Measurement of Myeloid Cell Immune Suppressive Activity

Luigi Dolcetti1, Elisa Peranzoni1, Vincenzo Bronte1

1 Istituto Oncologico Veneto, IRCCS, Padua, Italy
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.17
DOI:  10.1002/0471142735.im1417s91
Online Posting Date:  November, 2010
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit presents simple methods to assess the immunosuppressive properties of immunoregulatory cells of myeloid origin, such as myeloid‐derived suppressor cells (MDSCs), both in vitro and in vivo. These methods are general and could be adapted to test the impact of different suppressive populations on T cell activation, proliferation, and cytotoxic activity; moreover they could be useful to assess the influence exerted on immune suppressive pathways by genetic modifications, chemical inhibitors, and drugs. Curr. Protoc. Immunol. 91:14.17.1‐14.17.25. © 2010 by John Wiley & Sons, Inc.

Keywords: MDSC; immunosuppression; immunomagnetic sorting; thymidine incorporation; chromium release test; adoptive cell transfer

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Enrichment of Myeloid Cell Subsets for Measurement of Immunosuppressive Activity
  • Basic Protocol 2: Measuring Myeloid‐Induced Suppression of T Cell Proliferation In Vitro by Anti‐CD3/Anti‐CD28 Stimulation and Evaluation of [3H]Thymidine ([3H]TdR) Incorporation
  • Basic Protocol 3: Measuring Myeloid Cell–Induced Suppression of T Cell Cytotoxic Activity In Vitro: Inhibition of Antigen‐Induced Cytotoxic Activity of T Cells in Microcultures
  • Support Protocol 1: Interpretation of Results and Calculation of Lytic Units
  • Basic Protocol 4: Measuring Myeloid Cell–Dependent In Vivo Tolerance by Adoptive Transfer
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Enrichment of Myeloid Cell Subsets for Measurement of Immunosuppressive Activity

  Materials
  • Tumor‐bearing mice—e.g., 4T1 or C26GM tumor‐bearing Balb/c mice—or in vitro–generated MDSC (Marigo et al., )
  • 3% RPMI (see recipe)
  • Sorting buffer (see recipe)
  • 10% RPMI (see recipe)
  • Mouse FcR blocking reagent (Miltenyi Biotech)
  • Mouse anti‐Ly6G biotin (Miltenyi Biotech)
  • Anti‐biotin microbeads (Miltenyi Biotech)
  • Mouse anti‐Gr‐1 biotin (Miltenyi Biotech)
  • µMAC‐Streptavidin microbeads (Miltenyi Biotech)
  • Mouse/Human CD11b Microbeads (Miltenyi Biotech)
  • 10‐mm culture dish
  • 2‐ml syringe
  • 15‐ and 50‐ml conical tubes
  • Centrifuge
  • 100‐µm nylon‐mesh cell strainer
  • LS columns (Miltenyi Biotech)
  • Magnet for magnetic separations (Miltenyi Biotech)
  • Additional reagents and equipment for harvesting spleens from mice (unit 1.9) and determining cell number using trypan blue dye exclusion ( appendix 3B)
NOTE: All Miltenyi Biotech antibodies and beads should be used without predilution; all quantities below refer to the items as provided by Miltenyi Biotech.

Basic Protocol 2: Measuring Myeloid‐Induced Suppression of T Cell Proliferation In Vitro by Anti‐CD3/Anti‐CD28 Stimulation and Evaluation of [3H]Thymidine ([3H]TdR) Incorporation

  Materials
  • Anti‐CD3 (2C11, ATCC)
  • Anti‐CD28 (clone 37.5, ATCC)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Immunosuppressive (myeloid) cells, e.g., MDSCs ( protocol 1)
  • 10% RPMI (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • BALB/c mice
  • 3% RPMI (see recipe)
  • 10× red blood cell lysis buffer (see recipe)
  • [3H]TdR (PerkinElmer)
  • Serum‐free RPMI medium (e.g., Invitrogen)
  • 96% ethanol
  • MicroScint‐20 (PerkinElmer)
  • 96‐well flat‐bottom microtiter plate
  • 2‐ml syringe
  • 10‐mm culture dish
  • 50‐ml conical tubes (BD Falcon)
  • Centrifuge
  • 100‐µm nylon mesh cell strainer
  • Unifilter‐96 GF/C plate (PerkinElmer) with plate sticker
  • 96‐well U‐bottom microtiter plate (PerkinElmer)
  • Plate harvester: FilterMate 196 (Packard)
  • TopSeal‐A 96‐well microtiter plate adhesive sealers (PerkinElmer)
  • Scintillation counting device (TopCount, PerkinElmer)
  • Computer running spreadsheet program, e.g., Microsoft Excel
  • Additional reagents and equipment for preparing immunosuppressive cells ( protocol 1), harvesting of spleens from mice (unit 1.9), and counting viable cells by trypan blue exclusion ( appendix 3B)

