Monitoring Antigen‐Specific T Cells Using MHC‐Ig Dimers

Jonathan P. Schneck1, Jill E. Slansky1, Sean M. O'Herrin1, Tim F. Greten1

1 Johns Hopkins University, Baltimore, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 17.2
DOI:  10.1002/0471142735.im1702s35
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The lack of high affinity reagents has made distinguishing T cells on the basis of antigen specificity difficult to accomplish. This unit provides protocols that utilize innovations in molecular design to permit construction of soluble multivalent MHC complexes (MHC‐Ig dimers) with high avidity for cognate T cell receptors. MHC‐Ig dimers display stable binding properties when they interact with antigen‐specific T cells thus allowing their use in the staining of antigen‐specific T cells by flow cytometry. Methods for constructing and detecting these MHC‐Ig dimers are included along with protocols for applying their use for the quantitation of antigen‐specific T cells.

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

Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Construction of MHC Class I–Ig Dimers
  • Support Protocol 1: Quantification of MHC‐Ig Fusion Protein Concentration
  • Support Protocol 2: Large‐Scale Cultures of J558L Transfectants and Concentration of Supernatants
  • Support Protocol 3: Purification of MHC Class I–Ig Dimer by 5‐IODO‐4‐Hydroxy‐3‐Nitrophenylacetyl‐Sepharose Chromatography
  • Support Protocol 4: Preparation of 5‐IODO‐4‐Hydroxy‐ 3‐Nitrophenylacetyl‐Sepharose
  • Support Protocol 5: Peptide Loading of MHC‐Ig Chimeric Protein by Passive Exchange
  • Support Protocol 6: Peptide Loading by Alkaline Stripping
  • Support Protocol 7: Peptide Loading Under Mild Acidic Conditions
  • Support Protocol 8: Flow Cytometric Analysis of Antigen‐Specific T Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Construction of MHC Class I–Ig Dimers

  Materials
  • cDNA encoding MHC class I molecule of interest
  • MluI and XhoI restriction enzymes and buffers (New England Biolabs)
  • pXIg plasmid (available from the authors upon request)
  • 10× T4 DNA ligase buffer (unit 10.9) supplemented with 5 mM ATP
  • 1 Weiss unit/µl T4 DNA ligase (Life Technologies)
  • LB medium (unit 10.3) containing 50 µg/ml ampicillin
  • LB plates (unit 10.19) containing 50 µg/ml ampicillin and 30 µg/ml kanamycin
  • PvuI restriction enzyme and buffer
  • Plasmid containing genomic human β 2‐microglobulin (available from the authors upon request)
  • 3 M sodium acetate buffer, pH 5.2
  • Ethanol
  • J558L cells (available from the authors upon request)
  • Complete RPMI‐10 medium ( appendix 2A) containing 10mM HEPES
  • 10 mg/ml geneticin (G418; Life Technologies)
  • 25‐cm2 flask
  • Electroporator (e.g., Bio‐Rad Gene Pulser) and cuvettes for eukaryotic cells
  • 96‐well flat‐bottom plates (Falcon)
  • Additional reagents and equipment for PCR amplification (unit 10.20), agarose gel electrophoresis (unit 10.4), phenol/chloroform extraction and ethanol precipitation (unit 10.1), restriction enzyme digestion (unit 10.8), DNA gel purification and quantitation with ethidium bromide (unit 10.5), bacterial transformation by electroporation (unit 10.15), isolation of plasmid DNA (miniprep and maxiprep; unit 10.3), counting cells ( appendix 3A), trypan blue exclusion ( appendix 3B), and enzyme‐linked immunosorbant assay (ELISA; see protocol 2)

