Definition of the Nature and Hapten Threshold of the β-Lactam Antigen Required for T Cell Activation In Vitro and in Patients

Abstract

Covalent modification of protein by drugs may disrupt self-tolerance leading to lymphocyte activation. Determination of the threshold required for this process has not hitherto been possible. We have therefore performed quantitative mass spectrometric analyses to define the epitopes formed in tolerant and hypersensitive patients taking the β-lactam antibiotic piperacillin and the threshold required for T-cell activation. A hydrolysed piperacillin hapten was detected on 4 Lys residues of HSA isolated from tolerant patients. The level of modified Lys541 ranged from 2.6-4.8%. Analysis of plasma from hypersensitive patients revealed the same pattern and levels of modification 1-10 days after commencement of therapy. Piperacillin-responsive skin-homing CD4+ clones expressing an array of Vβ receptors were activated in a dose, time and processingdependent manner; analysis of incubation medium revealed that 2.6% of Lys541 in HSA was modified when T-cells were activated. Piperacillin-HSA conjugates that had levels and epitopes identical to those detected in patients were shown to selectively stimulate additional CD4+ clones, which expressed a more restricted Vβ repertoire. To conclude, the levels of piperacillin-HSA modification that activated T-cells are equivalent to the ones formed in hypersensitive and tolerant patients, which indicates that threshold levels of drug antigen are formed in all patients. Thus, the propensity to develop hypersensitivity is dependent on other factors such as on the presence of Tcells within an individual's repertoire that can be activated with the β-lactam hapten and/or an imbalance in immune regulation.

Immune pathomechanism and classification of drug hypersensitivity

“…An important area of DHR research will be tolerance mechanism . Exposure to beta‐lactam leads often to hapten‐carrier formation, but some persons develop no immune reaction, others a harmless IgG response, others T cells with or without symptoms (exanthems) and some even a potentially dangerous IgE response . There is normal tolerance mechanism present—and we should know its rules.…”

“…Hapten formation and generation of hapten‐carrier complexes occur during each penicillin therapy. Why some persons develop a B‐, others mainly a T‐cell reaction to the drug and why the vast majority do not react is still unclear. It is also a puzzle why—if an immune response develops—the clinical symptoms are based on IgE‐ or T‐cell‐mediated reactions.…”

Identification of T‐cell epitopes from benzylpenicillin conjugated to human serum albumin and implication in penicillin allergy

“…This study also identifies 2 peptides located on lysines 159 and 525 likely implicated in the immunization of allergic patients to penicillins. Recently, Meng et al also published work using piperacillin and showing that the level of adducts formed in the circulation of patients was not different from the one required to activate T cells using a Lys 541 peptide . These results suggested that a threshold level of drug‐protein adduct was exceeded in all patients exposed to a therapeutic drug course and a large proportion of patients were at risk to be “immunized” to beta‐lactams.…”

Identification and characterization of a naïve CD8+ T cell repertoire for benzylpenicillin

“…Protein haptenation by covalent conjugation of drugs such as benzylpenicillin (BP) is considered to be one of the key process for immunization and allergic reaction . Significant progress has been made in understanding the immunopathogenesis of allergic drug reactions, and drug‐specific memory T cells have been detected in drug allergic patients . However, little is known about the origin of patients’ immunization to drugs.…”