Airway proteomics reveals broad residual anti-inflammatory effects of prednisolone in mepolizumab-treated asthma.

BACKGROUND: Mepolizumab is an anti-IL-5 mAb treatment for severe eosinophilic asthma that reduces asthma exacerbations. Residual airway inflammation with mepolizumab therapy may lead to persistent exacerbations. Oral corticosteroids remain the main treatment for these residual exacerbations. OBJECTIVE: Our study aimed to explore the corticosteroid responsiveness of airway inflammation after mepolizumab treatment to find potentially treatable inflammatory mechanisms beyond the IL-5 pathway. METHODS: The MAPLE trial was a multicenter, randomized, double-blind, placebo-controlled, crossover study of 2 weeks of high-dose oral prednisolone treatment at stable state in 27 patients treated with mepolizumab for severe eosinophilic asthma. We analyzed paired sputum (n = 16) and plasma (n = 25) samples from the MAPLE trial using high-throughput Olink proteomics. We analyzed additional sputum proteins using ELISA. RESULTS: In patients receiving mepolizumab, prednisolone significantly downregulated sputum proteins related to type 2 inflammation and chemotaxis including IL-4, IL-5, IL-13, CCL24, CCL26, EDN, CCL17, CCL22, OX40 receptor, FCER2, and the ST2 receptor. Prednisolone also downregulated cell adhesion molecules, prostaglandin synthases, mast cell tryptases, MMP1, MMP12, and neuroimmune mediators. Neutrophilic pathways were upregulated. Type 2 proteins were also downregulated in plasma, combined with IL-12, IFN-γ, and IP-10. IL-10 and amphiregulin were upregulated. CONCLUSIONS: At stable state, prednisolone has broad anti-inflammatory effects on top of mepolizumab. These effects are heterogeneous and may be clinically relevant in residual exacerbations.

The effects of inhaled corticosteroids on healthy airways.

BACKGROUND: The effects of inhaled corticosteroids (ICS) on healthy airways are poorly defined. OBJECTIVES: To delineate the effects of ICS on gene expression in healthy airways, without confounding caused by changes in disease-related genes and disease-related alterations in ICS responsiveness. METHODS: Randomized open-label bronchoscopy study of high-dose ICS therapy in 30 healthy adult volunteers randomized 2:1 to (i) fluticasone propionate 500 mcg bd daily or (ii) no treatment, for 4 weeks. Laboratory staff were blinded to allocation. Biopsies and brushings were analysed by immunohistochemistry, bulk RNA sequencing, DNA methylation array and metagenomics. RESULTS: ICS induced small between-group differences in blood and lamina propria eosinophil numbers, but not in other immunopathological features, blood neutrophils, FeNO, FEV1, microbiome or DNA methylation. ICS treatment upregulated 72 genes in brushings and 53 genes in biopsies, and downregulated 82 genes in brushings and 416 genes in biopsies. The most downregulated genes in both tissues were canonical markers of type-2 inflammation (FCER1A, CPA3, IL33, CLEC10A, SERPINB10 and CCR5), T cell-mediated adaptive immunity (TARP, TRBC1, TRBC2, PTPN22, TRAC, CD2, CD8A, HLA-DQB2, CD96, PTPN7), B-cell immunity (CD20, immunoglobulin heavy and light chains) and innate immunity, including CD48, Hobit, RANTES, Langerin and GFI1. An IL-17-dependent gene signature was not upregulated by ICS. CONCLUSIONS: In healthy airways, 4-week ICS exposure reduces gene expression related to both innate and adaptive immunity, and reduces markers of type-2 inflammation. This implies that homeostasis in health involves tonic type-2 signalling in the airway mucosa, which is exquisitely sensitive to ICS.

Species-level, metagenomic and proteomic analysis of microbe-immune interactions in severe asthma.

