An inverse relationship between T cell receptor affinity and antigen dose during CD4⁺T cell responsesin vivoandin vitro

Abstract

Multimeric peptide͞class II MHC staining reagents were synthesized and shown to bind with appropriate specificity to T cell hybridomas. A small, expanded population of T cells detected with one of these reagents in peptideimmunized C57BL͞10 mice persisted for several months. This population expanded further on secondary immunization. Equating the extent of binding of this reagent to T cell receptor affinity, we saw little correlation of immunizing peptide dose to T cell receptor affinity at the peak of the primary response. However, there was an inverse relation between peptide dose and the apparent receptor affinity of the T cells that were present several months after a primary response or after a secondary stimulation either in vivo or in vitro.Tracking the cellular dynamics of antigen-specific T cell responses in vivo has been difficult, not only because the responding precursors occur at low frequency, but also because the peptide͞MHC ligand for the T cell receptor (TCR) is cell bound and of low affinity. These latter problems have been overcome by methods for producing soluble MHC molecules bearing single peptides (1-4) and for producing fluorescent multimeric versions of these molecules (5-10). The cooperative binding achieved with these multivalent ligands produces an avidity high enough that antigen-specific T cells can be detected by flow cytometry. In examining the binding of these types of reagents to T cells with receptors of known affinity for monovalent peptide͞MHC, we observed a direct correlation between the extent of multimer binding and receptor affinity (10).The phenomenon of affinity maturation is well established in B cell responses. With multiple immunizations of T celldependent antigens, B cells bearing Ig receptors of steadily increasing affinity grow to dominate the B cell pool (11, 12). These cells appear to arise by selection of somatically mutated receptors as antigen becomes limiting (13)(14)(15). Recent studies have demonstrated that T cells having higher average affinity for peptide͞MHC are selected after multiple exposures to antigen (16,17). Whether this affinity maturation is primarily the result of the experience of repeated exposure to antigen or a shaping of the T cell repertoire during antigen waning (18-20) is unknown.We examined these questions for CD4 ϩ T cells responding to a peptide presented by the mouse class II MHC molecule, IA b . We tracked the frequency and relative affinities of peptide͞MHC-binding T cells during both primary and secondary immune responses. Our results show that with repeated or prolonged exposure to antigen, limiting doses of antigen select for T cells with higher-affinity receptors.

Alternate interactions define the binding of peptides to the MHC molecule IAb

“…Our previous studies have shown that p3K binds very strongly to IA b and, when bound to soluble IA b , forms a very stable molecule (25,39). How is this stability maintained in the face of virtually no occupancy of the conventional side chain binding pockets?…”

“…In this version of the peptide (p3K), three positions in pE␣ were changed to Lys residues. We have shown previously that this peptide binds to IA b as stably as pE␣ does (39) and is very immunogenic in IA b -expressing mice (25,39).…”

“…For example, the alignment of the DO-11.10 reactive ovalbumin peptide described above that places amino acids with small side chains in the p1, p4, p6, and p9 pockets also puts a favorable Asn at p7. Another variant of pE␣ that is immunogenic in IA b -expressing mice and binds very well to IA b has Trp substituted at positions p2 and p8 (25,39), but otherwise has the same potential interactions with IA b as p3K and pE␣.…”

“…The production of soluble IA b containing a peptide attached to the ␤-chain N terminus via a flexible peptide linker has been described (25). In the original constructions, peptides included both the E␣-derived peptide, ASFEAQGALANIAVDKA (pE␣), which occupies about 10% of natural IA b , and an immunogenic variant, ASFEAQKAK-ANKAVDKA (p3K), in which three positions in the peptide (underlined) had lysines substituted for the E␣ peptide amino acids.…”

“…The T cell hybridomas, B3K05-06 and B3K05-08, were produced from C57BL͞6 mice, which were immunized as described (25). p3K and its variants were synthesized in the Molecular Resource Center of Nation Jewish Medical and Research Center.…”