 mM)
    100 U/ml penicillin-streptomycin (stock solution10,000 U/ml)
    10 mM HEPES (stock solution 1 M)
    50 μM 2-mercaptoethanol (stock solution 50 mM)
  2. MACS buffer (stored at 4 °C)
    Phosphate buffered saline (PBS) pH 7.2
    0.5% bovine serum albumin (BSA)
    2 mM EDTA
  3. TCCM
    1. To generate tumor cell conditioned medium (TCMM), subconfluent B16 melanoma cells were kept in RPMI 1640 medium with a reduced (3%) serum concentration for 48 h
    2. After that time, supernatants were collected, aliquoted and kept at -80 °C until further use (Youn et al., 2008)
  4. Collagenase 100 U/ml and 400 U/ml
    1. Prepare a stock solution of collagenase 8,000 U/ml in HBSS w/o Ca and Mg and keep this solution aliquoted (1 ml/tube) at -20 °C
    2. Starting from the stock solution, dilute collagenase to 100 U/ml in HBSS or 400 U/ml in HBSS and keep these solutions at 4 °C
  5. Nycodenz (stored at 4 °C)
    30.55 g histodenz into 100 ml Milli-Q water (final volume)
    Filter through a 0.22 µm filter and protect from light
  6. Nycodenz buffer (aliquoted and stored at -20 °C)
    9 g NaCl
    0.6055 g Tris
    0.22368 g KCl
    0.11167 g EDTA
    Dissolve in 900 ml Milli-Q water
    Adjust pH to 7.5
    Bring volume to 1 L
    Filter through a 0.22 µm filter, aliquot and store

Acknowledgments

This work was supported by the European Research Council (338954-DIDO; to Prof. Ursula Grohmann and Antonio Macchiarulo) and by Ministero dell’Istruzione, Università e Ricerca, Italy (FIRB RBAP11T3WB; to Prof. Ursula Grohmann, Vincenzo Bronte, and Silvio Bicciato; PRIN 2015C2PP7 to Claudia Volpi). This protocol was adapted from Mondanelli et al. (2017).

References

  1. Marvel, D. and Gabrilovich, D. I. (2015). Myeloid-derived suppressor cells in the tumor microenvironment: expect the unexpected. J Clin Invest 125(9): 3356-3364.
  2. Mondanelli, G., Bianchi, R., Pallotta, M. T., Orabona, C., Albini, E., Iacono, A., Belladonna, M. L., Vacca, C., Fallarino, F., Macchiarulo, A., Ugel, S., Bronte, V., Gevi, F., Zolla, L., Verhaar, A., Peppelenbosch, M., Mazza, E. M., Bicciato, S., Laouar, Y., Santambrogio, L., Puccetti, P., Volpi, C. and Grohmann, U. (2017). A relay pathway between arginine and tryptophan metabolism confers immunosuppressive properties on dendritic cells. Immunity 46(2): 233-244.
  3. Ostrand-Rosenberg, S., Sinha, P., Beury, D. W. and Clements, V. K. (2012). Cross-talk between myeloid-derived suppressor cells (MDSC), macrophages, and dendritic cells enhances tumor-induced immune suppression. Semin Cancer Biol 22(4): 275-281.
  4. Ugel, S., De Sanctis, F., Mandruzzato, S. and Bronte, V. (2015). Tumor-induced myeloid deviation: when myeloid-derived suppressor cells meet tumor-associated macrophages. J Clin Invest 125(9): 3365-3376.
  5. Youn, J. I., Nagaraj, S., Collazo, M. and Gabrilovich, D. I. (2008). Subsets of myeloid-derived suppressor cells in tumor-bearing mice. J Immunol 181(8): 5791-5802.

