Differentiation and Characterization of Tr1 Cells

Norio Chihara1, Asaf Madi1, Katarzyna Karwacz1, Amit Awasthi2, Vijay K. Kuchroo1

1 Evergrande Center for Immunologic Diseases and Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, 2 Translational Health Science and Technology Institute (an autonomous institute of Department of Biotechnology, Govt. of India), Faridabad
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 3.27
DOI:  10.1002/0471142735.im0327s113
Online Posting Date:  April, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Regulatory T cell–mediated suppression serves as a pivotal mechanism of negative regulation of immune‐mediated inflammation. Type 1 regulatory T cells (Tr1 cells) are an important subset of CD4+ T cells that prevent excessive inflammatory responses and maintain immune tolerance. The anti‐inflammatory role of Tr1 cells is mediated in part by their production of interleukin 10 (IL‐10), which dampens the function of both antigen‐presenting cells and antigen‐specific effector T cells. Additionally, Tr1 cells can kill effector and myeloid cells through the perforin‐granzyme B pathway. Adoptive transfer of in vitro differentiated Tr1 cells can be used to suppress autoimmune tissue inflammation in vivo. This unit describes the in vitro stimulation of naïve murine CD4+ T cells using IL‐27 to generate IL‐10–producing Tr1 cells. © 2016 by John Wiley & Sons, Inc.

Keywords: T cell differentiation; CD4; IL‐27; Tr1; IL‐10

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Generation of IL‐10 Producing CD4+ T Cells (Tr1 Cells) In Vitro
  • Support Protocol 1: Characterization of Tr1 cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1: Generation of IL‐10 Producing CD4+ T Cells (Tr1 Cells) In Vitro

  • C57BL/6 mice (e.g., Jackson Laboratory)
  • Dulbecco's phosphate‐buffered saline, pH 7.4 (DPBS; Thermo Fisher, cat. no. 14190250)
  • Clone medium (see recipe)
  • Magnetic cell sorting (MACS) buffer (see recipe)
  • Anti‐CD4 (L3T4) microbeads (Miltenyi Biotec, cat. no. 130‐049‐201), MACS beads to purify CD4+ cells
  • Fluorescent conjugated anti‐mouse CD44 (IM7; Biolegend, cat. no. 103030), CD62L (MEL‐14; Biolegend, cat. no. 104406), and CD4 (RM4‐5; Biolegend, cat.no. 100414) antibody
  • Anti‐mouse CD3 (145‐2C11; Bio X Cell, cat. no. BE0001‐1) and CD28 (37.51; Bio X Cell, cat. no. BE0015‐1) antibody
  • Recombinant IL‐27 protein (e‐Bioscience, cat. no. 14‐8271‐63)
  • 70‐μm cell strainer (Fisher Scientific, cat. no. 22‐363‐548)
  • Corning® Costar® 96 well flat bottom cell culture plate (Sigma, cat. no. CLS3595)
  • Additional reagents and equipment for preparation of cell suspensions from mouse lymph nodes or spleens and removal of red blood cells (unit 3.1; Kruisbeek, ), counting cells using a hemocytometer ( appendix 3A; Strober, ), fractionation of T cells using magnetic beads (unit 3.5; Thornton, ), and flow cytometry (Chapter 5).

