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. Author manuscript; available in PMC: 2011 Jun 1.
Published in final edited form as: J Immunol. 2010 Oct 27;185(11):6448–6460. doi: 10.4049/jimmunol.1000889

Figure 6. SMX-derived protein adduct formation in antigen presenting cells represents a functional antigen for lymphocytes and T-cell clones from hypersensitive human patients.

Figure 6

(A) Confocal microscopy imaging showing formation of cell membrane adducts when monocyte-derived dendritic cells are incubated with SMX-NO and the parent drug. SMX-NO derived adducts become internalized and are detected inside cells after 6 h. In contrast, intracellular cytoplasmic adducts are detected with dendritic cells exposed to the parent drug. Protein adducts were not detected with dendritic cells exposed to DMSO. (B) Concentration- and time-dependent protein adduct formation in SMX and SMX-NO treated dendritic cells and EBV-transformed B-cells from hypersensitive patients and healthy volunteers, quantified by ELISA using a specific anti-SMX antibody. (C) The quantity of SMX- and SMX-NO-derived protein adducts in EBV-transformed B-cells from hypersensitive patients remains constant for 3 days, the duration of a proliferation assay with T-cell clones. EBV-transformed B-cells pulsed with SMX for 16 h stimulated 9 out of 11 SMX-NO-responsive T cell clones. (D) Stimulation of a second independent panel of T-cell clones with EBV-transformed B-cells pulsed with SMX-NO for 16 h. (E) Enzyme inhibition with 1-aminobenzotriazole (ABT) and methimazole (meth) diminished proliferative response of drug-specific T cell clones to SMX-pulsed EBV-transformed B-cells. (F) SMX-pulsed lymphocytes generate a functional antigen that stimulates the proliferation of lymphocytes from hypersensitive patients. The quantity of SMX- and SMX-NO-derived protein adducts in lymphocytes from hypersensitive patients remains constant for 6 days, the duration of the lymphocyte transformation test. Lymphocytes from hypersensitive patients proliferated in presence of soluble SMX and SMX-NO and irradiated lymphocytes pulsed overnight with SMX-NO and the parent drug. In contrast, lymphocytes pulsed with SMX 1 h and used as a source of antigen did not stimulate a proliferative response.