Presenter Status
Post-Doctorial Research
Abstract Type
Basic Research
Primary Mentor or Principal Investigator
Dr. Jennifer Goldman
Presentation Type
Poster
Start Date
20-5-2026 11:00 AM
End Date
20-5-2026 12:00 PM
Abstract Text
Background: Trimethoprim-sulfamethoxazole (TMP-SMX) is a commonly prescribed antibiotic that can cause life-threatening but rare idiosyncratic adverse drug reactions (IADRs). The only identified biomarker is certain human leukocyte antigen (HLA) risk alleles, such as B*07:02 and C*07:02 for acute respiratory distress syndrome (ARDS). Additionally, B*14:01, B*35:01, and B*13:01 are known to cause tissue-specific reactions to TMP-SMX in certain populations. Other drugs have previously been shown to cause changes to peptides presented by HLAs, leading to T cell activation and immune responses. However, the link between IADR pathophysiology and HLA risk alleles for TMP-SMX or its metabolites is unknown.
Objectives/Goal: We aim to establish in vitro-based assays to study the mechanism by which TMP-SMX HLA risk alleles can cause IADRs. From HLA peptides isolated from monoallelic cell lines, we wish to perform mass spectrometry-based immunopeptidomics analysis. Ultimately, we aim to identify changes to the repertoire of peptides presented by different HLA risk alleles in response to TMP-SMX or its metabolites.
Methods/Design: To generate stable monoallelic cell lines, we transfected HEK293T cells with constructs encoding for hygromycin B (hygB) selection marker and B*07:02 or C*07:02, fused to Twin-Strep-Tag (TST) for downstream purification. HLA expression levels were confirmed and compared between groups using Western Blot analysis. After performing a titration study for hygB, we selected for stably transfected cells using an optimized dose of hygB over the course of several weeks. From these cells, clonal lines were developed, and relative levels of HLA expression were compared. Transfections for TST-fused B*14:01, B*35:01, and B*13:01 were similarly performed. Pull-down experiments were executed to purify HLA molecules from cell lysate, facilitated by TST interaction with magnetic beads. Immunopeptidomics analysis using mass spectrometry, with or without cell treatments with TMP-SMX or its metabolites, is an ongoing process.
Results: We have developed or are in the process of developing clonal monoallelic cell lines expressing B*07:02, C*07:02, B*14:01, B*35:01, or B*13:01 HLA risk alleles for TMP-SMX induced IADRs. Additionally, we have validated the strategy of using TST to isolate HLAs.
Conclusions: We are developing monoallelic cell lines to study the link between TMP-SMX induced IADR pathophysiology and previously identified HLA risk alleles. We hope that an understanding of the mechanism that connects HLA risk alleles, drug metabolism, and immune responses will better enable the prediction and prevention of IADRs.
Development of in vitro assays to study HLA risk alleles for TMP-SMX induced IADRs
Background: Trimethoprim-sulfamethoxazole (TMP-SMX) is a commonly prescribed antibiotic that can cause life-threatening but rare idiosyncratic adverse drug reactions (IADRs). The only identified biomarker is certain human leukocyte antigen (HLA) risk alleles, such as B*07:02 and C*07:02 for acute respiratory distress syndrome (ARDS). Additionally, B*14:01, B*35:01, and B*13:01 are known to cause tissue-specific reactions to TMP-SMX in certain populations. Other drugs have previously been shown to cause changes to peptides presented by HLAs, leading to T cell activation and immune responses. However, the link between IADR pathophysiology and HLA risk alleles for TMP-SMX or its metabolites is unknown.
Objectives/Goal: We aim to establish in vitro-based assays to study the mechanism by which TMP-SMX HLA risk alleles can cause IADRs. From HLA peptides isolated from monoallelic cell lines, we wish to perform mass spectrometry-based immunopeptidomics analysis. Ultimately, we aim to identify changes to the repertoire of peptides presented by different HLA risk alleles in response to TMP-SMX or its metabolites.
Methods/Design: To generate stable monoallelic cell lines, we transfected HEK293T cells with constructs encoding for hygromycin B (hygB) selection marker and B*07:02 or C*07:02, fused to Twin-Strep-Tag (TST) for downstream purification. HLA expression levels were confirmed and compared between groups using Western Blot analysis. After performing a titration study for hygB, we selected for stably transfected cells using an optimized dose of hygB over the course of several weeks. From these cells, clonal lines were developed, and relative levels of HLA expression were compared. Transfections for TST-fused B*14:01, B*35:01, and B*13:01 were similarly performed. Pull-down experiments were executed to purify HLA molecules from cell lysate, facilitated by TST interaction with magnetic beads. Immunopeptidomics analysis using mass spectrometry, with or without cell treatments with TMP-SMX or its metabolites, is an ongoing process.
Results: We have developed or are in the process of developing clonal monoallelic cell lines expressing B*07:02, C*07:02, B*14:01, B*35:01, or B*13:01 HLA risk alleles for TMP-SMX induced IADRs. Additionally, we have validated the strategy of using TST to isolate HLAs.
Conclusions: We are developing monoallelic cell lines to study the link between TMP-SMX induced IADR pathophysiology and previously identified HLA risk alleles. We hope that an understanding of the mechanism that connects HLA risk alleles, drug metabolism, and immune responses will better enable the prediction and prevention of IADRs.
Comments
Poster Board Number: 33