简介

骨髓来源的抑制细胞(MDSCs)具有抑制免疫应答的能力,并扩增其他免疫细胞即树突状细胞的调节特性。 在这里,我们描述了MDSC与鼠骨髓细胞分化的方案,并且从鼠脾中纯化CD11c +树突状细胞。 可以共培养MDSC和CD11c树突状细胞,并且可以通过特定的功能体内实验来评估MDSCs条件树突细胞的免疫调节表型,即皮肤试验作为延迟型超敏反应的量度 抗免疫原性较差的抗原。
【背景】骨髓来源的抑制细胞(MDSCs)是由早期分化阶段的巨噬细胞,粒细胞,树突状细胞和骨髓细胞的前体组成的骨髓细胞组(Youn等人,2008),其在肿瘤的淋巴组织中大量积累感染性小鼠以及感染性疾病,败血症和创伤的小鼠。这些细胞的主要特征是它们以Ag特异性和/或非特异性方式抑制T细胞应答的能力。这些细胞现在被认为是负责肿瘤相关免疫缺陷的主要细胞类型之一;涉及MDSC介导的免疫抑制的主要因素包括Arg1的高表达(Marvel和Gabrilovich,2015)。精氨酸酶1(Arg1)和吲哚胺2,3-双加氧酶1(IDO1)分别是催化L-精氨酸(L-Arg)和L-色氨酸(L-Trp)降解的免疫调节酶,导致局部氨基酸剥夺。此外,与Arg1不同,IDO1在树突细胞(DC)中也具有非酶信号传导活性(Mondanelli等,2017)。除了其固有的免疫抑制活性外,MDSC还可能扩增其他免疫细胞的调节特性,特别是在肿瘤微环境中。虽然建立了MDSC-巨噬细胞相互作用的一些机制(Ugel等,2015),MDSCs和DCs之间的串扰仍然不清楚(Ostrand-Rosenberg等,2012);为弥补这一差距,我们已经制定了该方案,并且我们证明了Arg1 + MDSCs通过Arg1代谢物(即多胺,如腐胺和亚精胺)赋予DCs IDO1依赖性免疫抑制表型(Mondanelli等,2017)。 Arg和Trp免疫调节途径在功能上整合,这种整合发生在(即DC)和细胞间(MDSCs和DCs)之间(Mondanelli等,2017)。

关键字:骨髓源性抑制细胞, 树突状细胞, 共培养

材料和试剂

  1. 培养皿(Corning,Falcon ®,目录号:351029)
  2. 15 ml Falcon管(Corning,Falcon ®,目录号:352096)
  3. 细胞过滤器(Corning,Falcon ®,目录号:352340)
  4. 5 ml无菌移液器(Corning,Falcon ®,目录号:357543)
  5. 10 ml无菌移液器(Corning,Falcon ®,目录号:357551)
  6. 200μl无菌提示(Biotix,Neptune ®,目录号:2102 NS)
  7. 1000μl无菌提示(Biotix,Neptune ®,目录号:2372 S)
  8. 24孔板(Corning,Falcon ®,目录号:353047)
  9. 50ml Falcon管(Corning,Falcon ®,目录号:352070)
  10. 10ml注射器(Terumo Medical,目录号:SS + 10S21381)
  11. 1 ml注射器,26 G½针(Terumo Medical,目录号:SS + 01H26131)
  12. 2 ml注射器柱塞(Terumo Medical,目录号:SS-02S2238)
  13. 6孔板(Corning,Falcon ®,目录号:353046)
  14. 具有0.4μm半透明高密度PET膜的24孔板的透气性支撑(Corning,Falcon ®,目录号:353495)
  15. LS列(Miltenyi Biotec,目录号:130-042-401)
  16. MS色谱柱(Miltenyi Biotec,目录号:130-042-201)
  17. 巴斯德吸管(Sigma-Aldrich,目录号:Z627992-1000EA)
  18. 6周龄的C57BL / 6雌性小鼠(C57BL / 6NCr1)(Charles River Laboratories,目录号:027)
  19. RPMI 1640培养基(Thermo Fisher Scientific,目录号:11875093)
  20. FCS(Thermo Fisher Scientific,目录号:A3160801)
  21. L-谷氨酰胺(Thermo Fisher Scientific,目录号:25030024)
  22. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  23. HEPES(Thermo Fisher Scientific,目录号:15630056)
  24. 2-巯基乙醇(Thermo Fisher Scientific,Gibco TM,目录号:31350010)
  25. 台盼蓝(Thermo Fisher Scientific,Gibco TM ,目录号:15250061)
  26. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:10010023)
  27. 牛血清白蛋白(BSA)(Rockland Immunochemicals,目录号:BSA-50)
  28. 乙二胺四乙酸(EDTA)(AppliChem,目录号:131669.1211)
  29. CD11b MicroBeads,人和小鼠(Miltenyi Biotec,目录号:130-049-601)
  30. CD11c MicroBeads UltraPure,Mouse(Miltenyi Biotec,目录号:130-108-338)
  31. HBSS,无钙,无镁(Thermo Fisher Scientific,目录号:14170088)
  32. 氯化钠(NaCl)(CARLO ERBA试剂,目录号:479687)
  33. Tris(Bio-Rad Laboratories,目录号:1610719)
  34. 氯化钾(KCl)(CARLO ERBA试剂,目录号:471177)
  35. 重组鼠GMCSF(PeproTech,目录号:315-03)
  36. 重组鼠IL-4(PeproTech,目录号:214-14)
  37. 来自<溶解性Clostridium histolyticum 的胶原酶(Sigma-Aldrich,目录号:C5138-1G)
  38. Histodenz(Sigma-Aldrich,目录号:D2158-100G)
  39. Nor-NOHA(Cayman Chemicals,目录号:10006861)
  40. MACS缓冲区(请参阅配方)
  41. RPMI培养基(参见食谱)
  42. TCCM(见配方)
  43. 胶原酶100 U / ml和400 U / ml(见配方)
  44. Nycodenz(见食谱)
  45. Nycodenz缓冲液(参见食谱)