Support Protocol 1: Quantification of MHC‐Ig Fusion Protein Concentration

  Materials
  • Antibody:
    •  Goat anti–mouse IgG1 (Southern Biotech)
    •  Anti–MHC class I monoclonal antibody (mAb), kappa chain (e.g., BB7.2, W6‐32, 30.5.7s, 20.8.4; ATCC)
  • Carbonate buffer: 5.26 g/liter anhydrous Na 2CO 3, pH 10.4 (with HCl)
  • Blocking buffer: 1% (v/v) FBS in carbonate buffer
  • Wash buffer: recipePBS ( appendix 2A) containing 1% (v/v) FBS and 0.5% (v/v) Tween 20
  • Diluent buffer: recipePBS ( appendix 2A) containing 1% (v/v) FBS
  • Standard: e.g., Mouse IgG1 λ standard (Pharmingen) or MHC‐Ig (Pharmingen)
  • Sample: supernatants containing MHC‐Ig (see protocol 1)
  • Goat anti–mouse λ light chain secondary antibody conjugated to horseradish peroxidase (anti‐mouse‐λ‐HRP; Southern Biotechnology)
  • Final wash buffer: recipePBS ( appendix 2A) containing 0.5% (v/v) Tween 20
  • Developing reagent: TMB One‐Step Substrate System (Dako)
  • 1 N H 2SO 4
  • 96‐well ELISA plates: high‐protein‐binding, ½ ‐area plates for enzyme immunoassay/radioimmunoassay(EIA/RIA; Costar)
  • 96‐well plate reader

Support Protocol 2: Large‐Scale Cultures of J558L Transfectants and Concentration of Supernatants

  Materials
  • J558L transfectants (see protocol 1)
  • Hybridoma SFM medium (Life Technologies)
  • Geneticin (G418; Life Technologies)
  • 10% (w/v) sodium azide (Sigma)
  • 75‐ and 175‐cm2 cell culture flasks
  • 0.45‐µm filter
  • Protein concentration system: e.g., Hollow Fiber Concentrator (A/G Technology) or Centriflo membrane cone (CF50A membrane; Amicon)

Support Protocol 3: Purification of MHC Class I–Ig Dimer by 5‐IODO‐4‐Hydroxy‐3‐Nitrophenylacetyl‐Sepharose Chromatography

  Materials
  • 5‐Iodo‐4‐hydroxy‐3‐nitrophenylacetyl (NIP)–Sepharose resin (see protocol 5) or 5‐ml NIP‐Sepharose column (Biosearch Technologies)
  • recipePBS ( appendix 2A)
  • Cell supernatant containing MHC‐Ig fusion protein (see protocol 3)
  • recipeElution buffer (see recipe)
  • recipePBS containing 0.02% (w/v) sodium azide (Sigma)
  • Centriflo cones (Amicon)
  • Additional reagents and equipment for ELISA (see protocol 2) or SDS‐PAGE (unit 8.4)

Support Protocol 4: Preparation of 5‐IODO‐4‐Hydroxy‐ 3‐Nitrophenylacetyl‐Sepharose

  Materials
  • N‐(3‐aminopropyl)‐1,3‐propanediamine (Aldrich)
  • Sepharose CL‐4B (Pharmacia)
  • 0.1 M NaOH
  • 2 M Na 2CO 3 (Sigma)
  • 0.1 M NaHCO 3, pH 9.5 (Sigma)
  • Cyanogen bromide (CNBr; Sigma)
  • 20 mM sodium azide (Sigma), aqueous and in recipePBS
  • 3% (w/v) NaHCO 3 (Sigma)
  • 3‐Nitro‐4‐hydroxy‐phenylacetyl‐aminocaproic acid succinimide ester (NIP‐CAP‐OSu; Biosearch Technologies, mol. wt. 423)
  • Dioxane (Aldrich)
  • 1‐liter fritted funnel
NOTE: All glassware and solutions should be endotoxin free. Bake glassware at 200°C for ≥4 hr or soak glassware in 0.1 N NaOH, wash with distilled water, and autoclave when possible. Make all solutions in treated glassware and autoclave immediately, if possible.