Basic Protocol 3: Measuring Myeloid Cell–Induced Suppression of T Cell Cytotoxic Activity In Vitro: Inhibition of Antigen‐Induced Cytotoxic Activity of T Cells in Microcultures

  Materials
  • Immunosuppressive cells, e.g., MDSCs ( protocol 1)
  • 10% RPMI (see recipe)
  • BALB/c mice
  • Transgenic CL‐4 mice on a BALB/c background bearing a αβ T cell receptor (TCR) recognizing the Kd‐restricted HA 512‐520 peptide of influenza hemagglutinin (HA) (Morgan et al., ); these mice were a gift from L. Sherman of The Scripps Research Institute, La Jolla, Calif.; also available from The Jackson Laboratory, with the name CBy.Cg‐Thy1a Tg(TcraCl4,TcrbCl4)1Shrm/ShrmJ
  • 3% RPMI (see recipe)
  • Red cell lysis buffer (see recipe)
  • 10 mg/ml HA 512‐520 peptide stock solution (available lyophilized from JPT Peptide Technologies, http://www.jpt.com/)
  • Subconfluent CT26 cell line (ATCC no. CRL‐2638™) maintained in culture in 75‐cm2 culture flasks: prepare a sufficient number of flasks containing CT26 cells, which have to be subconfluent on day 5
  • 0.25% trypsin/2 mM EDTA (e.g., Invitrogen)
  • 5% RPMI (see recipe)
  • 10% fetal bovine serum (FBS) in phosphate‐buffered saline (PBS; appendix 2A)
  • Fetal bovine serum (FBS)
  • 1 mCi/ml Na51CrO 4 (PerkinElmer, cat. no. NEZ‐030S)
  • 5% RPMI (see recipe) containing 1% (w/v) SDS
  • 96‐well flat bottom microplate
  • 10‐mm culture dish
  • 2‐ml syringe
  • 50‐ml conical tube
  • Centrifuge
  • γ‐irradiator
  • 100‐µm nylon mesh cell strainer
  • 12‐ml round‐bottom tubes
  • Microtiter plate carrier for centrifuge
  • LumaPlate (PerkinElmer)
  • Scintillation counting device (TopCount, PerkinElmer)
  • Additional reagents and equipment for preparing immunosuppressive cells ( protocol 1), harvesting spleens from mice (unit 1.9), and counting viable cells by trypan blue exclusion ( appendix 3B)

