Characterization of T-Cell Responses to SMX and SMX-NO in Co-Trimoxazole Hypersensitivity Patients Expressing HLA-B*13:01

Abstract

HLA-B*13:01-positive patients in Thailand can develop frequent co-trimoxazole hypersensitivity reactions. This study aimed to characterize drug-specific T cells from three co-trimoxazole hypersensitive patients presenting with either Stevens-Johnson syndrome or drug reaction with eosinophilia and systemic symptoms. Two of the patients carried the HLA allele of interest, namely HLA-B*13:01. Sulfamethoxazole and nitroso sulfamethoxazole specific T cell clones were generated from T cell lines of co-trimoxazole hypersensitive HLA-B*13:01-positive patients. Clones were characterized for antigen specificity and cross-reactivity with structurally related compounds by measuring proliferation and cytokine release. Surface marker expression was characterized via flow cytometry. Mechanistic studies were conducted to assess pathways of T cell activation in response to antigen stimulation. Peripheral blood mononuclear cells from all patients were stimulated to proliferate and secrete IFN-γ with nitroso sulfamethoxazole. All sulfamethoxazole and nitroso sulfamethoxazole specific T cell clones expressed the CD4+ phenotype and strongly secreted IL-13 as well as IFN-γ, granzyme B and IL-22. No secretion of IL-17 was observed. A number of nitroso sulfamethoxazole-specific clones cross-reacted with nitroso dapsone but not sulfamethoxazole whereas sulfamethoxazole specific clones cross-reacted with nitroso sulfamethoxazole only. The nitroso sulfamethoxazole specific clones were activated in both antigen processing-dependent and -independent manner, while sulfamethoxazole activated T cell responses via direct HLA binding. Furthermore, activation of nitroso sulfamethoxazole-specific, but not sulfamethoxazole-specific, clones was blocked with glutathione. Sulfamethoxazole and nitroso sulfamethoxazole specific T cell clones from hypersensitive patients were CD4+ which suggests that HLA-B*13:01 is not directly involved in the iatrogenic disease observed in co-trimoxazole hypersensitivity patients.

Keywords: T cell; co-trimoxazole; drug hypersensitivity; human leukocyte antigen; sulfamethoxazole.

Figure 1

ELISpot images of cytokine secretion by SMX-NO and SMX specific T cell clones. TCCs (5x10⁴) were cultured with irradiated autologous EBV-transformed B-cells (1x10⁴) in the presence or absence of SMX-NO (40 µM) or SMX (1 mM) in an ELISpot plate pre-coated for IFN-γ, granzyme B, IL-13, IL-17 and IL-22 for 48h (37˚C; 5% CO₂). Following incubation, the plate was developed according to the manufactures instructions visualized by ELISpot AID reader.

Figure 2

The proliferative response of SMX-NO specific T cell clones. (A) T cell clones (5x10⁴) were culture with autologous EBV-transformed B-cells (1x10⁴) and SMX-NO (40 µM) in the presence or absence of HLA class I and class II blocking antibodies for 48 hours (37°C, 5% CO₂). Following incubation, [3H]-thymidine (0.5 μCi) were added to measure proliferative response. (B) T cell activation of SMX-NO clones in the response of different HLA-B. T cell clones (5x10⁴) were cultured with SMX-NO (40 µM) and irradiated EBV-transformed B-cells (1x10⁴) from 9 different patients carrying HLA-B*13:01 (P1-3), -B*57:01 (P4-6) and other HLA-B (P7-9).

Figure 3

SMX-NO stimulates specific T cell via antigen processing-dependent and processing-independent pathways. Autologous EBV-transformed B-cells (1x10⁴) were incubated with T cell clones (5x10⁴) in the presence or absence of SMX-NO (40 µM) for 1 and 16 hours. For fixation assay, SMX-NO specific clones (5x10⁴) were cultured with either irradiated or glutaraldehyde-fixed autologous EBV-transformed B-cell (1x10⁴) in the presence of SMX-NO (40 µM) for 48 hours (37˚C; 5% CO₂). [³H]-thymidine (0.5 μCi) incorporation was used to measure proliferative response. (A) SMX-NO T cell clones are stimulated in the presence of glutaraldehyde-fixed APC (B) Glutaraldehyde-fixed APC reduced the proliferative response of SMX-NO T cell clones.

Figure 4

The proliferative response of SMX-NO and SMX specific T cell clones in the presence of glutathione (GSH) and enzyme inhibitors. (A) Autologous EBV-transformed B-cells (1x10⁴) were culture with T cell clones (5x10⁴) in the presence or absence of GSH (1 mM). For pulsing EBVs, T cell clones (5x10⁴) were culture with and without 2 h pulsed-antigen presenting cells (1x10⁴) in the presence or absence of SMX-NO (40 µM) or SMX (1 mM) for 48 hours (37˚C; 5% CO₂). After incubation, [3H]-thymidine (0.5 μCi) were added to measure proliferative response. (B) 16 h-enzyme inhibitor pulsed EBVs (1x10⁴) were incubated with T cell clones (5×10⁴) for 48 hours (5% CO₂ at 37˚C). For normal condition, Autologous EBV-transformed B-cells were cultured with T cell clones and enzyme inhibitors for 1 hour (5% CO₂ at 37˚C) and 40 µM nitroso sulfamethoxazole. Following incubation, the plate was developed according to the manufactures instructions visualized by ELISpot AID reader. Methimazole; Meth, 1-aminobenzotriazole; ABT.

Figure 5

T cell activation in response to antigen stimulation of SMX specific clone. Autologous EBV-transformed B-cells (1x10⁴) were incubated with SMX (1 mM) for 1 and 16 hours, and then incubated with T cell clones (5x10⁴) after three washing steps. For fixation assay, SMX specific T cell clones (1x10⁴) were cultured with either irradiated or glutaraldehyde-fixed autologous EBV-transformed B-cell (5x10⁴) in the presence of SMX (1 mM) for 48 hours (37°C, 5% CO₂). After incubation, [3H]-thymidine (0.5 μCi) incorporation was used to measure proliferative response.