The adaptive immune response starts when CD4+ T cells recognize peptide antigens presented by class II molecules of the Major Histocompatibility Complex (MHCII). Two outstanding features of MHCII molecules are their polymorphism and the ability of each allele to bind a large panoply of peptides. Our long-term goal is to formally describe the three stages of the peptide recognition process: 1) formation of a peptide-MHCII complex, 2) will the complex be selected for presentation, and 3) will the complex be recognized by a T cell. Predicting epitopes and T cell responses to them is important in two practical areas where MHCII plays a significant role, vaccination and trasnplantation. The goal of the present application is to characterize in detail the first of these three stages, i.e., the prediction of HA-DR (DR) binding peptides. The research proposed here will generate a thermodynamic description of the binding process sufficient to provide a basis for generating predictive algorithms. The ability to predict binding will be an important step in making progress overall. The MHCII and peptides represent a flexible binding system, featuring specificity and permissiveness. This flexibility is evidenced by thermodynamic phenomena, in particular the presence of cooperativity and the occurrence of isothermal entropy-enthalpy compensation (iEEC). Cooperativity is observed in systems where one interaction is affected by other interactions and is associated with protein folding. iEEC describes the ability of entropy (increased search of structural space) to compensate for poorer binding energy (enthalpy). This is the driving mechanism behind permissiveness, in that there is an affinity range in which a peptide with poor binding characteristics has a measurable probability of finding a conformation that allows binding. To include flexibility in a binding prediction model, a deeper understanding of the interaction between cooperativity and iEEC in determining the permissive specificity of peptide binding at the molecular level is needed. To apply our studies to all DR alleles we have to also measure the contribution of the polymorphic cassettes that define the different alleles. Therefore, in Aim 1 we will investigate the energetics involved in peptide/DR complex formation, deconvoluted into enthalpic and entropic components and we will correlate the extent of iEEC to cooperativity. In Aim 2 we will define the biophysical contribution of DR residues to affinity, folding, and energetics to outline a binding model valid for all HLA alleles. Peptide binding usually takes place in the presence of an adjunct molecule, HLA-DM (DM). We have evidence that DM affects peptide binding by setting a permissiveness threshold. Therefore in Aim 3 we will investigate the DR-peptide-DM interaction in thermodynamic terms, correlating DM activity to the thermodynamic signatures of various pDR complexes. In Aim 4 we outline an initial peptide-binding prediction model encompassing the structural and thermodynamic data derived in the first three aims and we propose to validate this approach by predicting binding peptides within randomly generated antigenic sequences.