Remarkably low affinity of CD4/peptide-major histocompatibility complex class II protein interactions

Abstract

The αβ T-cell coreceptor CD4 enhances immune responses more than 1 million-fold in some assays, and yet the affinity of CD4 for its ligand, peptide-major histocompatibility class II (pMHC II) on antigen-presenting cells, is so weak that it was previously unquantifiable. Here, we report that a soluble form of CD4 failed to bind detectably to pMHC II in surface plasmon resonance-based assays, establishing a new upper limit for the solution affinity at 2.5 mM. However, when presented multivalently on magnetic beads, soluble CD4 bound pMHC II-expressing B cells, confirming that it is active and allowing mapping of the native coreceptor binding site on pMHC II. Whereas binding was undetectable in solution, the affinity of the CD4/pMHC II interaction could be measured in 2D using CD4-and adhesion molecule-functionalized, supported lipid bilayers, yielding a 2D K d of ∼5,000 molecules/μm 2 . This value is two to three orders of magnitude higher than previously measured 2D K d values for interacting leukocyte surface proteins. Calculations indicated, however, that CD4/pMHC II binding would increase rates of T-cell receptor (TCR) complex phosphorylation by threefold via the recruitment of Lck, with only a small, 2-20% increase in the effective affinity of the TCR for pMHC II. The affinity of CD4/pMHC II therefore seems to be set at a value that increases T-cell sensitivity by enhancing phosphorylation, without compromising ligand discrimination. T cells with αβ T-cell receptors (TCRs) comprise functionally distinct subsets depending on which transcription factors and which of two coreceptors, CD8 or CD4, they express. CD8 + T cells respond to peptide agonists presented by major histocompatibility class I molecules (pMHC I) and are cytotoxic, whereas conventional CD4 + cells recognize peptide-MHC class II (pMHC II) and provide "help" defined by the cytokines they secrete (1). Cell adhesion assays explain this functionality insofar as CD8 and CD4 bind directly to pMHC I and pMHC II, respectively (2, 3). CD4 comprises two pairs of V-set and C2-set Ig superfamily domains, with early mutational data showing that the "top" two domains bind pMHC II (4). Crystal structures of cross-species and affinitymatured CD4/pMHC II complexes suggest that CD4 binds a pocket formed by the α2 and β2 domains of pMHC II (5, 6). The role of coreceptors in heightening T-cell responses is well established. For example, whereas CD4 + T-cells can respond to single pMHC II complexes presented by antigen-presenting cells (APCs), 30 or more complexes are required if CD4 is blocked (7).How CD4 achieves these effects, however, is incompletely understood. Coreceptors are pivotal in recruiting the kinase Lck to TCR/pMHC complexes (8, 9), but for reasons that are unclear coreceptor/pMHC interactions are extraordinarily weak. Traditionally, weak protein interactions are characterized using surface plasmon resonance (SPR) measurements, where one protein is tethered to the sensor surface and over it the other is passed at various concentrations. The SPR-based ...

Eleven Amino Acids of HLA-DRB1 and Fifteen Amino Acids of HLA-DRB3, 4, and 5 Include Potentially Causal Residues Responsible for the Risk of Childhood Type 1 Diabetes

“…2 OR, z score statistic, and P value computed under the null hypothesis. Table 6-Characterizing associations of four selected amino acids in DRB1 with IA-2A-positive and 1A-2A-negative patients and their differences consistent with or against this hypothesis (33,(36)(37)(38)(39)(40)(41)(42)(43). One matter complicating all analyses is the considerable in vitro instability of the human CD4 molecule, necessitating in many cases the use of mutant forms that form stable complexes with HLA-DR molecules to obtain crystallization of CD4-HLA-DR complexes (41,42).…”

Molecular mechanisms of T cell sensitivity to antigen

“…Therefore, CD8 can be effectively thought of as a bivalent molecule able to bind both pMHC and TCR to increase their association rate. It should be noted that the CD4/MHC‐II affinity is substantially lower than the CD8/MHC‐I affinity, and therefore, modulation of TCR/pMHC binding may be less important for CD4 (see below).…”

Dimensions and Interactions of Large T-Cell Surface Proteins

“…How the effective height of surface proteins affects protein distribution in cell-cell contacts will be discussed in the next section. However, protein exclusion from cell-cell contacts is also influenced by crowding effects, and it has been observed that proteins smaller than the gap size can also be excluded ( 16 , 18 , 23 , 65 , 66 ). This can be due to interactions with stationary adhesion molecules in the contact but also with mobile proteins on the contacting surface.…”