BACKGROUND: The airway microbiome in severe asthma has not been characterised at species-level by metagenomic sequencing, nor have the relationships between specific species and mucosal immune responses in 'type-2 low', neutrophilic asthma been defined. We performed an integrated species-level metagenomic data with inflammatory mediators to characterise prevalence of dominant potentially pathogenic organisms and host immune responses. METHODS: Sputum and nasal lavage samples were analysed using long-read metagenomic sequencing with Nanopore and qPCR in two cross-sectional adult severe asthma cohorts, Wessex (n = 66) and Oxford (n = 30). We integrated species-level data with clinical parameters and 39 selected airway proteins measured by immunoassay and O-link. RESULTS: The sputum microbiome in health and mild asthma displayed comparable microbial diversity. By contrast, 23% (19/81) of severe asthma microbiomes were dominated by a single respiratory pathogen, namely H. influenzae (n = 10), M. catarrhalis (n = 4), S. pneumoniae (n = 4) and P. aeruginosa (n = 1). Neutrophilic asthma was associated with H. influenzae, M. catarrhalis, S. pneumoniae and T. whipplei with elevated type-1 cytokines and proteases; eosinophilic asthma with higher M. catarrhalis, but lower H. influenzae, and S. pneumoniae abundance. H. influenzae load correlated with Eosinophil Cationic Protein, elastase and IL-10. R. mucilaginosa associated positively with IL-6 and negatively with FGF. Bayesian network analysis also revealed close and distinct relationships of H. influenzae and M. catarrhalis with type-1 airway inflammation. The microbiomes and cytokine milieu were distinct between upper and lower airways. CONCLUSIONS: This species-level integrated analysis reveals central, but distinct associations between potentially pathogenic bacteria and airways inflammation in severe asthma.

Defining the key intrahepatic gene networks in HCV infection driven by sex.

OBJECTIVE: The transcriptional response in the liver during HCV infection is critical for determining clinical outcomes. This issue remains relatively unexplored as tissue access to address this at scale is usually limited. We aimed to profile the transcriptomics of HCV-infected livers to describe the expression networks involved and assess the effect on them of major predictors of clinical outcome such as IFNL4 (interferon lambda 4) host genotype and sex. DESIGN: We took advantage of a large clinical study of HCV therapy accompanied by baseline liver biopsy to examine the drivers of transcription in tissue samples in 195 patients also genotyped genome-wide for host and viral single nucleotide polymorphisms. We addressed the role of host factors (disease status, sex, genotype, age) and viral factors (load, mutation) on transcriptional responses. RESULTS: We observe key modules of transcription which can be impacted differentially by host and viral factors. Underlying cirrhotic state had the most substantial impact, even in a stable, compensated population. Notably, sex had a major impact on antiviral responses in concert with IL28B (interleukin 28B)/IFNL4 genotype, with stronger interferon and humoral responses in females. Males tended towards a dominant cellular immune response. In both sexes, there was a strong influence of the underlying host disease status and of specific viral mutations, and sex-specific expression quantitative trait loci were also observed. CONCLUSION: These features help define the major influences on tissue responses in HCV infection, impacting on the response to treatment and with broader implications for responses in other sex-biased infections.

BreakAlign: a Perl program to align chimaeric (split) genomic NGS reads and allow visual confirmation of novel retroviral integrations.

BACKGROUND: Retroviruses replicate by integrating a DNA copy into a host chromosome. Detecting novel retroviral integrations (ones not in the reference genome sequence of the host) from genomic NGS data is bioinformatically challenging and frequently produces many false positives. One common method of confirmation is visual inspection of an alignment of the chimaeric (split) reads that span a putative novel retroviral integration site. We perceived the need for a program that would facilitate this by producing a multiple alignment containing both the viral and host regions that flank an integration. RESULTS: BreakAlign is a Perl program that uses blastn to produce such a multiple alignment. In addition to the NGS dataset and a reference viral sequence, the program requires either (a) the ~ 500nt host genome sequence that spans the putative integration or (b) coordinates of this putative integration in an installed copy of the reference human genome (multiple integrations can be processed automatically). BreakAlign is freely available from https://github.com/marchiem/breakalign and is accompanied by example files allowing a test run. CONCLUSION: BreakAlign will confirm and facilitate characterisation of both (a) germline integrations of endogenous retroviruses and (b) somatic integrations of exogenous retroviruses such as HIV and HTLV. Although developed for use with genomic short-read NGS (second generation) data and retroviruses, it should also be useful for long-read (third generation) data and any mobile element with at least one conserved flanking region.

Human Endogenous Retrovirus-K HML-2 integration within RASGRF2 is associated with intravenous drug abuse and modulates transcription in a cell-line model.

