Immune checkpoint therapy responders display early clonal expansion of tumor infiltrating lymphocytes.

Immune checkpoint therapy (ICT) causes durable tumour responses in a subgroup of patients, but it is not well known how T cell receptor beta (TCRß) repertoire dynamics contribute to the therapeutic response. Using murine models that exclude variation in host genetics, environmental factors and tumour mutation burden, limiting variation between animals to naturally diverse TCRß repertoires, we applied TCRseq, single cell RNAseq and flow cytometry to study TCRß repertoire dynamics in ICT responders and non-responders. Increased oligoclonal expansion of TCRß clonotypes was observed in responding tumours. Machine learning identified TCRß CDR3 signatures unique to each tumour model, and signatures associated with ICT response at various timepoints before or during ICT. Clonally expanded CD8+ T cells in responding tumours post ICT displayed effector T cell gene signatures and phenotype. An early burst of clonal expansion during ICT is associated with response, and we report unique dynamics in TCRß signatures associated with ICT response.

Multiomic single-cell sequencing defines tissue-specific responses in Stevens-Johnson Syndrome and Toxic epidermal necrolysis.

Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) is a rare but life-threatening cutaneous drug reaction mediated by human leukocyte antigen (HLA) class I-restricted CD8+ T-cells. To obtain an unbiased assessment of SJS/TEN cellular immunopathogenesis, we performed single-cell (sc) transcriptome, surface proteome, and TCR sequencing on unaffected skin, affected skin, and blister fluid from 17 SJS/TEN patients. From 119,784 total cells, we identified 16 scRNA-defined subsets, confirmed by subset-defining surface protein expression. Keratinocytes upregulated HLA and IFN-response genes in the affected skin. Cytotoxic CD8+ T-cell subpopulations of expanded and unexpanded TCRαß clonotypes were shared in affected skin and blister fluid but absent or unexpanded in SJS/TEN unaffected skin. SJS/TEN blister fluid is a rich reservoir of oligoclonal CD8+ T-cells with an effector phenotype driving SJS/TEN pathogenesis. This multiomic database will act as the basis to define antigen-reactivity, HLA restriction, and signatures of drug-antigen-reactive T-cell clonotypes at a tissue level.

Underrepresentation of activating KIR gene expression in single-cell RNA-seq data is due to KIR gene misassignment.

Standard single-cell RNA-sequencing alignment pipelines exhibit a propensity for misassigning killer immunoglobulin-like receptor (KIR) transcripts, thereby giving rise to inaccuracies in quantifying KIR expression. Alves et al. elucidated that these default workflows frequently misclassify activating KIR transcripts as inhibitory KIR expression, resulting in a skewed representation of the KIR repertoire.

High-resolution HLA genotyping in inclusion body myositis refines 8.1 ancestral haplotype association to DRB1*03:01:01 and highlights pathogenic role of arginine-74 of DRß1 chain.

OBJECTIVES: Inclusion body myositis (IBM) is a progressive inflammatory-degenerative muscle disease of older individuals, with some patients producing anti-cytosolic 5'-nucleotidase 1A (NT5C1A, aka cN1A) antibodies. Human Leukocyte Antigens (HLA) is the highest genetic risk factor for developing IBM. In this study, we aimed to further define the contribution of HLA alleles to IBM and the production of anti-cN1A antibodies. METHODS: We HLA haplotyped a Western Australian cohort of 113 Caucasian IBM patients and 112 ethnically matched controls using Illumina next-generation sequencing. Allele frequency analysis and amino acid alignments were performed using the Genentech/MiDAS bioinformatics package. Allele frequencies were compared using Fisher's exact test. Age at onset analysis was performed using the ggstatsplot package. All analysis was carried out in RStudio version 1.4.1717. RESULTS: Our findings validated the independent association of HLA-DRB1*03:01:01 with IBM and attributed the risk to an arginine residue in position 74 within the DRß1 protein. Conversely, DRB4*01:01:01 and DQA1*01:02:01 were found to have protective effects; the carriers of DRB1*03:01:01 that did not possess these alleles had a fourteenfold increased risk of developing IBM over the general Caucasian population. Furthermore, patients with the abovementioned genotype developed symptoms on average five years earlier than patients without. We did not find any HLA associations with anti-cN1A antibody production. CONCLUSIONS: High-resolution HLA sequencing more precisely characterised the alleles associated with IBM and defined a haplotype linked to earlier disease onset. Identification of the critical amino acid residue by advanced biostatistical analysis of immunogenetics data offers mechanistic insights and future directions into uncovering IBM aetiopathogenesis.