设备

  1. 剪刀(IsolabLaborgeräte,目录号:048.25.130)
  2. 无菌生物安全柜
  3. P20L移液器(Pipetman L)(Gilson,目录号:FA10003M)
  4. P200L移液器(Pipetman L)(Gilson,目录号:FA10005M)
  5. P1000L移液器(Pipetman L)(Gilson,目录号:FA10006M)
  6. 吸管控制器(Corning,Falcon ®,目录号:357471)
  7. 离心机(Eppendorf,型号:5810 R)
  8. 培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:BB15)
  9. -80°C冷冻(Thermo Fisher Scientific,Thermo Scientific TM,型号:Forma TM 88000系列)
  10. 光学显微镜(ZEISS Primostar)
  11. MiniMACS分离器(Miltenyi Biotec,目录号:130-042-102)
  12. MidiMACS分离器(Miltenyi Biotec,目录号:130-042-302)

程序

  1. MDSC与骨髓分离
    1. 取两只C57BL / 6小鼠的后肢切开骨头。
    2. 在无菌生物安全柜下,彻底清除骨骼中的肉体。将骨头放入陪替氏培养皿中,并使用10 ml注射器(26 G½针)在每只大腿骨内注射10 ml的完整RPMI培养基(见食谱),以提取骨髓。
    3. 吸出并排出培养基,直到骨髓细胞完全重新悬浮,使用相同的初始10ml RPMI。
    4. 将骨髓细胞悬浮液转移到配备40μm细胞过滤器的15 ml Falcon管中,然后取出细胞过滤器并离心(582 x 10 g,10 min,RT)。
    5. 弃去上清液,将细胞重新悬浮在10ml新鲜的完整RPMI中
    6. 计数细胞,以台盼蓝(从4条腿,约50×10 6 细胞)适当地稀释它们。
    7. 离心细胞悬浮液(582 x g,10分钟,RT)
    8. 排序CD11b + 单元格:
      1. 将细胞沉淀物重悬于80μl冰冷的MACS缓冲液(参见Recipes)/10μg/ ml总细胞中。
      2. 每10个总细胞中加入20μlCD11b微珠。
      3. 混匀,4℃孵育15分钟
      4. 通过每10个细胞加入1-2ml冰冷的MACS缓冲液洗涤细胞并离心(582 x g,10分钟,4℃)。
      5. 在500μlMACS缓冲液中重悬10个细胞。继续进行磁选。
      6. 将柱置于磁场中,并用适量的缓冲液冲洗:MS柱500μl(最多10μL),LS柱3 ml(10“细胞至10 9细胞)
      7. 将细胞悬液涂于柱上。收集未标记的细胞,通过并用适量的缓冲液洗涤柱。通过加入冰冷的MACS缓冲液进行洗涤步骤三次,每次一次柱储存器空(MS柱:3×500μl,LS柱:3×3ml)。收集总流出物(未标记的细胞分数)。
      8. 从分离器中取出色谱柱并将其放在合适的收集管上。
      9. 移取适量的缓冲液到柱上。用柱子提供的柱塞(MS:1 ml,LS:5 ml)立即用磁性标记的细胞冲洗成分。
      10. 将标记的细胞(CD11b + 细胞)离心(582×g,10分钟,RT)。通常,从50×10 6个细胞中,CD11b +细胞的回收率约为15×10 6个细胞。
    9. 在含有30%TCCM(参见食谱),20ng / ml GMCSF和20ng的完整RPMI中重新分选CD11b + 细胞(1×10 6细胞/ ml) / ml的IL-4。将GMCSF和IL-4重新悬浮于RPMI中并等分并储存于-80℃。
    10. 将细胞在24孔板中平皿1ml /孔。在37℃孵育细胞4天。
    11. 4天后更换培养基:通过将其移液到50ml Falcon管中回收细胞并离心(805×10 5分钟,RT)。弃上清,加入含有30%TCCM,20ng / ml GMCSF和20ng / ml IL-4的新RPMI。在24孔板中再次将细胞1ml /孔。
    12. 7天后,从脾脏分离CD11c + DC。