Support Protocol 5: Peptide Loading of MHC‐Ig Chimeric Protein by Passive Exchange

  Materials
  • 2 mg/ml peptide stock solution in H 2O or dimethyl sulfoxide (DMSO)
  • 1 mg/ml MHC‐Ig dimer protein (see protocol 4)
  • 1 mg/ml human β 2‐microglobulin (Sigma)
  • 1% (w/v) sodium azide (Sigma)

Support Protocol 6: Peptide Loading by Alkaline Stripping

  Materials
  • MHC‐Ig dimer protein (see protocol 4)
  • Peptide‐stripping buffer: 150 mM NaCl, 15 mM Na 2CO 3, pH 11.5
  • Peptide of interest in H 2O or dimethyl sulfoxide (DMSO)
  • Neutralizing buffer: 250 mM Tris⋅Cl, pH 6.8

Support Protocol 7: Peptide Loading Under Mild Acidic Conditions

  Materials
  • MHC‐Ig dimer protein (see protocol 4)
  • recipeCitrate/phosphate buffer (see recipe)
  • Peptide of interest in H 2O or dimethyl sulfoxide (DMSO)
  • Human β 2‐microglobulin (Sigma)
  • 2 M Tris base

Support Protocol 8: Flow Cytometric Analysis of Antigen‐Specific T Cells

  Materials
  • Cells to be stained
  • recipeFACS buffer (see recipe)
  • Peptide‐loaded MHC‐Ig (see Support Protocols protocol 65, protocol 76, or protocol 87)
  • Phycoerythrin‐labeled goat anti–mouse IgG1 (anti‐IgG1‐PE; Caltag or Southern Biotechnology) or biotin‐labeled goat anti–mouse IgG1 (anti‐IgG1‐biotin; Caltag)
  • Phycoerythrin‐labeled avidin (avidin‐PE, Caltag), optional (for anti‐IgG1‐biotin)
  • Anti‐CD8‐FITC (Sigma): fluorescein‐labeled anti–mouse CD8 or anti–human CD8 (clone UCHT‐4)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Altman, J.D., Moss, P.A.H., Goulder, P.J.R., Barouch, D.H., McHeyzer‐Williams, M.G., Bell, J.I., McMichael, A.J., and Davis, M.M. 1996. Phenotypic analysis of antigen‐specific T lymphocytes. Science 274:94‐96.
   Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K.(eds.) 2000. Current Protocols in Molecular Biology. John Wiley & Sons, New York.
   Boniface, J.J., Rabinowitz, J.D., Wulfing, C., Hampl, J., Reich, Z., Altman, J.D., Kantor, R.M., Beeson, G., McConnell, H.M., and Davis, M.M. 1998. Initiation of signal transduction through the T cell receptor requires the peptide multivalent engagement of MHC ligands. Immunity 9:459‐466.
   Bruggemann, M., Williams, G.T., Bindon, I., Clark, M.R., Walker, M.R., Waldman, H., and Neuberger, M.S. 1987. Comparison of the effector functions of human immunoglobulins using a matched set of chimeric antibodies. J. Exp. Med. 166:1351‐1361.
   Capon, D.J., Chamow, S.M., Mordenti, J., Marsters, S.A., Gregory, T., Mitsuya, H., Byrn, R.A., Lucas, C., Wurm, F.M., Groopman, J.E., Broder, S., and Smith, D.H. 1989. Designing CD4 immunoadhesins for AIDS therapy. Nature 337:525‐531.
   Carruth, L.M., Greten, T.F., Murray, C.E., Castro, M.G., Crone, S.N., Pavlat, W., Schneck, J.P., and Siliciano, R.F. 1999. An algorithm for evaluating human cytotoxic T lymphocyte responses to candidate AIDS vaccines. AIDS Res. Hum. Retroviruses 15:1021‐1034.
   Casares, S., Bona, C.A., and Brumeanu, T.D. 1997. Engineering and characterization of a murine MHC class II‐ immunoglobulin chimera expressing an immunodominant CD4 T viral epitope. Protein Eng. 10:1295‐1301.
   Casares, S., Zong, C.S., Radu, D.L., Miller, A., Bona, C.A., and Brumeanu, T.D. 1999. Antigen‐specific signaling by a soluble, dimeric peptide/MHC class II/Fc chimera leading to Th2 differentiation. J. Exp. Med. 190:543‐554.