Transcriptomic Profiling of Influenza A Virus-Infected Mouse Lung at Recovery Stage Using RNA Sequencing.

Influenza A virus (IAV) is known to cause mild to severe respiratory illness. Under some conditions, the infection can lead to pneumonia (viral or bacterial), acute respiratory distress syndrome, and other complications that can be fatal, especially in vulnerable populations such as the elderly, young children, and individuals with underlying health conditions. Despite previous studies, little is known about the host immune response and neuroimmune interactions in IAV infection. Using RNA sequencing, we performed transcriptomic analysis of murine lung tissue 21 days post infection (dpi) with IAV (H1N1) in order to find the differentially expression genes (DEGs) related to the host immune response and neuroimmune interactions inside the lung during recovery. Among 792 DEGs, 434 genes were up-regulated, whereas 358 genes were down-regulated. The most prominent molecular functions of the up-regulated genes were related to the immune response and tissue repair, whereas a large proportion of the down-regulated genes were associated with neural functions. Although further molecular/functional studies need to be performed for these DEGs, our results facilitate the understanding of the host response (from innate immunity to adaptive immunity) and neuroimmune interactions in infected lungs at the recovery stage of IAV infection. These genes might have potential uses as mechanistic/diagnostic biomarkers and represent possible targets for anti-IAV therapies.

Comparison of in vitro growth characteristics of Cryptosporidium hominis (IdA15G1) and Cryptosporidium parvum (Iowa-IIaA17G2R1 and IIaA18G3R1).

Cryptosporidium is a major cause of diarrhoeal disease and mortality in young children in resource-poor countries, for which no vaccines or adequate therapeutic options are available. Infection in humans is primarily caused by two species: C. hominis and C. parvum. Despite C. hominis being the dominant species infecting humans in most countries, very little is known about its growth characteristics and life cycle in vitro, given that the majority of our knowledge of the in vitro development of Cryptosporidium has been based on C. parvum. In the present study, the growth and development of two C. parvum isolates (subtypes Iowa-IIaA17G2R1 and IIaA18G3R1) and one C. hominis isolate (subtype IdA15G1) in HCT-8 cells were examined and compared at 24 h and 48 h using morphological data acquired with scanning electron microscopy. Our data indicated no significant differences in the proportion of meronts or merozoites between species or subtypes at either time-point. Sexual development was observed at the 48-h time-point across both species through observations of both microgamonts and macrogamonts, with a higher frequency of macrogamont observations in C. hominis (IdA15G1) cultures at 48-h post-infection compared to both C. parvum subtypes. This corresponded to differences in the proportion of trophozoites observed at the same time point. No differences in proportion of microgamonts were observed between the three subtypes, which were rarely observed across all cultures. In summary, our data indicate that asexual development of C. hominis is similar to that of C. parvum, while sexual development is accelerated in C. hominis. This study provides new insights into differences in the in vitro growth characteristics of C. hominis when compared to C. parvum, which will facilitate our understanding of the sexual development of both species.

Distinct CD3 + CD14 + T Cell-Monocytes are dynamic complexes that harbor HIV and are increased with glucose intolerance.

