Perfluorooctane sulfonate (PFOS) poses significant health and environmental risks due to its persistence and widespread use and has been linked to various adverse outcomes, such as liver toxicity. Although the molecular responses and toxicity effects of PFOS exposure have been extensively studied, considerable uncertainty remains regarding the causal mechanisms leading to PFOS-associated adverse effects. To help bridge this gap, we conducted CRISPR screens in HepG2/C3A human liver cells exposed to IC25 (170 µM) of PFOS to identify genes and pathways influencing PFOS-induced cytotoxicity. Using a genome-wide CRISPR knockout library targeting 18,819 genes, we identified 340 candidate genes that modulate PFOS-induced cytotoxicity when genetically disrupted (189 gene disruptions increased sensitivity and 151 gene disruptions increased resistance). From these candidate genes, we individually disrupted two candidate genes, SLC6A9 which encodes the glycine transporter GlyT1, and CPSF2, and confirmed increased resistance to PFOS exposure. Further, molecular docking analysis predicts that PFOS directly binds to GlyT1 and functional inhibition of GlyT1 also increases resistance to PFOS exposure. Gene-Disease outcome association analysis using the Comparative Toxicogenomics Database (CTD) indicated an enrichment of candidate genes associated with cancer-related and liver disease phenotypes. KEGG and STRING enrichment analyses found over representation of several biological pathways including DNA damage response and cell cycle. Lastly, cross-species conservation analysis using the top two validated gene targets found that their pathways were highly conserved in several environmentally relevant species. These findings provide new mechanistic and functional insights into PFOS-induced cytotoxicity, highlight potential molecular targets for toxicity mitigation, and establish a foundation for cross-species toxicogenomic modeling of PFOS health effects.

F. tularensis is a highly infectious Gram-negative bacterial pathogen that causes tularemia, a re-emerging zoonosis of public health concern. Here we identify respiratory complex I as a selective vulnerability in Francisella and define the mechanism of action of a pyrazole compound, tolfenpyrad, with species-specific antibacterial activity. Using F. novicida as a surrogate model, we demonstrated that tolfenpyrad selectively inhibits growth with no measurable effect on E. coli or P. aeruginosa . Tolfenpyrad rapidly suppressed oxygen consumption, depleted ATP, collapsed proton motive force, and induced reactive oxygen species, indicating disruption of bacterial metabolism. Biochemical assays demonstrated selective inhibition of NADH-dependent respiration and membrane-associated NADH oxidation, whereas succinate-driven respiration was unaffected. Moreover, the alternative NADH dehydrogenase ( ndh ) was not required for tolfenpyrad activity. Structural docking identified a potential tolfenpyrad-binding pocket within the membrane subunit NuoM. These findings reveal species-specific inhibition of Francisella complex I and establish respiratory metabolism as a promising antimicrobial target in these bacteria.

Importance:Drug reaction with eosinophilia and systemic symptoms (DRESS) is a severe, potentially fatal hypersensitivity syndrome with 3% to 10% mortality, and lamotrigine, a first-line treatment for bipolar and seizure disorders, is among the top 5 causative agents of DRESS in the United States and globally. Commercial panels test HLA-B*15:02 and HLA-A*31:01 for risk of carbamazepine-associated severe cutaneous adverse reactions; however, these are not validated for lamotrigine-induced DRESS, and current reports from these commercial assays could therefore provide false reassurance. No HLA associations with lamotrigine-induced DRESS have been established in US populations. Objective:To identify HLA genetic variants associated with lamotrigine-induced DRESS in a US population. Design, Setting, and Participants:This matched case-control study was conducted at 2 US academic medical centers (Vanderbilt University Medical Center and Mass General Brigham). Patients with lamotrigine-induced DRESS confirmed by Registry of Severe Cutaneous Adverse Reactions (RegiSCAR) criteria (score ≥4) were prospectively enrolled between April 2016 and June 2025. Lamotrigine-tolerant controls were matched 10:1 from the Vanderbilt BioVU biobank on sex, self-reported race, and age. HLA typing included class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DPB1, HLA-DQA1, HLA-DQB1, and HLA-DRB1) loci for case (high-resolution typing) and control (imputed from genotyping array data) participants. Main Outcomes and Measures:HLA class I and II alleles and haplotypes were tested using logistic regression with Bonferroni correction for their association with lamotrigine-induced DRESS. Results:This study included 29 patients with lamotrigine-induced DRESS (case participants; median [IQR] age, 33 [24-56] years; 25 female [86.2%]; 2 Asian [6.9%], 2 Black [6.9%], 23 White [79.3%], and 2 unknown race [6.9%]) and 290 matched control participants. HLA-A*32:01 was associated with lamotrigine-induced DRESS (41.4% [12 case participants] vs 4.1% [12 control participants]; odds ratio [OR], 16.4; 95% CI, 6.4-42.5; Bonferroni-corrected P < .001). No other alleles or class II loci showed significant associations after correction. The A*32:01 ~ B*44:02 haplotype was enriched (OR, 18.4; 95% CI, 4.6-83.8; Bonferroni-corrected P = .001) in lamotrigine-induced DRESS. Conclusions and Relevance:In this case-control study, HLA-A*32:01 was associated with lamotrigine-induced DRESS in a US population. Given that HLA-A*32:01 is not included in existing commercial pharmacogenomic panels, adding this marker could improve preprescription DRESS risk identification for lamotrigine.