HERV-K HML-2 (HK2) has been proliferating in the germ line of humans at least as recently as 250,000 years ago, with some integrations that remain polymorphic in the modern human population. One of the solitary HK2 LTR polymorphic integrations lies between exons 17 and 18 of RASGRF2, a gene that affects dopaminergic activity and is thus related to addiction. Here we show that this antisense HK2 integration (namely RASGRF2-int) is found more frequently in persons who inject drugs compared with the general population. In a Greek HIV-1-positive population (n = 202), we found RASGRF2-int 2.5 times (14 versus 6%) more frequently in patients infected through i.v. drug use compared with other transmission route controls (P = 0.03). Independently, in a United Kingdom-based hepatitis C virus-positive population (n = 184), we found RASGRF2-int 3.6 times (34 versus 9.5%) more frequently in patients infected during chronic drug abuse compared with controls (P < 0.001). We then tested whether RASGRF2-int could be mechanistically responsible for this association by modulating transcription of RASGRF2 We show that the CRISPR/Cas9-mediated insertion of HK2 in HEK293 cells in the exact RASGRF2 intronic position found in the population resulted in significant transcriptional and phenotypic changes. We also explored mechanistic features of other intronic HK2 integrations and show that HK2 LTRs can be responsible for generation of cis-natural antisense transcripts, which could interfere with the transcription of nearby genes. Our findings suggest that RASGRF2-int is a strong candidate for dopaminergic manipulation, and emphasize the importance of accurate mapping of neglected HERV polymorphisms in human genomic studies.

Impact of Interferon Lambda 4 Genotype on Interferon-Stimulated Gene Expression During Direct-Acting Antiviral Therapy for Hepatitis C.

New directly acting antivirals (DAAs) provide very high cure rates in most patients infected by hepatitis C virus (HCV). However, some patient groups have been relatively harder to treat, including those with cirrhosis or infected with HCV genotype 3. In the recent BOSON trial, genotype 3, patients with cirrhosis receiving a 16-week course of sofosbuvir and ribavirin had a sustained virological response (SVR) rate of around 50%. In patients with cirrhosis, interferon lambda 4 (IFNL4) CC genotype was significantly associated with SVR. This genotype was also associated with a lower interferon-stimulated gene (ISG) signature in peripheral blood and in liver at baseline. Unexpectedly, patients with the CC genotype showed a dynamic increase in ISG expression between weeks 4 and 16 of DAA therapy, whereas the reverse was true for non-CC patients. Conclusion: These data provide an important dynamic link between host genotype and phenotype in HCV therapy also potentially relevant to naturally acquired infection. (Hepatology 2018; 00:000-000).

Human MAIT cells show metabolic quiescence with rapid glucose-dependent upregulation of granzyme B upon stimulation.

Mucosal-associated invariant T (MAIT) cells are a well-characterized innate-like T cell population abundant in the human liver, peripheral tissues and blood. MAIT cells serve in the first line of defense against infections, through engagement of their T cell receptor, which recognizes microbial metabolites presented on MR1, and through cytokine-mediated triggering. Typically, they show a quiescent memory phenotype but can undergo rapid upregulation of effector functions including cytolysis upon stimulation. T cells profoundly change their cellular metabolism during their maturation and activation. We sought to determine how MAIT cell metabolism may facilitate both the long-term memory phase in tissue and the transition to rapid effector function. Here, we show, by flow cytometric metabolism assays and extracellular flux analysis that, despite an effector-memory profile, human MAIT cells are metabolically quiescent in a resting state comparable to naïve and central memory T cells. Upon stimulation, they rapidly increase uptake of glucose and show a concomitant upregulation of the effector molecules notably granzyme B, which is impaired by inhibition of glycolysis with 2-deoxyglucose. These findings suggest that MAIT cells share some metabolic characteristics of both resting and effector T cell subsets, with a rapid transition upon triggering. Metabolic programming of this cell type may be of interest in understanding and modulating their function in infectious diseases and cancer.

A novel murine cytomegalovirus vaccine vector protects against Mycobacterium tuberculosis.

Tuberculosis remains a global health problem so that a more effective vaccine than bacillus Calmette-Guérin is urgently needed. Cytomegaloviruses persist lifelong in vivo and induce powerful immune and increasing ("inflationary") responses, making them attractive vaccine vectors. We have used an m1-m16-deleted recombinant murine CMV (MCMV) expressing Mycobacterium tuberculosis Ag 85A to show that infection of mice with this recombinant significantly reduces the mycobacterial load after challenge with M. tuberculosis, whereas control empty virus has a lesser effect. Both viruses induce immune responses to H-2(d)-restricted epitopes of MCMV pp89 and M18 Ags characteristic of infection with other MCMVs. A low frequency of 85A-specific memory cells could be revealed by in vivo or in vitro boosting or after challenge with M. tuberculosis. Kinetic analysis of M. tuberculosis growth in the lungs of CMV-infected mice shows early inhibition of M. tuberculosis growth abolished by treatment with NK-depleting anti-asialo ganglio-N-tetraosylceramide Ab. Microarray analysis of the lungs of naive and CMV-infected mice shows increased IL-21 mRNA in infected mice, whereas in vitro NK assays indicate increased levels of NK activity. These data indicate that activation of NK cells by MCMV provides early nonspecific protection against M. tuberculosis, potentiated by a weak 85A-specific T cell response, and they reinforce the view that the innate immune system plays an important role in both natural and vaccine-induced protection against M. tuberculosis.