Persistent systemic inflammation in persons with HIV (PWH) is accompanied by an increased risk of metabolic disease. Yet, changes in the innate and adaptive immune system in PWH who develop metabolic disease remain poorly defined. Using unbiased approaches, we show that PWH with prediabetes/diabetes have a significantly higher proportion of circulating CD14 + monocytes complexed to T cells. The complexed CD3 + T cells and CD14 + monocytes demonstrate functional immune synapses, increased expression of proinflammatory cytokines, and greater glucose utilization. Furthermore, these complexes harbor more latent HIV DNA compared to CD14 + monocytes or CD4 + T cells. Our results demonstrate that circulating CD3 + CD14 + T cell-monocyte pairs represent functional dynamic cellular interactions that likely contribute to inflammation and, in light of their increased proportion, may have a role in metabolic disease pathogenesis. These findings provide an incentive for future studies to investigate T cell-monocyte immune complexes as mechanistic in HIV cure and diseases of aging. Highlights: Persons with HIV and diabetes have increased circulating CD3 + CD14 + T cell-monocyte complexes. CD3 + CD14 + T cell-monocytes are a heterogenous group of functional and dynamic complexes. We can detect HIV in T cell-monocyte complexes. The proportion of CD3 + CD14 + T cell-monocyte complexes is positively associated with blood glucose levels and negatively with plasma IL-10 and CD4 + T regulatory cells.

Tracking of activated cTfh cells following sequential influenza vaccinations reveals transcriptional profile of clonotypes driving a vaccine-induced immune response.

Introduction: A vaccine against influenza is available seasonally but is not 100% effective. A predictor of successful seroconversion in adults is an increase in activated circulating T follicular helper (cTfh) cells after vaccination. However, the impact of repeated annual vaccinations on long-term protection and seasonal vaccine efficacy remains unclear. Methods: In this study, we examined the T cell receptor (TCR) repertoire and transcriptional profile of vaccine-induced expanded cTfh cells in individuals who received sequential seasonal influenza vaccines. We measured the magnitude of cTfh and plasmablast cell activation from day 0 (d0) to d7 post-vaccination as an indicator of a vaccine response. To assess TCR diversity and T cell expansion we sorted activated and resting cTfh cells at d0 and d7 post-vaccination and performed TCR sequencing. We also single cell sorted activated and resting cTfh cells for TCR analysis and transcriptome sequencing. Results and discussion: The percent of activated cTfh cells significantly increased from d0 to d7 in each of the 2016-17 (p < 0.0001) and 2017-18 (p = 0.015) vaccine seasons with the magnitude of cTfh activation increase positively correlated with the frequency of circulating plasmablast cells in the 2016-17 (p = 0.0001) and 2017-18 (p = 0.003) seasons. At d7 post-vaccination, higher magnitudes of cTfh activation were associated with increased clonality of cTfh TCR repertoire. The TCRs from vaccine-expanded clonotypes were identified and tracked longitudinally with several TCRs found to be present in both years. The transcriptomic profile of these expanded cTfh cells at the single cell level demonstrated overrepresentation of transcripts of genes involved in the type-I interferon pathway, pathways involved in gene expression, and antigen presentation and recognition. These results identify the expansion and transcriptomic profile of vaccine-induced cTfh cells important for B cell help.

Uncovering the significance of expanded CD8+ large granular lymphocytes in inclusion body myositis: Insights into T cell phenotype and functional alterations, and disease severity.