Anti-hypertensive (anti-HTN) drugs are widely prescribed, but the features of associated drug-induced liver injury (DILI) are poorly defined. A total of 2207 patients enrolled in the Drug-Induced Liver Injury Network (DILIN) from 2003 to 2024 were adjudicated as definite, highly likely, or probable DILI. Of these, 44 cases were attributed to anti-HTN drugs. These were compared with non–anti-HTN DILI cases and population controls. Human Leukocyte Antigen (HLA) sequencing was performed, and allele frequencies were analyzed using Fisher’s Exact Test. Among the 44 anti-HTN DILI cases, 14 were caused by ACE inhibitors/ARBs, 13 by methyldopa, 11 by hydralazine, and 3 each by beta-blockers and calcium-channel blockers. Methyldopa DILI presented with hepatocellular injury in young women (prescribed mainly for pregnancy-related HTN). Non-methyldopa cases were 60% hepatocellular, 28% cholestatic, and 9% mixed. Severe outcomes included two liver transplants and two deaths (lisinopril and hydralazine). HLA-B∗35:01 was markedly enriched (50% vs. 13% in controls; OR 6.0; P = 1 × 10−8) and was linked to shorter latency (50 vs. 109 days; P = 0.02) but not severity. HLA-B∗35:01 was overrepresented across all drug classes, including 66% of methyldopa cases, 27% of hydralazine cases, and 42% of ACE/ARB cases. DILI from anti-HTN medications is very rare but strongly associated with HLA-B∗35:01, especially for methyldopa. Carriers display a shorter latency, potentially indicating an accelerated adaptive immune response, that warrants further exploration.

Neurolysin (Nln) is a peptidase recognized for its cerebroprotective function in acute ischemic stroke. This study aimed to identify small molecule activators of Nln as research tools to further explore the role of this enzyme in stroke and other neurological disorders. Building on our previous computational screen of ∼140,000 compounds from the National Cancer Institute Developmental Therapeutics Program database, we extended experimental testing to the top 100 candidates using an Nln enzymatic assay. A pyridine-piperazine derivative (Py-Pip) was identified as a hit molecule and was characterized in detail. Py-Pip concentration-dependently enhanced the hydrolysis of both synthetic and natural substrates (neurotensin, angiotensin I, and bradykinin) by rat Nln, and displayed comparable activating effects on human and mouse orthologs. Importantly, Py-Pip exhibited a favorable selectivity profile, showing no potentiation of homologous metallopeptidases or unrelated enzymes. Kinetic analysis revealed that Py-Pip increases the catalytic efficiency (Vmax/Km) of Nln via a nonessential activation mechanism, whereas competition assays with inhibitor dynorphin A(1-13) confirmed that Py-Pip acts at a distinct, nonoverlapping site. Direct binding was further validated by orthogonal biophysical techniques, including differential scanning fluorimetry, microscale thermophoresis, and biolayer interferometry, whereas circular dichroism spectroscopy indicated activator-induced secondary structural changes. These findings validate that Nln activity can be enhanced by small molecules and establish Py-Pip as a novel, nonpeptide scaffold for developing potent, "drug-like" activators to investigate Nln biology and therapeutic potential. SIGNIFICANCE STATEMENT: This study reports the discovery of a novel nonpeptide small molecule that selectively enhances the activity of neurolysin (Nln), a peptidase implicated in cerebroprotection. Unlike previous peptide-based activators, this molecule provides a stable, "drug-like" scaffold and a structural foundation for the development of potent Nln activators to probe Nln biology and therapeutic potential in ischemic stroke.

