Single cell transcriptomics reveal polyclonal memory T cell responses in abacavir patch test positive skin

Capsule Summary. Single-cell responses in HLA-B*57:01 abacavir patch test positive skin remote to the acute hypersensitivity reaction demonstrate polyclonal T-cell activation and proliferation characterized by a transcriptional and cellular response consistent with memory responses to altered peptides

Abacavir inhibits but does not cause self-reactivity to HLA-B*57:01-restricted EBV specific T cell receptors

Pre-existing pathogen-specific memory T cell responses can contribute to multiple adverse outcomes including autoimmunity and drug hypersensitivity. How the specificity of the T cell receptor (TCR) is subverted or seconded in many of these diseases remains unclear. Here, we apply abacavir hypersensitivity (AHS) as a model to address this question because the disease is linked to memory T cell responses and the HLA risk allele, HLA-B*57:01, and the initiating insult, abacavir, are known. To investigate the role of pathogen-specific TCR specificity in mediating AHS we performed a genome-wide screen for HLA-B*57:01 restricted T cell responses to Epstein-Barr virus (EBV), one of the most prevalent human pathogens. T cell epitope mapping revealed HLA-B*57:01 restricted responses to 17 EBV open reading frames and identified an epitope encoded by EBNA3C. Using these data, we cloned the dominant TCR for EBNA3C and a previously defined epitope within EBNA3B. TCR specificity to each epitope was confirmed, however, cloned TCRs did not cross-react with abacavir plus self-peptide. Nevertheless, abacavir inhibited TCR interactions with their cognate ligands, demonstrating that TCR specificity may be subverted by a drug molecule. These results provide an experimental road map for future studies addressing the heterologous immune responses of TCRs including T cell mediated adverse drug reactions

HLA-A*32:01 is strongly associated with vancomycin-induced drug reaction with eosinophilia and systemic symptoms

Background Vancomycin is a prevalent cause of the severe hypersensitivity syndrome drug reaction with eosinophilia and systemic symptoms (DRESS) which leads to significant morbidity and mortality and commonly occurs in the setting of combination antibiotic therapy which impacts future treatment choices. Variations in human leukocyte antigen (HLA) class I in particular have been associated with serious T-cell mediated adverse drug reactions which has led to preventive screening strategies for some drugs. Objective To determine if variation in the HLA region is associated with vancomycin-induced DRESS. Methods Probable vancomycin DRESS cases were matched 1:2 with tolerant controls based on sex, race, and age using BioVU, Vanderbilt’s deidentified electronic health record database. Associations between DRESS and carriage of HLA class I and II alleles were assessed by conditional logistic regression. An extended sample set from BioVU was utilized to conduct a time-to-event analysis of those exposed to vancomycin with and without the identified HLA risk allele. Results Twenty-three individuals met inclusion criteria for vancomycin-associated DRESS. 19/23 (82.6%) cases carried HLA-A*32:01 compared to 0/46 (0%) of the matched vancomycin tolerant controls (p=1x10-8) and 6.3% of the BioVU population (n=54,249) (p=2x10-16). Time-to-event analysis of DRESS development during vancomycin treatment among the HLA-A*32:01 positive group indicated that 19.2% developed DRESS and did so within four weeks. Conclusions HLA-A*32:01 is strongly associated with vancomycin DRESS in a population of predominantly European ancestry. HLA-A*32:01 testing could improve antibiotic safety, help implicate vancomycin as the causal drug and preserve future treatment options with co-administered antibiotics

Acute interactions between the MCMV m15 locus and asialo-GM1+ cells determines viral dissemination to and replication in the salivary glands

Murine cytomegalovirus (MCMV) expresses in excess of 170 open reading frames (ORFs ) (1), many of which have unknown function. In addition, it is becoming increasingly clear that MCMV, and other cytomegaloviruses, exhibit complex gene regulation, including the expression of alternative splice variants, the use of alternative start sites and the expression of multiple mRNA species from a single locus (2). Such complex regulation can complicate the study of gene function . We show here that the MCMV locus, composed of the m14 , m15 and m16 ORFs, expresses five overlapping mRNA transcripts that are all co -terminal with the predicted end of the m16 ORF . Functional inactivation of any one of these ORFs has minimal impact on the replication of MCMV . However, disruption of all five transcripts results in a complex in vivo phenotype . Disruption of all five transcripts leads to an increase in viral virulence during acute infection . Whilst slight, this elevated virulence is reproducibly seen in multiple mouse strains . Conversely, disruption of this locus also leads to significant viral attenuation, evidenced during chronic infection of the salivary glands . Attenuation at this site appears to be due to reduced viraemia . This complex in vivo phenotype is associated with heightened natural killer (NK) cell responses, including enhanced proliferation and IFNγ production. Depletion of NK cells, but not T cells, rescues viraemia and salivary gland replication . These data demonstrate that multiple transcripts can modulate, perhaps in a concerted fashion, the function of anti -viral NK cells . In addition, these data highlight the need to completely define the transcriptional regulation of a viral locus before assigning function to any particular gene