Introduction: Inclusion body myositis (IBM) is a progressive inflammatory myopathy characterised by skeletal muscle infiltration and myofibre invasion by CD8+ T lymphocytes. In some cases, IBM has been reported to be associated with a systemic lymphoproliferative disorder of CD8+ T cells exhibiting a highly differentiated effector phenotype known as T cell Large Granular Lymphocytic Leukemia (T-LGLL). Methods: We investigated the incidence of a CD8+ T-LGL lymphoproliferative disorder in 85 IBM patients and an aged-matched group of 56 Healthy Controls (HC). Further, we analysed the phenotypical characteristics of the expanded T-LGLs and investigated whether their occurrence was associated with any particular HLA alleles or clinical characteristics. Results: Blood cell analysis by flow cytometry revealed expansion of T-LGLs in 34 of the 85 (40%) IBM patients. The T cell immunophenotype of T-LGLHIGH patients was characterised by increased expression of surface molecules including CD57 and KLRG1, and to a lesser extent of CD94 and CD56 predominantly in CD8+ T cells, although we also observed modest changes in CD4+ T cells and γδ T cells. Analysis of Ki67 in CD57+ KLRG1+ T cells revealed that only a small proportion of these cells was proliferating. Comparative analysis of CD8+ and CD4+ T cells isolated from matched blood and muscle samples donated by three patients indicated a consistent pattern of more pronounced alterations in muscles, although not significant due to small sample size. In the T-LGLHIGH patient group, we found increased frequencies of perforin-producing CD8+ and CD4+ T cells that were moderately correlated to combined CD57 and KLRG1 expression. Investigation of the HLA haplotypes of 75 IBM patients identified that carriage of the HLA-C*14:02:01 allele was significantly higher in T-LGLHIGH compared to T-LGLLOW individuals. Expansion of T-LGL was not significantly associated with seropositivity patient status for anti-cytosolic 5'-nucleotidase 1A autoantibodies. Clinically, the age at disease onset and disease duration were similar in the T-LGLHIGH and T-LGLLOW patient groups. However, metadata analysis of functional alterations indicated that patients with expanded T-LGL more frequently relied on mobility aids than T-LGLLOW patients indicating greater disease severity. Conclusion: Altogether, these results suggest that T-LGL expansion occurring in IBM patients is correlated with exacerbated immune dysregulation and increased disease burden.

A Positively Selected fur-R88H Mutation Enhances Helicobacter pylori Fitness in a High-Salt Environment and Alters Fur-Dependent Regulation of Gene Expression.

Both Helicobacter pylori infection and a high-salt diet are risk factors for gastric cancer. We previously showed that a mutation in fur (encoding the ferric uptake regulator variant Fur-R88H) was positively selected in H. pylori strains isolated from experimentally infected Mongolian gerbils receiving a high-salt diet. In the present study, we report that continuous H. pylori growth in high-salt conditions in vitro also leads to positive selection of the fur-R88H mutation. Competition experiments with strains containing wild-type fur or fur-R88H, each labeled with unique nucleotide barcodes, showed that the fur-R88H mutation enhances H. pylori fitness under high-salt conditions but reduces H. pylori fitness under routine culture conditions. The fitness advantage of the fur-R88H mutant under high-salt conditions was abrogated by the addition of supplemental iron. To test the hypothesis that the fur-R88H mutation alters the regulatory properties of Fur, we compared the transcriptional profiles of strains containing wild-type fur or fur-R88H. Increased transcript levels of fecA2, which encodes a predicted TonB-dependent outer membrane transporter, were detected in the fur-R88H variant compared to those in the strain containing wild-type fur under both high-salt and routine conditions. Competition experiments showed that fecA2 contributes to H. pylori fitness under both high-salt and routine conditions. These results provide new insights into mechanisms by which the fur-R88H mutation confers a selective advantage to H. pylori in high-salt environments.

KIR Allelic Variation and the Remission of Atopic Dermatitis Over Time.

Atopic dermatitis (AD) is a common chronic skin disease. Although generally thought to be a disease of T-cell dysregulation, recent studies have suggested that immune dysregulation of NK cells is also important. Killer cell Ig-like receptors (KIRs) are involved with NK cell regulation. The Pediatric Eczema Elective Registry is a U.S. nationwide longitudinal cohort with up to 10 y of follow-up in which 655 children had DNA available for full allelic KIR sequencing. Every 6 mo, AD activity was reported by Pediatric Eczema Elective Registry children. Using generalized estimating equations, we evaluated the association of KIR allelic variation in concert with known HLA binding ligands and whether the child reported AD in "remission" (no skin lesions and not using AD medication). KIR2DS4*001:01 (odds ratio 0.53, 95% CI [0.32, 0.88]) and KIR2DL4*001:02 (0.54, [0.33, 0.89]) in the presence of C*04:01 had the largest effect on decreasing the likelihood of AD remission. The haplotype KIR 2DL4*001:02 ∼ 2DS4*001:01 ∼ 3DL2*002:01 (0.77, [0.60, 0.99]) was also associated with a decreased likelihood of AD remission. Our findings add to the general body of evidence of a growing literature on the importance of NK cells with respect to the immunopathogenesis and natural history of AD.