BACKGROUND: Co-trimoxazole is a leading global cause of severe cutaneous adverse drug reactions (SCAR) including Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/ TEN) and drug reaction with eosinophilia and systemic symptoms (DRESS). Co-trimoxazole-induced SCAR are associated with HLA class I alleles including HLA-B*13:01 and HLA-B*38:02 in Southeast Asian (SEA) populations. However, the global generalizability of these associations is unknown but critical for population-appropriate risk stratification and diagnosis. OBJECTIVE: To determine HLA risk factors associated with co-trimoxazole-induced SJS/TEN and DRESS in populations from the United States and South Africa. METHODS: We performed high-resolution HLA typing on dermatologist-adjudicated co-trimoxazole-induced patients with SCAR in the United States (n = 63) and South Africa (n = 26) compared with population controls. Peptide binding and docking analyses were performed using MHCcluster2.0 and CB-Dock2. RESULTS: In a multiple logistic regression model, HLA-B*44:03 (corrected P [Pc] < .001; odds ratio [OR] = 4.08), HLA-B*38:01 (Pc < .001; OR = 5.66), and HLA-C*04:01 (Pc = .003; OR = 2.50) were independently associated with co-trimoxazole-induced SJS/TEN in the United States. HLA-B*44:03 was also associated with co-trimoxazole-induced DRESS in South Africa (Pc = .019; OR = 10.69). Distinct HLA-B variants with shared peptide binding specificities (SPBS) and HLA-C*04:01 identified 94% and 78% of cotrimoxazole-induced SJS/TEN and DRESS in the United States, respectively. The SEA risk allele HLA-B*13:01, with SPBS to HLA-B*44:03, was identified in just one of 63 US patients with SCAR. CONCLUSIONS: HLA alleles with SPBS to SEA-related risk alleles, including HLA-B*44:03 (SPBS with HLA-B*13:01) and HLA-B*38:01 (SPBS with HLA-B*38:02) but also HLA-C*04:01, predisposed to co-trimoxazole-induced SCAR in the United States and South Africa. These findings provide biological plausibility and strategies for global risk prediction and diagnosis of co-trimoxazole-induced SCAR. (c) 2025 The Authors. Published by Elsevier Inc. on behalf of the American Academy of Allergy, Asthma & Immunology. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). (J Allergy Clin Immunol Pract 2025;13:3042-53)

BACKGROUND:Co-trimoxazole is a leading global cause of severe cutaneous adverse drug reactions (SCAR) including Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) and drug reaction with eosinophilia and systemic symptoms (DRESS). Co-trimoxazole-induced SCAR are associated with HLA class I alleles including HLA-B∗13:01 and HLA-B∗38:02 in Southeast Asian (SEA) populations. However, the global generalizability of these associations is unknown but critical for population-appropriate risk stratification and diagnosis. OBJECTIVE:To determine HLA risk factors associated with co-trimoxazole-induced SJS/TEN and DRESS in populations from the United States and South Africa. METHODS:We performed high-resolution HLA typing on dermatologist-adjudicated co-trimoxazole-induced patients with SCAR in the United States (n = 63) and South Africa (n = 26) compared with population controls. Peptide binding and docking analyses were performed using MHCcluster2.0 and CB-Dock2. RESULTS:In a multiple logistic regression model, HLA-B∗44:03 (corrected P [Pc] < .001; odds ratio [OR] = 4.08), HLA-B∗38:01 (Pc < .001; OR = 5.66), and HLA-C∗04:01 (Pc = .003; OR = 2.50) were independently associated with co-trimoxazole-induced SJS/TEN in the United States. HLA-B∗44:03 was also associated with co-trimoxazole-induced DRESS in South Africa (Pc = .019; OR = 10.69). Distinct HLA-B variants with shared peptide binding specificities (SPBS) and HLA-C∗04:01 identified 94% and 78% of co-trimoxazole-induced SJS/TEN and DRESS in the United States, respectively. The SEA risk allele HLA-B∗13:01, with SPBS to HLA-B∗44:03, was identified in just one of 63 US patients with SCAR. CONCLUSIONS:HLA alleles with SPBS to SEA-related risk alleles, including HLA-B∗44:03 (SPBS with HLA-B∗13:01) and HLA-B∗38:01 (SPBS with HLA-B∗38:02) but also HLA-C∗04:01, predisposed to co-trimoxazole-induced SCAR in the United States and South Africa. These findings provide biological plausibility and strategies for global risk prediction and diagnosis of co-trimoxazole-induced SCAR.

