© 2001 Nature Publishing Group http://immunol.nature.com ARTICLES The immunological synapse and CD28-CD80 interactions Shannon K. Bromley1,Andrea Iaboni2, Simon J. Davis2,Adrian Whitty3, Jonathan M. Green4, Andrey S. Shaw1,ArthurWeiss5 and Michael L. Dustin5,6 Published online: 19 November 2001, DOI: 10.1038/ni737 According to the two-signal model of T cell activation, costimulatory molecules augment T cell receptor (TCR) signaling, whereas adhesion molecules enhance TCR–MHC-peptide recognition.The structure and binding properties of CD28 imply that it may perform both functions, blurring the distinction between adhesion and costimulatory molecules. Our results show that CD28 on naïve T cells does not support adhesion and has little or no capacity for directly enhancing TCR–MHC- peptide interactions. Instead of being dependent on costimulatory signaling, we propose that a key function of the immunological synapse is to generate a cellular microenvironment that favors the interactions of potent secondary signaling molecules, such as CD28. The T cell receptor (TCR) interaction with complexes of peptide and as CD2 and CD48, which suggests that CD28 might have a dual role as major histocompatibility complex (pMHC) is central to the T cell an adhesion and a signaling molecule4. Coengagement of CD28 with response. However, efficient T cell activation also requires the partici- the TCR has a number of effects on T cell activation; these include pation of additional cell-surface receptors that engage nonpolymorphic increasing sensitivity to TCR stimulation and increasing the survival of ligands on antigen-presenting cells (APCs). Some of these molecules T cells after stimulation5. CD80-transfected APCs have been used to are involved in the “physical embrace” between T cells and APCs and assess the temporal relationship of TCR and CD28 signaling, as initiat- are characterized as adhesion molecules. With modified APCs, a dis- ed by natural ligands, and showed that CD28 interactions precede or tinct activity was identified that was required for effective T cell stim- occur simultaneously to TCR interactions6. There are a number of pos- © http://immunol.nature.com Group 2001 Nature Publishing ulation; it was termed costimulation. T cells stimulated in the absence sibilities for the mechanism by which costimulation is initiated by of costimulation were rendered nonresponsive to subsequent antigen CD28. CD28 recruits phosphatidylinositol-3-kinase (PI3K)7 and the stimulation. These observations led to the “two-signal model” of T cell pleckstrin homology domain–containing tyrosine kinase Itk8 and can activation, in which signal one is transduced by the TCR and enhanced activate the Src family kinase Lck through different motifs in the cyto- by adhesion molecules and signal two is generated by costimulatory plasmic domain of CD289. These biochemical activities are not unique molecules on the surface of the APC1,2. to CD28; they are shared by the TCR, which leads to the concept that The first costimulatory receptor to be identified was CD283. CD28 is CD28 intensifies and prolongs biochemical signals that are normally a homodimeric type 1 transmembrane protein that is a member of the generated by the TCR. CD28 and interleukin 1 signaling, which leads immunoglobulin (Ig) superfamily and has a single Ig-like domain. to enhanced cytokine production by T cells, is associated with CD28 interacts with CD80 (also known as B7-1) and CD86 (B7-2), enhanced activation of NF-κB10. which are expressed on the APC in response to activating signals that Compartmentalization of receptors at the interface between the T cell result from, for example, CD40 engagement. The CD28-ligand interac- and APC is correlated with T cell activation. The immunological tion is topologically similar to that of many adhesion molecules, such synapse is characterized by a ring of lymphocyte function–associated 1–intercellular adhesion molecule 1 (LFA-1–ICAM-1) interactions that surround a central cluster of TCR-pMHC interactions11,12; these are 20 pMHC referred to as a central supramolecular activation cluster (cSMAC)11. pMHC + B7 Figure 1. Costimulatory effect of GPI- CD28 is unusual among the nonpolymporphic receptors tested in that 15 CD80 on transgenic T cell activation. 2B4 T cells were incubated for 48 h on pla- its interaction with CD80 occupies the central cluster of the immuno- 10 12 3 nar phospholipid bilayers that contained 160 logical synapse and is colocalized with the engaged TCR . Thus, one molecules/µm2 GPI–ICAM-1 and various mechanism by which CD28 may enhance T cell activation is to stabi- 5 densities of GPI–I-Ek–MCC(91–103), with 3 lize the immunological synapse. Engagement of CD28 promotes the 2 10 cpm [ H]thymidine µ or without 160 molecules/ m of GPI- 13 0 CD80.T cells were then pulsed with 0.4 µCi cytoskeletal-dependent recruitment of cell surface proteins and lipid 0.01 0.1 1 10 100 14 of [3H]thymidine and collected 12 h later. rafts rich in kinases and adaptor proteins , which contribute to building I-E k -MCC(91–103) (molecules/µm2 ) Data are mean±s.e.m. of triplicate wells. the immunological synapse15. 