Presenting Exogenous Antigen to T Cells

Clifford V. Harding1, Lakshmi Ramachandra1

1 Case Western Reserve University, Cleveland, Ohio
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 16.2
DOI:  10.1002/0471142735.im1602s88
Online Posting Date:  February, 2010
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Antigen processing and presentation experiments can be done with a wide variety of antigen‐presenting cells (APCs). Most experiments will use one of the “professional” APC types: dendritic cells (DCs), macrophages, and B lymphocytes. Other types of cells may be used for antigen presentation in some circumstances. Each type of professional APC has an important antigen‐presentation function, but the different APC types contribute to different aspects of the immune response. Therefore, selection of an APC type for study must include consideration of the stage or aspect of immune response that is to be modeled in the experiment. An important technical distinction for some types of experiments is whether the APCs are adherent or nonadherent, since this dictates the procedures that must be used to wash the cells as the medium is changed. Curr. Protoc. Immunol. 88:16.2.1‐16.2.18. © 2010 by John Wiley & Sons, Inc.

Keywords: antigen presenting cells; antigen processing; antigen presentation; T lymphocyte; T hybridoma cells

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Table of Contents

  • Introduction
  • Basic Protocol 1: Antigen Processing and Presentation: A Simple Add‐Together System for Continuous Processing
  • Support Protocol 1: Detection of IL‐2 Using CTLL‐2 Cells and Alamar Blue
  • Alternate Protocol 1: Antigen Presentation by Fixed Adherent APCs to Detect Preprocessed Peptide
  • Alternate Protocol 2: Antigen Presentation by Fixed Nonadherent APCs to Detect Preprocessed Peptide
  • Alternate Protocol 3: Antigen Processing with Fixation After Incubation with Protein Antigen
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
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Basic Protocol 1: Antigen Processing and Presentation: A Simple Add‐Together System for Continuous Processing

  • Antigen‐presenting cells (APCs; unit 16.1), proliferating or nonproliferating, MHC‐compatible with T hybridoma cells; use macrophages or dendritic cells for particulate antigens.
  • Near‐confluent culture of T hybridoma cells
  • Complete DMEM‐10 medium ( appendix 2A)
  • Antigen stock solution (see recipe)
  • 96‐well flat‐bottom microtiter plates (round‐bottom for DCs)
  • Sorvall RT‐6000 centrifuge and HB‐1000 rotor (or equivalent) with tube and plate carriers
  • 8‐ or 12‐channel multichannel pipettor and sterile tips
  • Pipets, sterile
  • Additional reagents and equipment for counting viable cells ( appendix 3A& appendix 3B) and performing IL‐2 bioassay ( protocol 2 or unit 6.3)

Support Protocol 1: Detection of IL‐2 Using CTLL‐2 Cells and Alamar Blue

  • Supernatants to be assayed for IL‐2, in 96‐well plate
  • CTLL‐2 cells in active log‐phase growth (see unit 6.3)
  • DMEM medium, serum free (Hyclone)
  • Complete DMEM‐10 medium ( appendix 2A)
  • Murine IL‐2 (BD Biosciences or any other company) diluted to 2000 U/ml in complete DMEM‐10 medium (rat or human IL‐2 can also be used)
  • Alamar Blue (Invitrogen); keep at 4°C for short‐term use and at −80°C for long‐term storage
  • 15‐ml conical tubes (Falcon)
  • Sorvall RT6000 refrigerated tabletop centrifuge and HB‐1000 rotor (or equivalent)
  • Spectrophotometer (with filters at 600 and 570 nm or 595 and 550 nm)
  • Additional reagents and equipment for counting viable cells ( appendix 3A& appendix 3B)

Alternate Protocol 1: Antigen Presentation by Fixed Adherent APCs to Detect Preprocessed Peptide

  • 2% (w/v) paraformaldehyde in PBS (see recipe)
  • Lysine wash solution (see recipe)

Alternate Protocol 2: Antigen Presentation by Fixed Nonadherent APCs to Detect Preprocessed Peptide

  • Confluent cultures of nonadherent antigen‐presenting transformed B cells (e.g., CH‐27)
  • 2% (w/v) paraformaldehyde in PBS (see recipe)
  • Lysine wash solution (see recipe)
  • 15‐ and 50‐ml centrifuge tubes, sterile

Alternate Protocol 3: Antigen Processing with Fixation After Incubation with Protein Antigen

  • Complete DMEM‐10 medium lacking antibiotics (prepare as in appendix 2A, but omit antibiotics, for experiments using live bacteria)
  • Antigen: bacteria or bacteria expressing non‐endogenous protein
  • 2% (w/v) paraformaldehyde in PBS (see recipe)
  • Lysine wash solution (see recipe)
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Literature Cited

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