A day care teacher presented with complaints of headache, neck stiffness, and fever. Because of initial concerns about meningococcal meningitis, families of day care attendees were notified, and 10 children from the day care presented for evaluation. Cerebrospinal fluid from the teacher and nasal swabs from 4 children who were febrile were positive for enterovirus on reverse transcription polymerase chain reaction. A novel recombinant enterovirus was cultured from the teacher's cerebrospinal fluid and from 2 of the nasal swabs. The amino-terminal portion of the recombinant virus was derived from echovirus 6, with the carboxy-terminal portion originating from coxsackievirus B1; recombinant segments were most closely related to similar segments from strains isolated in France. Recombination occurred within the C2 gene associated with virus replication and virion morphogenesis. Structural modeling predicted that the recombinant protein was capable of forming hexameric and heptameric assemblies. Our data highlight the potential for recombination among enteroviruses, leading to modifications within viral proteins that may affect virulence.

Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer-related deaths by 2030, with most patients presenting with advanced, unresectable disease. Despite advances in chemotherapy, the 5-year survival rate remains low, underscoring the need for novel therapies. This study builds on the discovery that palmatine, a natural compound inhibits pancreatic cancer growth and enhances gemcitabine efficacy. This was done by using computational docking to explore palmatine's interaction with the STAT3 linker domain. A screening of 139,735 compounds from the National Cancer Institute Developmental Therapeutics Program identified compounds with superior STAT3 binding, with Striatal B emerging as the lead. In vitro tests confirmed the ability of Striatal B to significantly inhibit pancreatic cancer cell growth and potentiate gemcitabine effects, while computational modeling indicated effective binding to STAT3. Striatal B reduced STAT3 activation and epithelial-mesenchymal transition markers and modulated mechanical properties, suggesting a mechanism of action that involves altering cell mechanics, potentially providing a promising new therapeutic avenue for PDAC treatment.

Sjögren’s Disease (SjD) is an autoimmune disease characterized by dry mouth and eyes, affecting approximately 4 million Americans. SjD is a complex condition with a multifactorial etiology and a diverse patient population in terms of disease activity, severity, and response to therapy. To better comprehend the etiologies and develop effective treatments, it is crucial to utilize appropriate animal models to unravel the different aspects of the autoimmune process. In this study, we have been examining the Non-Obese Diabetic (NOD)-DR3 mouse model, an NOD-congenic animal with human HLA class II expression, specifically the DR3(DRw17) transgene. The DR3 transgene expresses the HLA-DR3 genotype, comprising HLA-DRA0101 and HLA-DRB10301. Our research revealed that NOD-DR3 mice develop SjD phenotypes characterized by decreased salivary flow rates, elevated levels of anti-nuclear autoantibodies, changes in salivary gland proinflammatory immune cell populations and their cytokines, and increased focal scores. A notable finding is that NOD-DR3 mice exhibit sexually dimorphic disease phenotypes, mirroring patterns observed in human patients. Lastly, employing machine learning algorithms targeting DR3, we have identified and tested novel drug compounds. Some compounds alleviated the clinical signs of SjD in the mouse model. In summary, our research demonstrates the potential of this inaugural humanized mouse model of SjD and identifies DR3 as a target for treating the disease. Supported by the National Institute of Dental and Craniofacial Research (NIDCR) (DE028544, DE028544-02S1) Therapeutic Approaches to Autoimmunity (THER)

Background: Drug-induced hypersensitivity such as anaphylaxis is an important cause of drug-related morbidity and mortality. Cefaclor is a leading cause of drug induced type I hypersensitivity in Korea, but little is yet known about genetic biomarkers to predict this hypersensitivity reaction. We aimed to evaluate the possible involvement of genes in cefaclor induced type I hypersensitivity. Methods: Whole exome sequencing (WES) and HLA genotyping were performed in 43 patients with cefaclor induced type I hypersensitivity. In addition, homology modeling was performed to identify the binding forms of cefaclor to HLA site. Results: Anaphylaxis was the most common phenotype of cefaclor hypersensitivity (90.69%). WES results show that rs62242177 and rs62242178 located in LIMD1 region were genome-wide significant at the 5 × 10−8 significance level. Cefaclor induced type I hypersensitivity was significantly associated with HLA-DRB1∗04:03 (OR 4.61 [95% CI 1.51–14.09], P < 0.002) and HLA-DRB1∗14:54 (OR 3.86 [95% CI 1.09–13.67], P < 0.002). Conclusion: LIMD1, HLA-DRB1∗04:03 and HLA-DRB1∗14:54 may affect susceptibility to cefaclor induced type I hypersensitivity. Further confirmative studies with a larger patient population should be performed to ascertain the role of HLA-DRB1 and LIMD1 in the development of cefaclor induced hypersensitivity.