Association of KIR2DL5, KIR2DS5, and KIR2DS1 allelic variation and atopic dermatitis.

Natural killer cells (NK) have been associated with the pathophysiology of atopic dermatitis (AD). NK function is regulated by killer cell Ig-like receptor family (KIR) receptors that interact with HLA ligands. The study goal was to focus on allelic variation in genes KIR2DL5, KIR2DS5, and KIR2DS1 with respect to AD. This was a case-control study of individuals with (n = 313) and without (n = 176) AD. Associations were estimated using logistic regression. The prevalence of KIR2DL5 was 52.5% (95% CI 48.0,57.0), KIR2DS5 was 33.0% (28.8,37.3), and KIR2DS1 was 33.6% (29.4,38.0). The presence of the KIR2DL5*001:01 increased the odds of having AD by about 86% (odds ratio (OR): 1.86(1.23,2.82) p = 0.003). The risk for individuals homozygous for KIR2DL5*001:01 was even greater (OR: 2.16 (95% CI 1.31,3.53) p = 0.0023). The odds of having AD with KIR2DL5*001:01 was similar in Whites and Blacks. Allelic variation in KIR2DS5 and KIR2DS1 was not associated with AD. There is no known HLA binding ligand for KIR2DL5. The effect of KIR2DL5*001:01 increased in the presence of HLA-B*-21TT leader sequence (2.46(1.37,4.41) p = 0.0025) and the HLA-C2 ligand (2.07 (1.37,4.41, p = 0.000002). Our study shows an independent association of the KIR2DL5*001:01 with AD and is the first study to associate AD with KIR allelic variation.

CD4+ T cells drive an inflammatory, TNF-α/IFN-rich tumor microenvironment responsive to chemotherapy.

While chemotherapy remains the first-line treatment for many cancers, it is still unclear what distinguishes responders from non-responders. Here, we characterize the chemotherapy-responsive tumor microenvironment in mice, using RNA sequencing on tumors before and after cyclophosphamide, and compare the gene expression profiles of responders with progressors. Responsive tumors have an inflammatory and highly immune infiltrated pre-treatment tumor microenvironment characterized by the enrichment of pathways associated with CD4+ T cells, interferons (IFNs), and tumor necrosis factor alpha (TNF-α). The same gene expression profile is associated with response to cyclophosphamide-based chemotherapy in patients with breast cancer. Finally, we demonstrate that tumors can be sensitized to cyclophosphamide and 5-FU chemotherapy by pre-treatment with recombinant TNF-α, IFNγ, and poly(I:C). Thus, a CD4+ T cell-inflamed pre-treatment tumor microenvironment is necessary for response to chemotherapy, and this state can be therapeutically attained by targeted immunotherapy.

Adaptation to HLA-associated immune pressure over the course of HIV infection and in circulating HIV-1 strains.

