Synthetic antigenic determinants of clavulanic acid induce dendritic cell maturation and specific T cell proliferation in patients with immediate hypersensitivity reactions

Abstract

Background: Immediate drug hypersensitivity reactions (IDHRs) to clavulanic acid (CLV) have increased in the last decades due to a higher consumption alongside amoxicillin (AX). Due to its chemical instability, diagnostic procedures to evaluate IDHRs to CLV are difficult, and current in vitro assays do not have an optimal sensitivity. The inclusion of the specific metabolites after CLV degradation, which are efficiently recognised by the immune system, could help to improve sensitivity of in vitro tests.

Methods: Recognition by dendritic cells (DCs) of CLV and the synthetic analogues of two of its hypothesised antigenic determinants (ADs) was evaluated by flow cytometry in 27 allergic patients (AP) and healthy controls (HC). Their ability to trigger the proliferation of T cells was also analysed by flow cytometry.

Results: The inclusion of synthetic analogues of CLV ADs, significantly increased the expression of maturation markers on DCs from AP compared to HC. A different recognition pattern could be observed with each AD, and, therefore, the inclusion of both ADs achieves an improved sensitivity. The addition of synthetic ADs analogues increased the proliferative response of CD4⁺ Th2 compared to the addition of native CLV. The combination of results from both ADs increased the sensitivity of proliferative assays from 19% to 65% with a specificity higher than 90%.

Conclusions: Synthetic ADs from CLV are efficiently recognised by DCs with ability to activate CD4⁺ Th2 cells from AP. The combination of analogues from both ADs, significantly increased the sensitivity of DC maturation and T-cell proliferation compared to native CLV.

Keywords: antigenic determinants; clavulanic acid; immediate drug hypersensitivity reactions; maturation; proliferation.

FIGURE 1

Dendritic cell maturation induced by clavulanic acid and its antigenic determinants. (A–E) Violin plots represent maturation index (MI) of (A) CCR7, (B) CD40, (C) CD80, (D) CD83, and (E) CD86 from allergic patients and healthy controls. (F–J) Bars represent % of positivity based on MI ≥2 of (F) CCR7, (G) CD40, (H) CD80, (I) CD83, and (J) CD86 from allergic patients and healthy controls. Comparisons in terms of positivity by X ² test and MI by Mann–Whitney test (*p < .05; **p < .01; ***p < .001; ****p < .0001)

FIGURE 2

Maturation positivity induced by clavulanic acid and its antigenic determinants. (A–E) Bars represent % of positive cases (maturation index ≥2) of (A) CCR7, (B) CD40, (C) CD80, (D) CD83, and (E) CD86 in allergic patients and healthy controls after the combination of synthetic analogues from both antigenic determinants. (F–J) Parts of whole represent % of positive cases (maturation index ≥2) for (F) CCR7, (G) CD40, (H) CD80, (I) CD83, (J) CD86 in allergic patients with positive response. Comparisons in terms of positivity by X ² test (*p < .05; **p < .01; ***p < .001)

FIGURE 3

T cell proliferative response induced by clavulanic acid and its antigenic determinants. (A–C) Bars and standard deviation represent proliferation index (PI) of (A) CD3⁺T cells, (B) CD4⁺T cells, (C) CD4⁺Th2 cells. (D, E) Bars represent % of positivity based on PI ≥2 of (D) CD3⁺T cells, (E) CD4⁺T cells, (F) CD4⁺Th2 cells from allergic patients and healthy controls. Comparisons in terms of positivity by X ² test and proliferation index by Mann–Whitney test (*p < .05; **p < .01; ***p < .001)

FIGURE 4

T cell proliferation positivity induced by clavulanic acid and its antigenic determinants. (A–C) Bars represent % of positive cases (proliferation index ≥2) of (A) CD3⁺T cells, (B) CD4⁺T cells, (C) CD4⁺Th2 cells in allergic patients and healthy controls after the combination of synthetic analogues from both antigenic determinants. (D–F) Parts of whole represent % of positive cases (proliferation index ≥2) of (D) CD3⁺T cells, (E) CD4⁺T cells, (F) CD4⁺Th2 cells in allergic patients with positive response. (G) Bars represent the % of positivity from clavulanic acid and synthetic analogues from each antigenic determinant by combining the proliferative results of CD3⁺T, CD4⁺T, and CD4⁺Th2 cells. (H) Diagram represent the % of sensitivity (sens.) and the % of contribution (contr.) regarding the total positive cases with the most relevant synthetic analogues in the proliferative response of CD4⁺Th2 cells. Comparisons in terms of positivity by X ² test (*p < .05; **p < .01; ***p < .001)