ABSTRACTA daycare teacher presented to a local Emergency Department (ED) with complaints of headache, neck stiffness, and fever. Preliminary analysis of cerebral spinal fluid (CSF) raised concerns about meningococcal meningitis and prompted notification of the county health department, which then notified the daycare. Ten children presented to a University referral hospital for evaluation, four of whom were febrile. CSF from the teacher and nasal swabs from the febrile children were all RT-PCR positive for enterovirus. A novel recombinant enterovirus was cultured from the teacher’s CSF and from two of the nasal swabs. The amino-terminal portion of the recombinant virus was derived from an Echovirus E6 with the carboxy-terminal portion originating from a Coxsackievirus B1; recombinant segments were most closely related to similar segments from strains isolated in France. Recombination occurred within the C-2 gene which encodes a multifunctional protein that functions as an RNA-stimulated ATPase associated with virus replication and virion morphogenesis. Structural modeling predicted that the recombinant protein was capable of forming hexameric and heptameric assemblies. Our data highlight the ongoing potential for recombination among enteroviruses groups, leading to modifications within viral proteins which may impact virulence.Brief Article SummaryAfter a daycare teacher was diagnosed with meningitis, ten children were referred by the county health department to a University Hospital for evaluation. Cerebral Spinal Fluid (CSF) from the teacher and nasal swabs from symptomatic children were positive for a novel recombinant enterovirus, with the amino-terminal portion of the virus derived from Echovirus E6 and the carboxy-terminal portion originating from a Coxsackievirus B1.

Pre-B cell leukemia homeobox 1 (PBX1) controls chromatin accessibility to a large number of genes in various cell types. Its dominant negative splice isoform, PBX1D, which lacks the DNA and Hox-binding domains, is expressed more frequently in the CD4+ T cells from lupus-prone mice and patients with systemic lupus erythematosus than healthy control subjects. PBX1D overexpression in CD4+ T cells impaired regulatory T cell homeostasis and expanded inflammatory CD4+ T cells. In this study, we showed that PBX1 message expression is downregulated by activation in CD4+ T cells as well as in B cells. PBX1D protein was less stable than the normal isoform, PBX1B, and it is degraded through the ubiquitin-proteasome-dependent pathway. The DNA binding domain lacking in PBX1D has two putative ubiquitin binding sites, K292 and K293, that are predicted to be in direct contact with DNA. Mutation of K292-293 reduced PBX1B stability to a level similar to PBX1D and abrogated DNA binding. In addition, contrary to PBX1B, PBX1D is retained in the cytoplasm without the help of the cofactors MEIS or PREP1, indicating a different requirement for nuclear translocation. Overall, these findings suggest that multiple post-transcriptional mechanisms are responsible for PBX1D loss of function and induction of CD4+ T cell inflammatory phenotypes in systemic lupus erythematosus.

Sjögren’s Disease (SjD) is a chronic, systemic autoimmune disease characterized by lymphocytic infiltration and the development of dry eyes and mouth as the result of the secretory dysfunction of the lacrimal and salivary glands. In recent years, infectious agents have been shown to be associated with SjD. Several studies indicated that SARS-CoV-2 infection induces autoantibodies production, patients also experienced various autoimmune disorders. This study used SARS-CoV-2 WA1/2020 to infect hACE2 mice to investigate the relationship between SARS-CoV-2 infection and SjD. Infected mice showed dry eyes, decreased salivary flow, lymphocytic infiltration of the lacrimal and salivary glands, elevated antinuclear antibodies (ANA), specifically anti-SSB/La, and apoptotic cells near the acinar cells. Notably, counting of apoptotic cells showed a significantly higher number in the lacrimal glands of mice infected with a low viral load than high viral load. We confirmed with clinical data that we observed the elevation of SjD-specific autoantibodies (ANA, anti-SSB/Ro52, and anti-SSA/La) in sera from COVID-19 patients. Salivary gland biopsies of patients diagnosed with SjD several months after SARS-CoV-2 infection also showed lymphocytic infiltration and fibrosis. Lastly, to identify whether monoclonal antibodies produced by COVID-19 patients are reactive against nuclear antigens, antibodies tested for greater antiviral binding capacity were shown to be more responsive. Overall, by observing SjD-like phenotypes in mouse models and patients, our study confirms a direct pathogenic role of SARS-CoV-2 in SjD. It also provides a different pilot for the study of the mechanisms of autoimmune responses induced by viral infections. This research was funded by the National Institutes of Health (NIH), National Institute of Dental and Craniofacial Research (NIDCR), Division of Extramural Research (DE028544, PI-Nguyen; DE028544-02S1, PI-Nguyen), and the NIH/NIDCR Division of Intramural Research (Z01-DE000704, PI-Warner; Z01-DE00695, PI-Chiorini).