Unfixed endogenous retroviral insertions in the human population.

UNLABELLED: One lineage of human endogenous retroviruses (HERVs), HERV-K(HML2), is upregulated in many cancers, some autoimmune/inflammatory diseases, and HIV-infected cells. Despite 3 decades of research, it is not known if these viruses play a causal role in disease, and there has been recent interest in whether they can be used as immunotherapy targets. Resolution of both these questions will be helped by an ability to distinguish between the effects of different integrated copies of the virus (loci). Research so far has concentrated on the 20 or so recently integrated loci that, with one exception, are in the human reference genome sequence. However, this viral lineage has been copying in the human population within the last million years, so some loci will inevitably be present in the human population but absent from the reference sequence. We therefore performed the first detailed search for such loci by mining whole-genome sequences generated by next-generation sequencing. We found a total of 17 loci, and the frequency of their presence ranged from only 2 of the 358 individuals examined to over 95% of them. On average, each individual had six loci that are not in the human reference genome sequence. Comparing the number of loci that we found to an expectation derived from a neutral population genetic model suggests that the lineage was copying until at least ∼250,000 years ago. IMPORTANCE: About 5% of the human genome sequence is composed of the remains of retroviruses that over millions of years have integrated into the chromosomes of egg and/or sperm precursor cells. There are indications that protein expression of these viruses is higher in some diseases, and we need to know (i) whether these viruses have a role in causing disease and (ii) whether they can be used as immunotherapy targets in some of them. Answering both questions requires a better understanding of how individuals differ in the viruses that they carry. We carried out the first careful search for new viruses in some of the many human genome sequences that are now available thanks to advances in sequencing technology. We also compared the number that we found to a theoretical expectation to see if it is likely that these viruses are still replicating in the human population today.

GATA1-mutant clones are frequent and often unsuspected in babies with Down syndrome: identification of a population at risk of leukemia.

Transient abnormal myelopoiesis (TAM), a preleukemic disorder unique to neonates with Down syndrome (DS), may transform to childhood acute myeloid leukemia (ML-DS). Acquired GATA1 mutations are present in both TAM and ML-DS. Current definitions of TAM specify neither the percentage of blasts nor the role of GATA1 mutation analysis. To define TAM, we prospectively analyzed clinical findings, blood counts and smears, and GATA1 mutation status in 200 DS neonates. All DS neonates had multiple blood count and smear abnormalities. Surprisingly, 195 of 200 (97.5%) had circulating blasts. GATA1 mutations were detected by Sanger sequencing/denaturing high performance liquid chromatography (Ss/DHPLC) in 17 of 200 (8.5%), all with blasts >10%. Furthermore low-abundance GATA1 mutant clones were detected by targeted next-generation resequencing (NGS) in 18 of 88 (20.4%; sensitivity ∼0.3%) DS neonates without Ss/DHPLC-detectable GATA1 mutations. No clinical or hematologic features distinguished these 18 neonates. We suggest the term "silent TAM" for neonates with DS with GATA1 mutations detectable only by NGS. To identify all babies at risk of ML-DS, we suggest GATA1 mutation and blood count and smear analyses should be performed in DS neonates. Ss/DPHLC can be used for initial screening, but where GATA1 mutations are undetectable by Ss/DHPLC, NGS-based methods can identify neonates with small GATA1 mutant clones.

Dysregulated immunologic landscape of the early host response in melioidosis.

Melioidosis, a neglected tropical infection caused by Burkholderia pseudomallei, commonly presents as pneumonia or sepsis with mortality rates up to 50% despite appropriate treatment. A better understanding of the early host immune response to melioidosis may lead to new therapeutic interventions and prognostication strategies to reduce disease burden. Whole blood transcriptomic signatures in 164 patients with melioidosis and in 70 patients with other infections hospitalized in northeastern Thailand enrolled within 24 hours following hospital admission were studied. Key findings were validated in an independent melioidosis cohort. Melioidosis was characterized by upregulation of interferon (IFN) signaling responses compared with other infections. Mortality in melioidosis was associated with excessive inflammation, enrichment of type 2 immune responses, and a dramatic decrease in T cell-mediated immunity compared with survivors. We identified and independently confirmed a 5-gene predictive set classifying fatal melioidosis (validation cohort area under the receiver operating characteristic curve 0.83; 95% CI, 0.67-0.99). This study highlights the intricate balance between innate and adaptive immunity during fatal melioidosis and can inform future precision medicine strategies for targeted therapies and prognostication in this severe infection.

