Abstract
CD2 is a cell adhesion molecule that mediates T-cell activation by binding to its ligand CD58 on antigen-presenting cells. Interaction between CD2 and CD58 or leukocyte function-associated antigen-3 (LFA-3) helps to optimize immune recognition facilitating contact between T lymphocytes and antigen-presenting cells. Modulation or inhibition of this interaction has been shown to be therapeutically useful in the treatment of autoimmune diseases. Antibodies and small molecules including peptides have been designed to modulate or disrupt the cell adhesion interactions due to CD2 and CD58. E-rosetting assay is a widely used method applied in the study of the modulation of CD2–CD58 interaction, which is either labor-intensive or radio-hazardous. In this chapter, we describe two methods that are used to study cell adhesion inhibition: (a) E-rosetting Assay and (b) Lymphocyte-epithelial assay. The second method, lymphocyte-epithelial assay, is a rapid and sensitive heterotypic cell adhesion assay for studying cell adhesion inhibition. The method relies on the CD2 expression on the surface of Jurkat cells and the CD58 expression on the surface of Caco-2 cells, which were confirmed by flow cytometry and ELISA studies respectively. This heterotypic cell adhesion assay described typically takes less than 4 h to perform, allows the evaluation of inhibitory activity of peptides/small molecules to modulate CD2–CD58 interaction in real cell system.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Davis, S.J., Ikemizu, S., Wild, M.K., van der Merwe, P.A. (1998) CD2 and the nature of protein interactions mediating cell-cell recognition. Immunol Rev. 163: 217–236.
Van der Merwe, P.A., Davis, S.J. (2003) Molecular interactions mediating T cell antigen recognition. Annu Rev Immunol. 21: 659–684.
Kim, M., Sun, Z.Y., Byron, O., Campbell, G., Wagner, G., Wang, J., Reinherz, E.L. (2001) Molecular dissection of the CD2-CD58 counter-receptor interface identifies CD2 Tyr86 and CD58 Lys34 residues as the functional “hot spot”. J Mol Biol. 312: 711–720.
Bachmann, M.F., Barner, M., Kopf, M. (1999) CD2 sets quantitative thresholds in T cell activation. J Exp Med. 190: 1383–1391
Mojcik, C.F., Shevach, E. (1997) Adhesion molecules: a rheumatologic perspective. Arthritis Rheum. 6: 991–1004.
Takahashi, H., Soderstrom, K., Nilsson, E., Kiessling, R., Patarroyo, M. (1992) Integrins and other adhesion molecules on lymphocytes from synovial fluid and peripheral blood of rheumatoid arthritis patients. Eur J Immunol. 22: 2879–2885.
Qin, L.H., Chavin, K.D., Lin, J.X., Yagita, H., Bromberg, J.S. (1994) Anti-CD2 receptor and anti-CD2 ligand (CD48) antibodies synergize to prolong allograft survival. J Exp Med. 179: 341–346.
Hirahara, H., Tsuchida, M., Wanatabe, T., Haga, M., Matsumoto, Y., Abo, T., Eguchi, S. (1995) Long-term survival of cardiac allografts in rats treated before and after surgery with monoclonal antibody to CD2. Transplantation 59: 85–90.
Mrowietz, U. (2002) Treatment targeted to cell surface epitopes. Clin Exp Derm. 27: 591–596.
Braun, J., Sieper, J. (2003). Role of novel biological therapies in psoriatic arthritis: effects on joints and skin. Biodrugs 17: 187–199.
Aruffo, A., Hollenbaugh, D. (2001) Therapeutic intervention with inhibitors of co-stimulatory pathways in autoimmune disease. Curr Opin Immunol. 13: 683–686.
Wang, J., Smolyar, A., Tan, K., Liu, J., Kim, M., Sun, Z.J., Wagner, G., Reinherz, E.L. (1999) Structure of a heterophilic adhesion complex between the human CD2 and CD58 (LFA-3) counterreceptors. Cell 97: 791–803.
Branco, L., Barren, P., Mao, S.Y., Pfarr, D., Kaplon, R., Psotema, C., Langermann, S., Koenig, S., Johnson, S. (1999) Selective deletion of antigen-specific, activated T cells by a humanized mAb to CD2 (MEDI-507) is mediated by NK cells. Transplantation 68: 1588–1596.
Hunig, T. (1985) The cell surface molecule recognized by the erythrocyte receptor of T lymphocytes identification and partial characterization using a monoclonal antibody. J Exp Med. 162: 890–901.
Albert-Wolf, M., Meuer, S.C., Wallich, R. (1991) Dual function of recombinant human CD58: inhibition of T-cell adhesion and activation via the CD2 pathway. Int Immunol. 3: 1335–1347.
Liu, J., Ying, J., Chow, V.T., Hruby, V.J., Satyanarayanajois, S.D. (2005). Structure-activity studies of peptides from the “hot spot” region of human CD2 protein: development of peptides for immunomodulation. J Med Chem. 48: 6236–6249.
Rink, T.J., Tsien, R.Y., Pozzan, T. (1982) Cytoplasmic pH and free Mg2+ in lymphocytes. J Cell Biol. 95: 189–196.
Hahn, W.C., Burakoff, S.J., Bierer, B.E. (1993) Signal transduction pathways involved in T cell receptor-induced regulation of CD2 avidity for CD58. J Immunol. 150: 2607–2619.
Hollo, Z., Homolya, L., Davis, C.W., Sarkadi, B. (1994) Calcein accumulation as a fluorometric functional assay of the multidrug transporter. Biochim Biophys Acta 1191: 384–388.
Homoloya, L., Hollo, Z., Germann, U.A., Pastan, I., Gottesmann, M.M., Sarkadi, B. (1993) Fluorescent cellular indicators are extruded by the multidrug resistance protein. J Biol Chem. 268: 21493–21496.
Liu, J., Chow, V.T., Jois, S.D. (2004). A novel, rapid and sensitive heterotypic cell adhesion assay for CD2-CD58 interaction, and its application for testing inhibitory peptides. J Immunol Methods 291: 39–49.
Acknowledgments
Ms. Sharon Ronald was supported by funding from Louisiana Biomedical Research Network grant (LBRN/INBRE Grant Number P20RR016456, Bioinformatics, Biostatistics and Computational Biology Core, from the National Center for Research Resources. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health). Part of the work was supported by funding from Louisiana Board of Regents (LEQSF(2009-12)-RD-A-23) to SJ.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Satyanarayanajois, S.D., Ronald, S., Liu, J. (2011). Heterotypic Cell Adhesion Assay for the Study of Cell Adhesion Inhibition. In: Satyanarayanajois, S. (eds) Drug Design and Discovery. Methods in Molecular Biology, vol 716. Humana Press. https://doi.org/10.1007/978-1-61779-012-6_14
Download citation
DOI: https://doi.org/10.1007/978-1-61779-012-6_14
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-011-9
Online ISBN: 978-1-61779-012-6
eBook Packages: Springer Protocols