  2. CD11c + 与脾脏隔离的DCs
    1. 开始之前,准备2个胶原酶溶液;对于每个脾脏,如食谱部分所述,制备1ml每种胶原酶溶液(15ml 100和15ml 400U / ml)(参见食谱)。
    2. 从15只C57BL / 6小鼠取脾。
    3. 将脾脏置于培养皿中,每只脾注射1ml 100U / ml胶原酶溶液(即,15只脾,使用15ml 100U / ml胶原酶溶液),使用10 ml注射器,26 G½针,直到脾脏变色(脾脏颜色应从红色渐变为白色)。重复此步骤。
    4. 回收细胞悬浮液,并将其过滤在配有40μm细胞过滤器的50ml Falcon管中,已经含有5ml完整的RPMI。将此上清液保持在室温。
    5. 在37℃下将脾脏用1ml / 400U / ml胶原酶溶液孵育30分钟。
    6. 在10ml注射器中回收胶原酶溶液,并使用2 ml注射器柱塞匀浆脾脏。
    7. 使用先前回收的胶原酶溶液将脾脏完全重悬,直到所有脾细胞均处于溶液状态。确保细胞均匀重悬,然后将细胞悬浮液通过细胞过滤器过滤到已经含有用100U / ml胶原酶处理的脾脏回收的第一细胞悬液的相同的50ml Falcon管上。
    8. 取出细胞过滤器并离心(582 x g,10分钟,RT)。
    9. 弃去上清液,并将细胞沉淀重悬于15ml(1ml /脾)由54%Nycodenz(参见食谱)和46%Nycodenz缓冲液(参见食谱)组成的溶液中。将获得的细胞悬浮液分成三个15ml Falcon管(5ml /管)
    10. 仔细覆盖每个含有5ml细胞悬浮液的管中的3ml RPMI培养基(图1A)
    11. 离心机为1,811 x g,15分钟,4℃无刹车。
    12. 使用巴斯德吸管在两相界面处恢复环,并将细胞转移到新的50ml Falcon管中(图1B)。


      图1.通过密度梯度从脾细胞中分离树突状细胞。 :一种。在离心之前,脾细胞的密度梯度重悬于Nycodenz(下相)和中等RPMI(上相)。 B.离心后脾细胞的密度梯度:界面处的环含有CD11c +树突状细胞。

