NMBA-specific memory T cell quantification by CD154 expression in anaphylaxis diagnosis

Perioperative anaphylaxis is an acute systemic hypersensitivity reaction occurring during general anesthesia and frequently triggered by neuromuscular blocking agents (NMBA). It is considered to rely on specific IgE antibodies against an allergen and histamine release by mast cells and basophils. However, data from animal models and clinical study results (NASA study) suggest an alternative pathway dependent on specific IgG antibodies and involving platelet-activating factor. Both IgE and IgG pathways imply the existence of NMBA-specific T cells that are necessary for isotype switch and antibody production. Drug-specific T cells have been well documented mostly in antibiotic allergy, but definitive evidence of NMBA-specific memory T cells as well as data on their clinical relevance is lacking. Thirty-two (32) patients with a suspicion of NMBA-induced anaphylaxis from 10 French anesthesia departments were prospectively included and their PBMC isolated during an allergology visit (NASA study). Twenty (20) healthy blood donors were used as controls. As the original antigen driving sensitization is not known, we constructed and characterized by MALDI-TOF bioconjugates of NMBA (suxamethonium) with human serum albumin. PBMC were cultured for 48h with these bioconjugates and a cytokine cocktail to differentiate and activate antigen-presenting cells. After 48h, T cells activation was quantified by CD154 expression (flow cytometry) and cytokine production (INFγ, IL-5, IL-13) was measured by Fluorospot. CD154+ T cells could be detected at higher levels in patients as compared to controls (p<0.0001). ROC curve analysis of percentage CD4+/CD154+ memory T cells showed an area under curve of 0.98 with a 96% sensitivity and 98% specificity at optimal cut-off. Cytokine production analysis showed mainly INFγ-producing T cells. There was no link between CD154+ memory T cells levels and anaphylaxis severity, tryptase or histamine concentrations, or skin test results. Patients injected with suxamethonium had higher CD154+ T cells than patients injected with another NMBA. Interestingly, memory T cells could be detected in 8/9 patients with negative NMBA skin tests, of which 4 had a severe reaction. We characterize for the first time INFγ producing NMBA-specific T cells and show they may represent an interesting additional diagnostic tool in perioperative anaphylaxis particularly when usual tests are negative.