   Corr, M., Slanetz, A.E., Boyd, L.F., Jelonek, M.T., Khiko, S., Al‐Ramadi, B.K., Kim, Y.S., Maher, S.E., Bothwell, A.L.M., and Margulies, D.H. 1994. T cell receptor‐MHC class I peptide interactions: Affinity, kinetics and specificity. Science 265:946‐949.
   Cullen, C.M., Jameson, S.C., DeLay, M., Cottrell, C., Becken, E.T., Choi, E., and Hirsch, R. 1999. A divalent major histocompatibility complex/IgG1 fusion protein induces antigen‐specific T cell activation in vitro and in vivo. Cell. Immunol. 192:54‐62.
   Dal Porto, J., Johansen, T.E., Catipovic, B., Parfitt, D.J., Tuveson, D., Gether, U., Kozlowski, S., Fearon, D., and Schneck, J.P. 1993. A soluble divalent class I major histocompatibilty complex molecule inhibits alloreactive T cells at nanomolar concentrations. Proc. Natl. Acad. Sci. U.S.A. 90:6671‐6675.
   Gascoigne, N.R.J., Goodnow, C.C., Dudzik, K.I., Oi, V.T., and Davis, M.M. 1987. Secretion of a chimeric T‐cell receptor‐immunoglobulin protein. Proc. Natl. Acad. Sci. U.S.A. 84:2936‐2940.
   Goldstein, J., Mostowsky, H., Tung, J., Hon, H., Brunswick, M., and Kozlowski, S. 1997. Naive alloreactive CD8 T cells are activated by purified major histocompatibility complex class I and antigenic peptide. Eur. J. Immunol. 27:871‐878.
   Goldstein, J.S., Chen, T., Brunswick, M., Mostowsky, H., and Kozlowski, S. 1998. Purified MHC class I and peptide complexes activate naive CD8+ T cells independently of the CD28/B7 and LFA‐1/ICAM‐1 costimulatory interactions. J. Immunol. 160:3180‐3187.
   Greten, T.F., Slansky, J.E., Kubota, R., Soldan, S.S., Jaffee, E.M., Leist, T.P., Pardoll, D.M., Jacobson, S., and Schneck, J.P. 1998. Direct visualization of antigen‐specific T cells: HTLV‐1 Tax11‐19‐specific CD8+ T cells are activated in peripheral blood and accumulate in cerebrospinal fluid from HAM/TSP patients. Proc. Natl. Acad. Sci. U.S.A. 95:7568‐7573.
   Hamad, A.R.A., O'Herrin, S.M., Lebowitz, M.S., Srikrishnan, A., Bieler, J., Schneck, J., and Pardoll, D. 1998. Potent T cell activation with dimeric peptide‐major histocompatibility complex class II ligand: The role of CD4 coreceptor. J. Exp. Med. 188:1633‐1640.
   Lebowitz, M.S., O'Herrin, S.M., Hamad, A.‐R.A., Fahmy, T., Marguet, D., Barnes, N.C., Pardoll, D., Bieler, J.G., and Schneck, J.P. 1999. Soluble, high affinity dimers of T‐cell receptors and class II major histocompatability complexes: Biochemical probes for analysis and modulation of immune responses. Cell. Immunol. 192:175‐184.
   Lepley, D.M., Gillanders, W.E., Myers, N.B., Robinson, R.A., Beisel, K.W., Wisecarver, J.L., Pirruccello, S.J., Lee, D.R., Hansen, T.H., and Rubocki, R.J. 1997. Biochemical and functional characterization of soluble multivalent MHC Ld/Fcγ1 and Ld/Fµ chimeric proteins loaded with specific peptides. Transplantation 63:765‐774.
   Murali‐Krishna, K., Altman, J.D., Suresh, M., Sourdive, D.J., Zajac, A.J., Miller, J.D., Slansky, J., and Ahmed, R. 1998. Counting antigen‐specific CD8 T cells: A reevaluation of bystander activation during viral infection. Immunity 8:177‐187.
   Selin, L.D., Lin, M.Y., Kraemer, K.A., Pardoll, D.M., Schneck, J.P., Varga, S.M., Santolucito, P.A., Pinto, A.K., and Welsch, R.A. 1999. Attrition of T cell memory: Selective loss of LCMU epitope‐specific memory CD8 T cells following infections with heterologous viruses. Immunity 11:733‐742.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library