Adaptation to human leukocyte antigen (HLA)-associated immune pressure represents a major driver of human immunodeficiency virus (HIV) evolution at both the individual and population level. To date, there has been limited exploration of the impact of the initial cellular immune response in driving viral adaptation, the dynamics of these changes during infection and their effect on circulating transmitting viruses at the population level. Capturing detailed virological and immunological data from acute and early HIV infection is challenging as this commonly precedes the diagnosis of HIV infection, potentially by many years. In addition, rapid initiation of antiretroviral treatment following a diagnosis is the standard of care, and central to global efforts towards HIV elimination. Yet, acute untreated infection is the critical period in which the diversity of proviral reservoirs is first established within individuals, and associated with greater risk of onward transmissions in a population. Characterizing the viral adaptations evident in the earliest phases of infection, coinciding with the initial cellular immune responses is therefore relevant to understanding which changes are of greatest impact to HIV evolution at the population level. In this study, we utilized three separate cohorts to examine the initial CD8+ T cell immune response to HIV (cross-sectional acute infection cohort), track HIV evolution in response to CD8+ T cell-mediated immunity over time (longitudinal chronic infection cohort) and translate the impact of HLA-driven HIV evolution to the population level (cross-sectional HIV sequence data spanning 30 years). Using next generation viral sequencing and enzyme-linked immunospot interferon-gamma recall responses to peptides representing HLA class I-specific HIV T cell targets, we observed that CD8+ T cell responses can select viral adaptations prior to full antibody seroconversion. Using the longitudinal cohort, we uncover that viral adaptations have the propensity to be retained over time in a non-selective immune environment, which reflects the increasing proportion of pre-adapted HIV strains within the Western Australian population over an approximate 30-year period.

A low-cost, sensitive and specific PCR-based tool for rapid clinical detection of HLA-B*35 alleles associated with delayed drug hypersensitivity reactions.

HLA (HLA) alleles are risk factors for CD8+ T-cell-mediated drug hypersensitivity reactions. However, as most HLA associations are incompletely predictive and/or involve risk alleles at low frequency, costly sequence-based typing can elude an economically productive cost: benefit ratio for clinical validation studies and diagnostic and/or preventative screening. Hence rapid and low-cost detection assays are now required, both for single alleles but also across risk loci associated with broader multi-disease risk; exemplified by associations with diverse alleles in HLA-B*35, including HLA-B*35:01 and green tea- or co-trimoxazole-induced liver injury. Here, we developed a cost-effective (<$10USD) qPCR assay for rapid (<2.5 h) clinical detection of HLA-B*35 alleles. The assay was validated using 430 DNA samples with previous American society for histocompatibility and immunogenetics-accredited sequence-based high-resolution HLA typing, positively detecting all HLA-B*35 allelic variants in our cohort, and as expected by primer design, the six samples that expressed low-frequency B*78:01. The assay did not result in positive detection for any negative control allele. With expected detection of B*35 and B*78, our assay sensitivity (95% CI, 95.07%-100.00%) and specificity (95% CI, 98.97%-100.00%) of 100% using as low as 10 ng of DNA provides a reliable HLA-B*35 screening tool for clinical validation and HLA-risk-based prevention and diagnostics.

Increased H3K27 trimethylation contributes to cone survival in a mouse model of cone dystrophy.

Inherited retinal diseases (IRDs) are a heterogeneous group of blinding disorders, which result in dysfunction or death of the light-sensing cone and rod photoreceptors. Despite individual IRDs (Inherited retinal disease) being rare, collectively, they affect up to 1:2000 people worldwide, causing a significant socioeconomic burden, especially when cone-mediated central vision is affected. This study uses the Pde6ccpfl1 mouse model of achromatopsia, a cone-specific vision loss IRD (Inherited retinal disease), to investigate the potential gene-independent therapeutic benefits of a histone demethylase inhibitor GSK-J4 on cone cell survival. We investigated the effects of GSK-J4 treatment on cone cell survival in vivo and ex vivo and changes in cone-specific gene expression via single-cell RNA sequencing. A single intravitreal GSK-J4 injection led to transcriptional changes in pathways involved in mitochondrial dysfunction, endoplasmic reticulum stress, among other key epigenetic pathways, highlighting the complex interplay between methylation and acetylation in healthy and diseased cones. Furthermore, continuous administration of GSK-J4 in retinal explants increased cone survival. Our results suggest that IRD (Inherited retinal disease)-affected cones respond positively to epigenetic modulation of histones, indicating the potential of this approach in developing a broad class of novel therapies to slow cone degeneration.

Deep Sequencing Analysis of Individual HIV-1 Proviruses Reveals Frequent Asymmetric Long Terminal Repeats.

