No abstract

T lymphocyte receptor CTLA-4 binds costimulatory molecules CD80 (B7-1) and CD86 (B7-2) with high avidity and negatively regulates T cell activation. CTLA-4 functions at the cell surface, yet is primarily localized in intracellular vesicles. Here, we demonstrate cycling of CTLA-4 between intracellular stores and the cell surface. Intracellular vesicles containing CTLA-4 overlapped with endocytic compartment(s) and with perforin-containing secretory granules. Cell surface expression of CTLA-4 was rapidly increased by raising intracellular calcium levels. During T cell activation, intracellular and cell surface CTLA-4 became focused towards sites of TCR activation. Cycling and directional control of CTLA-4 expression may regulate its functional interaction with APCs bearing peptide-MHC complexes of appropriate specificity and avidity.

SummaryT cell costimulation by molecules on the antigen presenting cell (APC) is required for optimal T cell proliferation. The B7 molecule on APC binds the T lymphocyte receptor CD28, triggering increased interleukin 2 (IL-2) production and subsequent T cell proliferation. CTLA-4 is a predicted T cell membrane receptor homologous to CD28, which also binds the B7 counter receptor, but whose distribution and function are unknown. Here we have developed monodonal antibodies (mAbs) specific for CTLA-4 and have investigated these questions, mAbs were produced that bound CTLA-4 but not CD28, and that blocked binding of CTLA-4 to B7. CTLA-4 expression as measured by these mAbs was virtually undetectable on resting T cells, but was increased several hundred-fold during T cell activation. On activated lymphocytes, CTLA-4 was expressed equally on CD4 + and CD8 + T cell subsets and was coexpressed with CD25, CD28, and CD45RO. CTLA-4 expression was lower than that of CD28, reaching a maximum of "~1/30-50 the level of CD28. Despite its lower expression, CTLA-4 was responsible for much of the B7 binding by large activated T cells. Anti-CTLA-4 mAb 11D4 and anti-CD28 mAb 9.3 acted cooperatively to inhibit T cell adhesion to B7, and to block T cell proliferation in primary mixed lymphocyte culture. When coimmobilized with anti T cell receptor (TCR) mAb, anti-CTLA-4 mAbs were less effective than anti-CD28 mAb 9.3 at costimulating proliferation of resting or activated T cells. However, coimmobilized combinations of anti-CD28 and anti-CTLA-4 were synergistic in their ability to augment anti-TCR-induced proliferation of preactivated CD4 + T cells. These results indicate that CTLA-4 is coexpressed with CD28 on activated T lymphocytes and cooperatively regulates T cell adhesion and activation by B7.

SummaryT cell surface receptors CD28 and CTLA-4 are homologous members of the immunoglobulin superfamily (IgSF), each comprising a single V-like extracellular domain. CD28 and CTLA-4 bind to the B7-1 and B7-2 counter-receptors on antigen presenting cells (APCs), thereby triggering a costimulatory pathway important for optimal T cell activation in vitro and in vivo. Soluble forms of CD28 and CTLA-4 in which the V-like extracellular domains were fused to Ig constant domains (CD28Ig and CTLA4Ig), have been used to study their interactions with B7-1 and B7-2, with CTLA4Ig binding B7-1 more strongly than CD28Ig (~20-fold higher avidity). We have now, by site-specific and homologue mutagenesis, identified regions in CTLA4Ig important for strong binding to B7-1. A hexapeptide motif (MYPPPY) in the complementarity determining region 3 (CDR3)-like region is fully conserved in all CD28 and CTLA-4 family members. Alanine scanning mutagenesis through the motif in CTLA4Ig and at selected residues in CD28Ig reduced or abolished binding to B7-1. Chimeric molecules HS4, HS4-A, and HS4-B were constructed in which CDR3-like regions of CTLA-4, COOH-terminally extended to include nonconserved residues, were grafted onto CD28Ig. These homologue mutants showed stronger binding to B7-1 than did CD28Ig. Grafting of the CDRl-like region of CTLA-4, which is not conserved in CD28 and is predicted to be spatially adjacent to CDR3, into HS4 and HS4-A, resulted in chimeric molecules (HS7 and HSS) which bound B7-1 even better. Inclusion of the CDR2-1ike domain of CTLA-4 into HS7 and HS8 did not further increase binding. Thus, the MYPPPY motifs of CTLA4Ig and CD28Ig are important for their binding to B7-1, but the increased strength of this binding by CTLA4Ig is mediated by nonconserved residues in the CDR1-and CDR3-analogous regions. FLor T cells to respond to an antigenic stimulus, multiple activation signals are required from the APC (1, 2). An antigen-specific signal occurs when the TCR binds to antigenic peptides presented by MHC molecules. An important nonspecific costimulatory signal is delivered to the T cell when B7-related receptors on APCs bind to CD28 and/or CTLA-4. There are at least two homologous B7 family members found on APCs, B7-1 (also called B7 or CD80) and B7-2, both of which can deliver costimulatory signals to T cells via CD28 and/or CTLA-4 (3-7). Costimulation through CD28 or CTLA-4 is essential for T cell activation since a soluble Ig fusion protein of CTLA-4 (CTLA4Ig) blocks T cell-dependent immune responses in vitro and in vivo (5,8,9). Failure to deliver this second signal may lead to clonal inactivation or T cell anergy (10-12).CD28 and CTLA-4 both contain a single Ig V-like extracellular ligand binding domain which share •25% sequence homology (13-15). The B7 ligands are also members of the Ig superfamily (IgSF) 1 but have, in contrast to CD28 and CTLA-4, two Ig domains in their extraceUular region, an NH2-terminal V-like domain and a C-like domain (3, 6, 7). CTLA4Ig binds to B7-1 with "~20 fold higher avidity tha...

T lymphocyte receptors CD28 and CTLA-4 bind costimulatory molecules CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells and regulate T cell activation. While distinct functional roles have been ascribed to each of these molecules, little is known about how they interact. To better characterize these interactions, we have used surface plasmon resonance to perform equilibrium and kinetic binding analyses of extracellular fragments of CD28/CTLA-4/CD80/CD86. We show that CTLA-4 and CD28 binding are both characterized by rapid kinetic on-rates and rapid dissociation rates. Native disulfide-linked homodimers of CD28 and CTLA-4 bound with two kinetically distinct binding sites, one of high avidity and slow dissociation and one of low avidity and more rapid dissociation. Monomeric CTLA-4 bound only with low affinity and rapid dissociation. Therefore, covalent dimerization of CTLA-4 is required for its high avidity binding. Oligomerization of CD80/CD86 is also required for high avidity CTLA-4 binding since CTLA-4 bound with low avidity to monomeric CD86. This contrasts with the ability of CD80/CD86 on antigen-presenting cells to bind CTLA4Ig with high avidity and predicts their organization as oligomers or clusters that permit multivalent binding. Thus, covalent receptor dimerization and ligand oligomerization are two key features of the CD28/CTLA-4/CD80/CD86 receptor system that control ligand binding and may regulate signal transduction by controlling the duration of receptor occupancy.