    13. 加入10ml RPMI培养基,并以582 x g,10分钟RT离心。弃去上清液,将细胞沉淀重悬于10ml完整的RPMI中
    14. 计数细胞并再次以582 x g,10分钟RT离心。通常,一个脾脏的恢复是约100×10 6个细胞。
    15. 将细胞沉淀物重悬于8.5μl冰冷的MACS缓冲液中,每10次总共细胞。
    16. 每10个细胞总共加入1.5μlCD11c微珠
    17. 混匀,4℃孵育15分钟
    18. 通过加入1-2毫升冰冷的MACS缓冲液/ 10 7细胞洗涤细胞,并以582 x g,10分钟RT进行离心。
    19. 在500μl冰冷的MACS缓冲液中重悬10个细胞。继续进行磁选。
    20. 将柱置于磁场中,并用适量的缓冲液冲洗:MS柱500μl(最多至10个细胞),LS柱3 ml(从10μg/至10 9 单元格)。
    21. 将细胞悬液涂于柱上。丢弃未标记的细胞,通过适当量的缓冲液洗涤柱。通过加入冰冷的MACS缓冲液进行洗涤步骤三次,每次一次柱储存器空(MS柱:3×500μl,LS柱:3×3ml)。收集总流出物(未标记的细胞分数)。
    22. 从分离器中取出色谱柱并将其放在合适的收集管上。
    23. 移取适量的缓冲液到柱上。用柱子提供的柱塞(MS:1 ml,LS:5 ml)立即用磁性标记的细胞冲洗成分。
    24. 将标记的细胞(CD11c + 细胞)(582 x g,10分钟,RT)离心,弃去上清液,将细胞重悬于10ml完整的RPMI中并计数。通常,CD11c + 细胞的恢复是来自一个脾脏的约1×10 6个细胞。
    25. 在完全RPMI中重悬细胞(1.5×10 6细胞/ ml),并在6孔板中将4ml /孔板平板。在37℃,5%CO 2 孵育CD11c + 细胞过夜。

  3. MDSCs和CD11c + DCs的共同培养
    对于以1:2比例的MDSC和CD11c + DC的共培养物:
    1. 恢复和计数CD11c + 单元格。在700μl完整的RPMI中重悬1×10 6个细胞。板细胞在24孔板中。考虑每个共培养样品( ie ,CD11c + 单独1×10 CD11c + ,与用Nor-NOHA处理的MDSC共培养的未处理的MDSC和CD11c +共同培养的CD11c + / sup同时抑制Arg1酶活性)。
    2. 将含有CD11c + 细胞的每个孔放入填充有100μl完整RPMI(无MDSC))的transwell,并在37℃下孵育1小时。
    3. 同时,用合适的刺激(即,有和没有150μMNor-NOHA的培养物)孵育MDSC 1×10 6细胞/ ml,以抑制Arg1酶活性) h,然后回收和离心MDSC(582 xg,10分钟,RT)。
    4. 在100μl完整的RPMI中重悬MDSC 0.5×10 6个细胞。
    5. 将100μlMDSCs悬浮液加入适当的transwell中,并在37℃下孵育过夜
    6. 恢复MDSC和CD11c + 细胞和上清液并进行所需的分析(即,,使用皮肤试验的延迟型超敏反应测定法(Mondanelli等人, em>,2017),以评估与MDSC共培养后CD11c DC的功能表型。

数据分析

通常,MDSC与DCs的共培养必须重复至少三次以获得统计学上相关的数据。使用非配对学生的测试用于体外分析,使用至少三个来自每组2-3次实验的值(Mondanelli等人 ,2017)。

笔记

  1. 所有步骤都应在无菌条件下进行。即使从安乐死的小鼠中分离腿部和脾脏,强烈建议使用高压消毒的解剖工具和喷雾乙醇。
  2. 所有介质应在37°C使用前加温。
  3. 通常,MDSC的纯度为约60%,CD11c 细胞的纯度为约95%。更详细地说,TCCM和细胞因子刺激后超过60%的MDSCs是CD11b和Gr1阳性细胞。 CD11c + 细胞为90-95%CD11c +, 95%MHC I-A + ,&gt; 95%B7-2 + ,&lt; 0.1%CD3 + ,并且看起来由90-95%的CD8 +,5-10%CD8 +,和1-5% B220 + PDCA + ( ie ,浆细胞样DC或pDC)细胞。
  4. 如文献报道,有几种MDSCs分化方法,包括使用GCSF,GMCSF和IL-13,或GMCSF和IL-6,或通过PGE2。我们使用TCCM,GMCSF和IL-4,因为在这些特定的MDSC中,我们评估了Arg1的最高表达和功能活性。
  5. BMDC也可用于本协议;为了使用我们用于确定Arg1表达和功能的相同的DC(Mondanelli等人,2017),我们使用脾脏DCs与MDSC共同培养。