Effective strategies to eliminate human immunodeficiency virus type 1 (HIV-1) reservoirs are likely to require more thorough characterizations of proviruses that persist on antiretroviral therapy (ART). The rarity of infected CD4+ T-cells and related technical challenges have limited the characterization of integrated proviruses. Current approaches using next-generation sequencing can be inefficient and limited sequencing depth can make it difficult to link proviral sequences to their respective integration sites. Here, we report on an efficient method by which HIV-1 proviruses and their sites of integration are amplified and sequenced. Across five HIV-1-positive individuals on clinically effective ART, a median of 41.2% (n = 88 of 209) of amplifications yielded near-full-length proviruses and their 5'-host-virus junctions containing a median of 430 bp (range, 18 to 1,363 bp) of flanking host sequence. Unexpectedly, 29.5% (n = 26 of 88) of the sequenced proviruses had structural asymmetries between the 5' and 3' long terminal repeats (LTRs), commonly in the form of major 3' deletions. Sequence-intact proviruses were detected in 3 of 5 donors, and infected CD4+ T-cell clones were detected in 4 of 5 donors. The accuracy of the method was validated by amplifying and sequencing full-length proviruses and flanking host sequences directly from peripheral blood mononuclear cell DNA. The individual proviral sequencing assay (IPSA) described here can provide an accurate, in-depth, and longitudinal characterization of HIV-1 proviruses that persist on ART, which is important for targeting proviruses for elimination and assessing the impact of interventions designed to eradicate HIV-1. IMPORTANCE The integration of human immunodeficiency virus type 1 (HIV-1) into chromosomal DNA establishes the long-term persistence of HIV-1 as proviruses despite effective antiretroviral therapy (ART). Characterizing proviruses is difficult because of their rarity in individuals on long-term suppressive ART, their highly polymorphic sequences and genetic structures, and the need for efficient amplification and sequencing of the provirus and its integration site. Here, we describe a novel, integrated, two-step method (individual proviral sequencing assay [IPSA]) that amplifies the host-virus junction and the full-length provirus except for the last 69 bp of the 3' long terminal repeat (LTR). Using this method, we identified the integration sites of proviruses, including those that are sequence intact and replication competent or defective. Importantly, this new method identified previously unreported asymmetries between LTRs that have implications for how proviruses are detected and quantified. The IPSA method reported is unaffected by LTR asymmetries, permitting a more accurate and comprehensive characterization of the proviral landscape.

Positive Selection of Mutations in the Helicobacter pylori katA 5' Untranslated Region in a Mongolian Gerbil Model of Gastric Disease.

To evaluate potential effects of gastric inflammation on Helicobacter pylori diversification and evolution within the stomach, we experimentally infected Mongolian gerbils with an H. pylori strain in which Cag type IV secretion system (T4SS) activity is controlled by a TetR/tetO system. Gerbils infected with H. pylori under conditions in which Cag T4SS activity was derepressed had significantly higher levels of gastric inflammation than gerbils infected under conditions with repressed Cag T4SS activity. Mutations in the 5' untranslated region (UTR) of katA (encoding catalase) were detected in strains cultured from 8 of the 17 gerbils infected with Cag T4SS-active H. pylori and none of the strains from 17 gerbils infected with Cag T4SS-inactive H. pylori. Catalase enzymatic activity, steady-state katA transcript levels, and katA transcript stability were increased in strains with these single nucleotide polymorphisms (SNPs) compared to strains in which these SNPs were absent. Moreover, strains harboring these SNPs exhibited increased resistance to bactericidal effects of hydrogen peroxide, compared to control strains. Experimental introduction of the SNPs into the wild-type katA 5' UTR resulted in increased katA transcript stability, increased katA steady-state levels, and increased catalase enzymatic activity. Based on site-directed mutagenesis and modeling of RNA structure, increased katA transcript levels were correlated with higher predicted thermal stability of the katA 5' UTR secondary structure. These data suggest that high levels of gastric inflammation positively select for H. pylori strains producing increased levels of catalase, which may confer survival advantages to the bacteria in an inflammatory gastric environment.

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.