1Department of Pathology and Immunology and 4Department of Medicine,Washington University School of Medicine, 660 S. Euclid Ave, St. Louis MO, USA. 2Nuffield Department of Medicine,The University of Oxford, Oxford, UK. 3Biogen Inc, Cambridge, MA. 5Howard Hughes Medical Institute, Division of Rheumatology, Department of Medicine, University of California at San Francisco, San Francisco, CA, USA. 6Department of Pathology and the Program in Molecular Pathogenesis, New York University School of Medicine and The Skirball Institute of Biomolecular Medicine, 540 First Ave, New York, NY, USA. Correspondence should be addressed to M. L. D. ([email protected]). http://immunol.nature.com • december 2001 • volume 2 no 12 • nature immunology 1159 © 2001 Nature Publishing Group http://immunol.nature.com ARTICLES Figure 2. Naïve T cells do not adhere complex formed between I-Ek and the peptide formed by amino acids 80 to CD80 substrates. 2B4 T cells were (aa) 91–103 of moth cytochrome c, termed MCC(91–103) (which is settled onto planar phospholipid bilayers recognized as an agonist pMHC by T cells expressing the 2B4 TCR)12. 60 that contained 500 molecules/µm2 of CD48 or GPI-CD80 preincubated at 37 In this system the live T cells from 2B4 TCR–transgenic mice interact 40 °C. Images of the input cells were with the functional planar bilayer substrate, which essentially replaces 12 Adhesion (%) 20 acquired. After 20 min, the bilayers were the APC . Previously in vitro–primed effector T cells were used for gently washed with a laminar flow and the immunological synapse formation studies with bilayers12,21. 0 percentage of input cells that adhered to CD48 CD80 Immunological synapse formation in naïve T cells has not been report- Bilayer the bilayers was determined. Data are rep- resentative of two experiments. ed with the minimal bilayer system; however, optimal proliferation of naïve T cells in response to planar bilayers that contained agonist pMHC required CD28 cross-linking with anti-CD2822. CD28 could alternatively enhance TCR signaling through its func- We examined here the role of CD28-CD80 interactions in TCR- tion as an adhesion molecule within the immunological synapse16. The pMHC recognition and immunological synapse formation. When TCR must engage rare antigenic ligands presented on the surface of an CD28 was highly expressed, as it was on some Jurkat subclones, it APC, but this interaction is of low affinity. In addition, the TCR and mediated adhesion with a high 2D affinity but unexpectedly low max- MHC molecules are small compared to surrounding abundant cell sur- imum binding. When CD28 was presented on the cell and CD80 was face molecules such as integrins, CD43 and CD45. The distance presented in the planar bilayer substrate, only one-third of the CD28 spanned by the interaction of CD28 with CD80 or CD86 (∼15 nm), interacted with CD80 in the contact area. In comparison, when CD2 which occurs between the T cell and APC membranes, is similar to that is presented on Jurkat cells and CD58 is presented on the bilayer, over spanned by the TCR-pMHC interaction4,17. Thus, the interaction two-thirds of the CD2 interacts with CD58 in the contact area18. Low between CD28 and CD80 could generate the appropriate spacing for lateral mobility of CD28 was confirmed by fluorescence photo- TCR to efficiently interact with pMHC4. The interaction of CD2 on T bleaching recovery experiments and attributed to sequences in the cells with CD58 on the APC has a high two-dimensional (2D) affini- cytoplasmic domain of CD28. In cells that expressed physiological ty4,18. This is consistent with the creation of an ordered contact area in amounts of CD28, this low mobility prevented the effective interac- which CD2-CD58 interactions hold the membranes at a uniform dis- tion of CD28 with CD80. However, CD28-CD80 interactions were tance and effectively concentrate the respective binding sites in an atto- detected in contacts formed by CD2-CD48 and were focused within liter (10–18 l) volume. Thus, CD2 promotes TCR engagement in a size- the central cluster of immunological synapses formed by naïve T dependent manner, which suggests that topological mechanisms might cells. In contrast, engagement of CD28 was not required for synapse be important in T cell stimulation19. Whether CD28-CD80 interactions formation nor did it determine the density of MHC molecules that enhance TCR interactions with pMHC as part of its activity has not accumulated within the central cluster of the synapse. Rather than been tested directly. regulating the TCR-MHC interaction in the manner of an adhesion © http://immunol.nature.com Group 2001 Nature Publishing Immunological synapses can be studied with the supported planar molecule, it appeared instead that TCR engagement and immunolog- bilayers system20.