Support Protocol 1: Interpretation of Results and Calculation of Lytic Units

  Materials
  • Thy1.1+ transgenic CL‐4 mice
  • 3% RPMI (see recipe)
  • CD8α+ T cells isolation kit (Miltenyi Biotech)
  • Thy1.2+ BALB/c mice
  • RPMI‐1640 medium, serum free
  • Dendritic cell (DC) preparation (unit 3.7) started 5 days before beginning this protocol
  • 10% RPMI (see recipe)
  • 1 mg/ml LPS stock solution (Sigma, cat. no. L‐4516)
  • 10 mg/ml HA 512‐520 peptide stock solution (available lyophilized from JPT Peptide Technologies)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Immunosuppressive (myeloid) cells, e.g., MDSCs ( protocol 1)
  • 3% RPMI (see recipe)
  • BD Golgi Stop (BD Biosciences)
  • FcR blocking 2.4G2 (BD Pharmingen)
  • Anti–mouse CD8 PE/Cy5 (clone: 53‐6.7, Biolegend, http://www.biolegend.com/)
  • Anti–mouse Thy1.1 PE (clone: HIS51, eBioscience)
  • BD Cytofix/Cytoperm kit (BD Biosciences)
  • Anti–mouse IFN‐γ FITC (BD Pharmingen) or rat IgG1 FITC (BD Pharmingen)
  • 10‐mm culture dish
  • 2‐ml syringe
  • 50‐ml conical tubes
  • Centrifuge
  • Infrared heat lamp
  • 1‐ml syringe
  • 18‐G and 26‐G needles
  • 48‐well culture plate
  • 100‐µm nylon mesh cell strainer
  • 12‐ml and 4‐ml round‐bottom tubes
  • 96 well U‐bottom microtiter plate
  • Microtiter plate carrier for centrifuge
  • Additional reagents and equipment for culturing dendritic cells (unit 3.7), harvesting spleens and other lymphoid organs from mice (unit 1.9), counting viable cells by trypan blue exclusion ( appendix 3B), flow cytometry (Chapter 5), injection of mice (unit 1.6), preparing immunosuppressive cells ( protocol 1), euthanasia of mice (unit 1.8), and flow cytometric IFN‐γ intracellular staining (unit 6.24)
NOTE: Five days before starting, set up a dendritic cell (DC) culture from mouse bone marrow as described in Basic Protocol 2 of unit 3.7.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Azarov, I., Huang, K.T., Basu, S., Gladwin, M.T., Hogg, N., and Kim‐Shapiro, D.B. 2005. Nitric oxide scavenging by red blood cells as a function of hematocrit and oxygenation. J. Biol. Chem. 280:39024‐39032.
   Bronte, V. and Zanovello, P. 2005. Regulation of immune responses by L‐arginine metabolism. Nat. Rev. Immunol. 5:641‐654.
   Bronte, V. 2009. Myeloid‐derived suppressor cells in inflammation: Uncovering cell subsets with enhanced immunosuppressive functions. Eur. J. Immunol. 39:2670‐2672.
   Bronte, V., Apolloni, E., Cabrelle, A., Ronca, R., Serafini, P., Zamboni, P., Restifo, N.P., and Zanovello, P. 2000. Identification of a CD11b(+)/Gr‐1(+)/CD31(+) myeloid progenitor capable of activating or suppressing CD8(+) T cells. Blood 96:3838‐3846.
   Bronte, V., Cingarlini, S., Marigo, I., De Santo, C., Gallina, G., Dolcetti, L., Ugel, S., Peranzoni, E., Mandruzzato, S., and Zanovello, P. 2006. Leukocyte infiltration in cancer creates an unfavorable environment for antitumor immune responses: A novel target for therapeutic intervention. Immunol. Invest. 35:327‐357.
   Dolcetti, L., Marigo, I., Mantelli, B., Peranzoni, E., Zanovello, P., and Bronte, V. 2008. Myeloid‐derived suppressor cell role in tumor‐related inflammation. Cancer Lett. 267:216‐225.
   Dolcetti, L., Peranzoni, E., Ugel, S., Marigo, I., Fernandez Gomez, A., Mesa, C., Geilich, M., Winkels, G., Traggiai, E., Casati, A., Grassi, F., and Bronte, V. 2009. Hierarchy of immunosuppressive strength among myeloid‐derived suppressor cell subsets is determined by GM‐CSF. Eur. J. Immunol. 40:22‐35.
   Gabrilovich, D.I. and Nagaraj, S. 2009. Myeloid‐derived suppressor cells as regulators of the immune system. Nat. Rev. Immunol. 9:162‐174.
   Gabrilovich, D.I., Velders, M.P., Sotomayor, E.M., and Kast, W.M. 2001. Mechanism of immune dysfunction in cancer mediated by immature Gr‐1+ myeloid cells. J. Immunol. 166:5398‐5406.