OBJECTIVES Sjögren's Disease (SjD) is a chronic and systemic autoimmune disease characterized by lymphocytic infiltration and the development of dry eyes and dry mouth resulting from the secretory dysfunction of the exocrine glands. SARS-CoV-2 may trigger the development or progression of autoimmune diseases, as evidenced by increased autoantibodies in patients and the presentation of cardinal symptoms of SjD. The objective of the study was to determine whether SARS-CoV-2 induces the signature clinical symptoms of SjD. METHODS The ACE2-transgenic mice were infected with SARS-CoV-2; SjD profiling was conducted. COVID-19 patients' sera were examined to detect the presence of autoantibodies. Clinical evaluations of convalescent COVID-19 subjects, including minor salivary gland (MSG) biopsies, were collected. Lastly, monoclonal antibodies generated from single B cells of patients were interrogated for ACE2/spike inhibition and nuclear antigens. RESULTS Mice infected with the virus showed a decreased saliva flow rate, elevated antinuclear antibodies (ANAs) with anti-SSB/La, and lymphocyte infiltration in the lacrimal and salivary glands. Sera of COVID-19 patients showed an increase in ANA, anti-SSA/Ro52, and anti-SSB/La. The male patients showed elevated levels of anti-SSA/Ro52 compared to female patients, and female patients had more diverse ANA patterns. Minor salivary gland biopsies of convalescent COVID-19 subjects showed focal lymphocytic infiltrates in four of six subjects, and 2 of 6 subjects had focus scores >2. Lastly, we found that monoclonal antibodies produced in recovered patients can block ACE2/spike interaction and recognize nuclear antigens. CONCLUSION Overall, our study shows a direct association between SARS-CoV-2 and SjD. Hallmark features of SjD salivary glands were histologically indistinguishable from convalescent COVID-19 subjects.The results potentially implicate that SARS-CoV-2 could be an environmental trigger for SjD.

Supplementary Data from A Novel Inhibitor of DNA Polymerase β Enhances the Ability of Temozolomide to Impair the Growth of Colon Cancer Cells

Previous studies have shown that cysteine-reactive drug metabolites bind covalently with protein to activate patient T cells. However, the nature of the antigenic determinants that interact with HLA and whether T cell stimulatory peptides contain the bound drug metabolite has not been defined. Because susceptibility to dapsone hypersensitivity is associated with the expression of HLA-B*13:01, we have designed and synthesized nitroso dapsone-modified, HLA-B*13:01 binding peptides and explored their immunogenicity using T cells from hypersensitive human patients. Cysteine-containing 9-mer peptides with high binding affinity to HLA-B*13:01 were designed (AQDCEAAAL [Pep1], AQDACEAAL [Pep2], and AQDAEACAL [Pep3]), and the cysteine residue was modified with nitroso dapsone. CD8+ T cell clones were generated and characterized in terms of phenotype, function, and cross-reactivity. Autologous APCs and C1R cells expressing HLA-B*13:01 were used to determine HLA restriction. Mass spectrometry confirmed that nitroso dapsone-peptides were modified at the appropriate site and were free of soluble dapsone and nitroso dapsone. APC HLA-B*13:01-restricted nitroso dapsone-modified Pep1-(n = 124) and Pep3-responsive (n = 48) CD8+ clones were generated. Clones proliferated and secreted effector molecules with graded concentrations of nitroso dapsone-modified Pep1 or Pep3. They also displayed reactivity against soluble nitroso dapsone, which forms adducts in situ, but not with the unmodified peptide or dapsone. Cross-reactivity was observed between nitroso dapsone-modified peptides with cysteine residues in different positions in the peptide sequence. These data characterize a drug metabolite hapten CD8+ T cell response in an HLA risk allele -restricted form of drug hypersensitivity and provide a framework for structural analysis of hapten HLA binding interactions. The Journal of Immunology, 2023, 210: 1031-1042.