Support Protocol 1: Characterization of Tr1 cells

  • Tr1 cells and culture supernatants (see protocol 1)
  • RNAeasy mini kit (Qiagen, cat. no. 74134)
  • iScript cDNA synthesis kit (BioRad, cat. no. 1708891)
  • IL‐10 Taqman probe (Life Technologies, cat. no. Mm00439614_m1)
  • TaqMan Fast Universal PCR Master Mix (Life Technologies, cat. no. 4366072)
  • Culture medium (see recipe)
  • 0.1 mg/ml PMA stock (Sigma, cat. no. P8139)
  • 1 mg/ml ionomycin stock (Sigma, cat. no. I0634)
  • BD GolgiStop (BD Biosciences, cat. no. 554724)
  • 7‐amino‐actinomycin D (7AAD; Biolegend, cat. no. 420404) or propidium iodide (Biolegend, cat. no. 421301)
  • Fixation/Permeabilization Solution Kit (BD Biosciences, cat. no. 554714)
  • Fluorescent‐conjugated anti‐mouse IL‐10 antibody (Biolegend, cat. no. 505008)
  • Fluorescent‐conjugated anti‐mouse IFN‐γ antibody (Biolegend, cat. no. 505810)
  • Purified rat anti‐mouse IL‐10 antibody (BD Pharmingen, cat. no. 551215), capture antibody
  • Coating buffer (0.1 M NaHPO 4, pH 9.0;)
  • Tween 20 (Sigma, cat. no. P1379)
  • 10% BSA Diluent/Blocking Solution (KPL, cat. no. 50‐61‐10)
  • Recombinant IL‐10 (Biolegend, cat. no. 575809), ELISA standard
  • Biotin‐labeled rat anti‐mouse IL‐10 antibody (BD Pharmingen, cat. no. 554423), secondary antibody
  • 2.9 mg/ml avidin peroxidase stock (Sigma, cat. no. A7419)
  • 3,3′,5,5′‐Tetramethylbenzidine (TMB) buffer (KPL, cat. no. 52‐00‐00)
  • Sulfuric acid (H 3PO 4) stop solution (KPL, cat. no. 50‐85‐04)
  • Corning 96‐well V‐bottom plate (Thermo Scientific, cat. no. 249570)
  • Corning 96‐well EIA/RIA plate (Corning, cat. no. 3368)
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Awasthi, A., Carrier, Y., Peron, J.P., Bettelli, E., Kamanaka, M., Flavell, R.A., Kuchroo, V.K., Oukka, M., and Weiner, H.L. 2007. A dominant function for interleukin 27 in generating interleukin 10‐producing anti‐inflammatory T cells. Nat. Immunol. 8:1380‐1389. doi: 10.1038/ni1541.
  Barrat, F.J., Cua, D.J., Boonstra, A., Richards, D.F., Crain, C., Savelkoul, H.F., de Waal‐Malefyt, R., Coffman, R.L., Hawrylowicz, C.M., and O'Garra, A. 2002. In vitro generation of interleukin 10‐producing regulatory CD4(+) T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)‐ and Th2‐inducing cytokines. J. Exp. Med. 195:603‐616.
   Fitzgerald, D.C., Ciric, B., Touil, T., Harle, H., Grammatikopolou, J., Das Sarma, J., Gran, B., Zhang, G.X., and Rostami, A. 2007a. Suppressive effect of IL‐27 on encephalitogenic Th17 cells and the effector phase of experimental autoimmune encephalomyelitis. J. Immunol. 179:3268‐3275.
  Fitzgerald, D.C., Zhang, G.X., El‐Behi, M., Fonseca‐Kelly, Z., Li, H., Yu, S., Saris, C.J., Gran, B., Ciric, B., and Rostami, A. 2007b. Suppression of autoimmune inflammation of the central nervous system by interleukin 10 secreted by interleukin 27‐stimulated T cells. Nat. Immunol. 8:1372‐1379. doi: 10.1038/ni1540.
  Gagliani, N., Magnani, C.F., Huber, S., Gianolini, M.E., Pala, M., Licona‐Limon, P., Guo, B., Herbert, D.R., Bulfone, A., Trentini, F., Di Serio, C., Bacchetta, R., Andreani, M., Brockmann, L., Gregori, S., Flavell, R.A., and Roncarolo, M.G. 2013. Coexpression of CD49b and LAG‐3 identifies human and mouse T regulatory type 1 cells. Nat. Med. 19:739‐746. doi: 10.1038/nm.3179.
  Groux, H., O'Garra, A., Bigler, M., Rouleau, M., Antonenko, S., de Vries, J.E., and Roncarolo, M.G. 1997. A CD4+ T‐cell subset inhibits antigen‐specific T‐cell responses and prevents colitis. Nature 389:737‐742. doi: 10.1038/39614.
  Jiang, Y., Lueders, J., Glatfelter, A., Gooden, C., and Bittner, M. 2001. Profiling human gene expression with cDNA microarrays. Curr. Protoc. Mol. Biol, 53:22.3.1‐22.3.26. doi: 10.1002/0471142727.mb2203s53.
  Kruisbeek, A. M. 2001. Isolation of mouse mononuclear cells. Curr. Protoc. Immunol. 39:3.1.1–3.1.5. doi: 10.1002/0471142735.im0301s39.
  Levings, M.K., Sangregorio, R., Galbiati, F., Squadrone, S., de Waal Malefyt, R., and Roncarolo, M.G. 2001. IFN‐alpha and IL‐10 induce the differentiation of human type 1 T regulatory cells. J. Immunol. 166:5530‐5539.
   Maynard, C.L., Harrington, L.E., Janowski, K.M., Oliver, J.R., Zindl, C.L., Rudensky, A.Y., and Weaver, C.T. 2007. Regulatory T cells expressing interleukin 10 develop from Foxp3+ and Foxp3‐ precursor cells in the absence of interleukin 10. Nat. Immunol. 8:931‐941.
  Strober, W. 2001. Monitoring cell growth. Curr. Protoc. Immunol. 21:A.3A.1‐A.3A.2. doi: 10.1002/0471142735.ima03as21.
  Stumhofer, J.S., Silver, J.S., Laurence, A., Porrett, P.M., Harris, T.H., Turka, L.A., Ernst, M., Saris, C.J., O'Shea, J.J., and Hunter, C.A. 2007. Interleukins 27 and 6 induce STAT3‐mediated T cell production of interleukin 10. Nat. Immunol. 8:1363‐1371. doi: 10.1038/ni1537.
  Thornton, A.M. 2003. Fractionation of T and B cells using magnetic beads. Curr. Protoc. Immunol. 55:3.5A.1‐3.5A.11. doi: 10.1002/0471142735.im0305as57.
  Villarino, A.V., Stumhofer, J.S., Saris, C.J., Kastelein, R.A., de Sauvage, F.J., and Hunter, C.A. 2006. IL‐27 limits IL‐2 production during Th1 differentiation. J. Immunol. 176:237‐247. doi: 10.4049/jimmunol.176.1.237.
  Yosef, N., Shalek, A.K., Gaublomme, J.T., Jin, H., Lee, Y., Awasthi, A., Wu, C., Karwacz, K., Xiao, S., Jorgolli, M., Gennert, D., Satija, R., Shakya, A., Lu, D.Y., Trombetta, J.J., Pillai, M.R., Ratcliffe, P.J., Coleman, M.L., Bix, M., Tantin, D., Park, H., Kuchroo, V.K., and Regev, A. 2013. Dynamic regulatory network controlling TH17 cell differentiation. Nature 496:461‐468. doi: 10.1038/nature11981.
PDF or HTML at Wiley Online Library