   Gallina, G., Dolcetti, L., Serafini, P., De Santo, C., Marigo, I., Colombo, M.P., Basso, G., Brombacher, F., Borrello, I., Zanovello, P., Bicciato, S., and Bronte, V. 2006. Tumors induce a subset of inflammatory monocytes with immunosuppressive activity on CD8+ T cells. J. Clin. Invest. 116:2777‐2790.
   Greifenberg, V., Ribechini, E., Rossner, S., and Lutz, M.B. 2009. Myeloid‐derived suppressor cell activation by combined LPS and IFN‐gamma treatment impairs DC development. Eur. J. Immunol. 39:2865‐2876.
   Huang, B., Pan, P.Y., Li, Q., Sato, A.I., Levy, D.E., Bromberg, J., Divino, C.M., and Chen, S.H. 2006. Gr‐1+CD115+ immature myeloid suppressor cells mediate the development of tumor‐induced T regulatory cells and T‐cell anergy in tumor‐bearing host. Cancer Res. 66:1123‐1131.
   Kryczek, I., Zou, L., Rodriguez, P., Zhu, G., Wei, S., Mottram, P., Brumlik, M., Cheng, P., Curiel, T., Myers, L., Lackner, A., Alvarez, X., Ochoa, A., Chen, L., and Zou, W. 2006. B7‐H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma. J. Exp. Med. 203:871‐881.
   Loke, P., MacDonald, A.S., Robb, A., Maizels, R.M., and Allen, J.E. 2000. Alternatively activated macrophages induced by nematode infection inhibit proliferation via cell‐to‐cell contact. Eur. J. Immunol. 30:2669‐2678.
   Marigo, I., Bosio, E., Solito, S., Mesa, C., Fernandez, A., Dolcetti, L., Ugel, S., Sonda, N., Bicciato, S., Falisi, E., Calabrese, F., Basso, G., Zanovello, P., Cozzi, E., Mandruzzato, S., and Bronte V. 2010. Tumor‐induced tolerance and immune suppression depend on the C/EBPbeta transcription factor. Immunity 32:790‐802.
   Morgan, D.J., Liblau, R., Scott, B., Fleck, S., McDevitt, H.O., Sarvetnick, N., Lo, D., and Sherman, L.A. 1996. CD8(+) T cell‐mediated spontaneous diabetes in neonatal mice. J. Immunol. 157:978‐983.
   Movahedi, K., Guilliams, M., Van den Bossche, J., Van den Bergh, R., Gysemans, C., Beschin, A., De Baetselier, P., and Van Ginderachter, J.A. 2008. Identification of discrete tumor‐induced myeloid‐derived suppressor cell subpopulations with distinct T cell‐suppressive activity. Blood 111:4233‐4244.
   Nagaraj, S. and Gabrilovich, D.I. 2008. Tumor escape mechanism governed by myeloid‐derived suppressor cells. Cancer Res. 68:2561‐2563.
   Ribechini, E., Leenen, P.J., and Lutz, M.B. 2009. Gr‐1 antibody induces STAT signaling, macrophage marker expression and abrogation of myeloid‐derived suppressor cell activity in BM cells. Eur. J. Immunol. 39:3538‐3551.
   Rodriguez, P.C., Quiceno, D.G., Zabaleta, J., Ortiz, B., Zea, A.H., Piazuelo, M.B., Delgado, A., Correa, P., Brayer, J., Sotomayor, E.M., Antonia, S., Ochoa, J.B., and Ochoa, A.C. 2004. Arginase I production in the tumor microenvironment by mature myeloid cells inhibits T‐cell receptor expression and antigen‐specific T‐cell responses. Cancer Res. 64:5839‐5849.
   Rodriguez, P.C., Hernandez, C.P., Quiceno, D., Dubinett, S.M., Zabaleta, J., Ochoa, J.B., Gilbert, J., and Ochoa, A.C. 2005. Arginase I in myeloid suppressor cells is induced by COX‐2 in lung carcinoma. J. Exp. Med. 202:931‐939.
   Serafini, P., De Santo, C., Marigo, I., Cingarlini, S., Dolcetti, L., Gallina, G., Zanovello, P., and Bronte, V. 2004. Derangement of immune responses by myeloid suppressor cells. Cancer Immunol. Immunother. 53:64‐72.
   Sica, A. and Bronte, V. 2007. Altered macrophage differentiation and immune dysfunction in tumor development. J. Clin. Invest. 117:1155‐1166.
   Solinas, G., Germano, G., Mantovani, A., and Allavena, P. 2009. Tumor‐associated macrophages (TAM) as major players of the cancer‐related inflammation. J. Leukoc. Biol. 86:1065‐1073.
   Yang, R., Cai, Z., Zhang, Y., Yutzy, W.H., Roby, K.F., and Roden, R.B. 2006. CD80 in immune suppression by mouse ovarian carcinoma‐associated Gr‐1+CD11b+ myeloid cells. Cancer Res. 66:6807‐6815.
   Youn, J.I., Nagaraj, S., Collazo, M., and Gabrilovich, D.I. 2008. Subsets of myeloid‐derived suppressor cells in tumor‐bearing mice. J. Immunol. 181:5791‐5802.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library