Identification of B Cell and T Cell Epitopes Using Synthetic Peptide Combinatorial Libraries

Clemencia Pinilla1, Jon R. Appel1, Valeria Judkowski1, Richard A. Houghten1

1 Torrey Pines Institute for Molecular Studies, San Diego, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 9.5
DOI:  10.1002/0471142735.im0905s99
Online Posting Date:  November, 2012
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Abstract

This unit presents a combinatorial library method that consists of the synthesis and screening of mixture‐based synthetic combinatorial libraries of peptide molecules. The protocols employ peptide libraries to identify peptides recognized by MAbs and T cells. The first protocol uses a positional scanning peptide library made up of hexapeptides to identify antigenic determinants recognized by MAbs. The 120 mixtures in the hexapeptide library are tested for their inhibitory activity in a competitive ELISA. The second protocol uses a decapeptide library to identify T cell peptide ligands. The 200 mixtures of the decapeptide library are tested for their ability to induce T cell activation. Support protocols cover optimization of the assay conditions for each MAb or T cell, to achieve the best level of sensitivity and reproducibility, and preparation of a hexapeptide library, along with deconvolution approaches. Curr. Protoc. Immunol. 99:9.5.1‐9.5.16. © 2012 by John Wiley & Sons, Inc.

Keywords: combinatorial libraries; monoclonal antibodies; T cells; peptides

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Screening a Peptide Library for Antibody Inhibition
  • Basic Protocol 2: Screening a Peptide Library to Identify CD4+ or CD8+ T Cell Ligands
  • Support Protocol 1: Optimizing Antigen and Antibody Concentrations for Screening Assay
  • Support Protocol 2: Preparing a Positional Scanning Peptide Library
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Screening a Peptide Library for Antibody Inhibition

  Materials
  • Peptide or protein antigen of interest
  • 1% (w/v) BSA/PBS (see recipe for PBS)
  • Hexapeptide positional scanning library (Table 9.5.1): 120 peptide mixtures at 5 mg/ml (see protocol 4; for information on the availability of these libraries, contact C. Pinilla at cpinilla@tpims.org)
  • Monoclonal antibody (MAb) against antigen of interest
  • Horseradish peroxidase (HRPO)‐conjugated anti‐mouse antibody specific for the isotype of the MAb (Calbiochem; use in accordance with manufacturer's instructions)
  • Developing solution (see recipe)
  • 4 N sulfuric acid
  • 96‐well microtiter plates made of high‐binding‐capacity polystyrene (e.g., A/2 area plates, Costar; or Immunolon 4 plates, Dynatech)
  • Moist chamber: sealed Tupperware box lined with wet paper towels
  • 96‐well microtiter plate reader with 492‐nm filter
  • Additional reagents and equipment for optimizing antigen and antibody concentrations for screening assay (see protocol 3) and peptide synthesis (see protocol 4 and unit 9.1)

Basic Protocol 2: Screening a Peptide Library to Identify CD4+ or CD8+ T Cell Ligands

  Materials
  • T cell clone (TCC)
  • Decapeptide positional scanning library (prepared in a manner similar to hexapeptide library in protocol 4; for information on the availability of these libraries, contact C. Pinilla at cpinilla@tpims.org)
  • Antigen‐presenting cells (APC) and appropriate growth medium
  • [3H]‐thymidine
  • Na 2 51CrO 4
  • ELISA kit to measure cytokine production, such as gamma‐interferon or GM‐CSF (PharMingen)
  • 96‐well U‐bottom microtiter plates (Costar)
  • Microtiter plate reader
  • Cell harvester
  • Liquid scintillation counter for proliferation CD4+ assays
  • Gamma counter for cytotoxicity assays (alternatively, a sensitive fluorometer for the detection of released fluorescence from labeled target cells)

Support Protocol 1: Optimizing Antigen and Antibody Concentrations for Screening Assay

  Materials
  • Peptide or protein antigen—one sample dissolved in PBS and one in bicarbonate buffer
  • Phosphate‐buffered saline (PBS: see recipe)
  • Bicarbonate buffer (see recipe)
  • 1% (w/v) BSA/PBS (see recipe for PBS)
  • MAb specific for peptide or protein antigen
  • Horseradish peroxidase (HRPO)‐conjugated secondary antibody specific for isotype of MAb (Calbiochem; diluted according to supplier's instructions in 1% BSA/PBS)
  • Developing solution (see recipe)
  • 4 N sulfuric acid
  • 96‐well microtiter plate made of high‐binding‐capacity polystyrene (e.g., A/2, Costar; or Immunolon 4, Dynatech)
  • Moist box: sealed Tupperware box lined with wet paper towels
  • Microtiter plate reader with 492‐nm filter

Support Protocol 2: Preparing a Positional Scanning Peptide Library

  Materials
  • Methylbenzhydrylamine (MBHA) polystyrene resin (Sigma‐Aldrich)
  • N‐α‐t‐Boc‐amino acids with side‐chain protecting groups
  • Polypropylene mesh pockets (Synthesis Division)
  • Additional reagents and equipment for peptide synthesis (units 9.1& 9.6) and ninhydrin test (unit 9.6)
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Figures