食谱

注意:所有缓冲液和培养基应无菌。

  1. RPMI媒体
    加入RPMI 1640培养基10%FCS(Thermo Fisher Scientific)
    2mM L-谷氨酰胺(储备溶液200mM)
    100 U / ml青霉素 - 链霉素(储液10,000 U / ml)
    10mM HEPES(储备溶液1M)
    50μM2-巯基乙醇(储备溶液50mM)
  2. MACS缓冲区(存储在4°C)
    磷酸盐缓冲盐水(PBS)pH 7.2
    0.5%牛血清白蛋白(BSA)
    2 mM EDTA
  3. TCCM
    1. 为了产生肿瘤细胞条件培养基(TCMM),将亚汇合的B16黑素瘤细胞保持在具有降低(3%)血清浓度的RPMI 1640培养基中48小时
    2. 之后,收集上清液,等分并保存在-80℃,直到进一步使用(Youn等人,2008)
  4. 胶原酶100U / ml和400U / ml
    1. 在HBSS w / o Ca和Mg中制备8,000 U / ml胶原酶的储备溶液,并将此溶液在-20°C下等分(1 ml /管)
    2. 从储备溶液开始,将HBSS中的胶原酶稀释至100 U / ml,HBSS中将400 U / ml稀释,并将其保存在4°C
  5. Nycodenz(储存于4°C)
    将30.55克组胺化至100ml Milli-Q水(最终体积)中 过滤0.22μm过滤器,防止光线
  6. Nycodenz缓冲液(等分并储存于-20℃)
    9克NaCl
    0.6055克Tris
    0.22368克KCl
    0.11167g EDTA
    溶解在900毫升Milli-Q水中 将pH调节至7.5
    将音量调至1 L
    过滤0.22μm过滤器,等分并储存

致谢

这项工作得到欧洲研究委员会(338954-DIDO; Ursula Grohmann教授和Antonio Macchiarulo)以及意大利Ricerca大学dell'Istruzione大学(FIRB RBAP11T3WB; Ursula Grohmann教授,Vincenzo Bronte教授和Silvio教授的支持) Bicciato; PRIN 2015C2PP7至Claudia Volpi)。该协议由Mondanelli等人改编而成。 (2017)。

参考

  1. Marvel,D. and Gabrilovich,DI(2015)。&nbsp; 骨髓衍生的抑制细胞在肿瘤微环境中:预期意想不到。 J Clin Invest 125(9):3356-3364。
  2. Mondanelli,G.Bianchi,R.,Pallotta,MT,Orabona,C.,Albini,E.,Iacono,A.,Belladonna,ML,Vacca,C.,Fallarino,F.,Macchiarulo,A.,Ugel, S.,Bronte,V.,Gevi,F.,Zolla,L.,Verhaar,A.,Peppelenbosch,M.,Mazza,EM,Bicciato,S.,Laouar,Y.,Santambrogio,L.,Puccetti,P 。,Volpi,C.和Grohmann,U.(2017)。&nbsp;
  3. Ostrand-Rosenberg,S.,Sinha,P.,Beury,DW and Clements,VK(2012)。&nbsp;
  4. Ugel,S.,De Sanctis,F.,Mandruzzato,S.and Bronte,V.(2015)。&nbsp; J Clin Invest 125(9):3365 -3376。
  5. Youn,JI,Nagaraj,S.,Collazo,M.and Gabrilovich,DI(2008)。&nbsp; 荷瘤小鼠中骨髓来源的抑制细胞的亚群 .J Immunol 181(8):5791-5802。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Mondanelli, G. and Volpi, C. (2017). Differentiation of Myeloid-derived Suppressor Cells from Murine Bone Marrow and Their Co-culture with Splenic Dendritic Cells. Bio-protocol 7(18): e2558. DOI: 10.21769/BioProtoc.2558.
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