Videos

Literature Cited

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   La Rosa, C., Krishnan, R., Markel, S., Schneck, J.P., Houghten, R., Pinilla, C., and Diamond, D. 2001. Enhanced immune activity of cytotoxic T‐lymphocyte epitope analogues derived from positional scanning synthetic combinatorial libraries. Blood 97:1776‐1786.
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   Nino‐Vasquez, J.J., Allicotti, G., Borras, E., Wilson, D.B., Valmori, D., Simon, R., Martin, R., and Pinilla, C. 2004. A powerful combination: The use of positional scanning libraries and biometrical analysis to identify cross‐reactive T cell epitopes. Mol. Immunol. 40:1063‐1074.
   Ostresh, J.M., Winkle, J.H., Hamashin, V.T., and Houghten, R.A. 1994. Peptide libraries: Determination of relative reaction rates of protected amino acids in competitive couplings. Biopolymers 34:1684‐1689.
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   Pinilla, C., Rubio‐Godoy, V., Dutoit, V., Guillaume, P., Simon, R., Zhao, Y., Houghten, R., Cerottini, J.‐C., Romero, P., and Valmori, D. 2001. Combinatorial peptide libraries as an alternative approach to the identification of ligands for tumor reactive cytolytic T lymphocytes. Cancer Res. 61:5153‐5160.
   Pinilla, C., Appel, J.R., Borras, E., and Houghten, R.A. 2003. Advances in the use of synthetic combinatorial chemistry: Mixture‐based libraries. Nat. Med. 9:118‐122.
   Rubio‐Godoy, V., Ayyoub, M., Dutoit, V., Servis, C., Schink, A., Rimoldi, D., Romero, P., Cerottini, J.‐C., Simon, R., Zhao, Y., Houghten, R.A., Pinilla, C., and Valmori, D. 2002a. Combinatorial peptide library based identification of peptide ligands for tumor‐reactive cytolytic T lymphocytes of unknown specificity. Eur. J. Immunol. 32:2292‐2299.
   Rubio‐Godoy, V., Dutoit, V., Zhao, Y., Simon, R., Guillaume, P., Houghten, R., Romero, P., Cerottini, J.C., Pinilla, C., and Valmori, D. 2002b. Positional scanning‐synthetic peptide library‐based analysis of self‐ and pathogen‐derived peptide cross‐reactivity with tumor‐reactive Melan‐A‐specific CTL. J. Immunol. 169:5696‐5707.
   Rubio‐Godoy, V., Pinilla, C., Dutoit, V., Borras, E., Simon, R., Zhao, Y., Cerottini, J.‐C., Romero, P., Houghten, R.A., and Valmori, D. 2002c. Towards synthetic combinatorial peptide libraries in positional scanning format (PS‐SCL)‐based identification of CD8+ tumor‐reactive T‐cell ligands: A comparative analysis of PS‐SCL recognition by a single tumor‐reactive CD8+ CTL. Cancer Res. 62:2058‐2063.
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   Sospedra, M., Zhao, Y., Giulianotti, M., Simon, R., Pinilla, C., and Martin, R. 2010. Combining positional scanning peptide libraries, HLA‐DR transfectants and bioinformatics to dissect the epitope spectrum of HLA class II cross‐restricted CD4+ T cell clones. J. Immunol. Methods 353:93‐101.
   Stewart, J.M. and Young, J.D. 1984. Solid Phase Peptide Synthesis, 2nd ed. Pierce Chemical Co., Rockford, Illinois.
   Tam, J.P., Heath, W.F., and Merrifield, R.B. 1983. SN2 deprotection of synthetic peptides with a low concentration of HF in dimethyl sulfide: Evidence and application in peptide synthesis. J. Am. Chem. Soc. 105:6442‐6455.
   Venturini, S., Allicotti, G., Zhao, Y., Simon, R., Burton, D.R., Pinilla, C., and Poignard, P. 2006. Identification of peptides from human pathogens able to cross‐activate an HIV‐1‐gag‐specific CD4(+) T cell clone. Eur. J. Immunol. 36:27‐36.
   Wilson, D.B., Pinilla, C., Wilson, D.H., Schroder, K., Boggiano, C., Judkowski, V., Kaye, J., Hemmer, B., Martin, R., and Houghten, R.A. 1999. Immunogenicity. I. Use of peptide libraries to identify epitopes that activate clonotypic CD4+ T cells and induce T cell responses to native peptide ligands. J. Immunol 163:6424‐6434.
   Zhao, Y., Gran, B., Pinilla, C., Markovic‐Plese, S., Hemmer, B., Tzou, A., Whitney, L.W., Biddison, W.E., Martin, R., and Simon, R. 2001a. Combinatorial peptide libraries and biometric score matrices permit the quantitative analysis of specific and degenerate interactions between clonotypic T‐cell receptors and MHC‐peptide ligands. J. Immunol. 167:2130‐2141.
   Zhao, Y., Grover, L., and Simon, R. 2001b. TEST: A web‐based T‐cell epitope search tool. Proceedings of the 14th IEEE Symposium on Computer‐Based Medical Systems, 493‐497.
Key References
   Houghten et al., 1999. See above.
  Both references above include reviews of the synthesis and use of mixture‐based combinatorial libraries.
   Houghten et al., 2008. See above.
  Both references above include reviews of the use of combinatorial libraries to study antibody, MHC, and T cell specificity.
   Pinilla et al., 1999. See above.
  Identification of peptides from immunoprevalent vaccinia proteins recognized by CD4+ T cells derived from smallpox vaccinated human subjects. GM‐CSF is found to be a sensitive readout to measure the activation of T cells in response to mixtures of positional scanning libraries.
   Borras et al., 2002. See above.
   Judkowski et al., 2011. See above.
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