Differentiation and activation of equine monocyte-derived dendritic cells are not correlated with CD206 or CD83 expression.

Dendritic cells (DC) are the main immune mediators inducing primary immune responses. DC generated from monocytes (MoDC) are a model system to study the biology of DC in vitro, as they represent inflammatory DC in vivo. Previous studies on the generation of MoDC in horses indicated that there was no distinct difference between immature and mature DC and that the expression profile was distinctly different from humans, where CD206 is expressed on immature MoDC whereas CD83 is expressed on mature MoDC. Here we describe the kinetics of equine MoDC differentiation and activation, analysing both phenotypic and functional characteristics. Blood monocytes were first differentiated with equine granulocyte-macrophage colony-stimulating factor and interleukin-4 generating immature DC (iMoDC). These cells were further activated with a cocktail of cytokines including interferon-γ) but not CD40 ligand to obtain mature DC (mMoDC). To determine the expression of a broad range of markers for which no monoclonal antibodies were available to analyse the protein expression, microarray and quantitative PCR analysis were performed to carry out gene expression analysis. This study demonstrates that equine iMoDC and mMoDC can be distinguished both phenotypically and functionally but the expression pattern of some markers including CD206 and CD83 is dissimilar to the human system.

Analysis of GATA1 mutations in Down syndrome transient myeloproliferative disorder and myeloid leukemia.

Children with Down syndrome (DS) up to the age of 4 years are at a 150-fold excess risk of developing myeloid leukemia (ML-DS). Approximately 4%-5% of newborns with DS develop transient myeloproliferative disorder (TMD). Blast cell structure and immunophenotype are similar in TMD and ML-DS. A mutation in the hematopoietic transcription factor GATA1 is present in almost all cases. Here, we show that simple techniques detect GATA1 mutations in the largest series of TMD (n = 134; 88%) and ML-DS (n = 103; 85%) cases tested. Furthermore, no significant difference in the mutational spectrum between the 2 disorders was seen. Thus, the type of GATA1 sequence mutation is not a reliable tool and is not prognostic of which patients with TMD are probable to develop ML-DS.

A conserved population of MHC II-restricted, innate-like, commensal-reactive T cells in the gut of humans and mice.

Interactions with commensal microbes shape host immunity on multiple levels and play a pivotal role in human health and disease. Tissue-dwelling, antigen-specific T cells are poised to respond to local insults, making their phenotype important in the relationship between host and microbes. Here we show that MHC-II restricted, commensal-reactive T cells in the colon of both humans and mice acquire transcriptional and functional characteristics associated with innate-like T cells. This cell population is abundant and conserved in the human and murine colon and endowed with polyfunctional effector properties spanning classic Th1- and Th17-cytokines, cytotoxic molecules, and regulators of epithelial homeostasis. T cells with this phenotype are increased in ulcerative colitis patients, and their presence aggravates pathology in dextran sodium sulphate-treated mice, pointing towards a pathogenic role in colitis. Our findings add to the expanding spectrum of innate-like immune cells positioned at the frontline of intestinal immune surveillance, capable of acting as sentinels of microbes and the local cytokine milieu.

Cardiac iron overload in transfusion-dependent patients with myelodysplastic syndromes.

Transfusion-dependent myelodysplastic (MDS) patients are prone to iron overload. We evaluated 43 transfused MDS patients with T2* magnetic resonance imaging scans. 81% had liver and 16·8% cardiac iron overload. Liver R2* (1000/T2*), but not cardiac R2*, was correlated with number of units transfused (r=0·72, P<0·0001) and ferritin (r=0·53, P<0·0001). The area under the curve of a time-ferritin plot was found to be much greater in patients with cardiac iron loading (median 53·7x10(5) Megaunits vs. 12·2x10(5) Megaunits, P=0·002). HFE, HFE2, HAMP or SLC40A1 genotypes were not predictors of iron overload in these patients.

Author Correction: Hypoxic gene expression in chronic hepatitis B virus infected patients is not observed in state-of-the-art in vitro and mouse infection models.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Hypoxic gene expression in chronic hepatitis B virus infected patients is not observed in state-of-the-art in vitro and mouse infection models.

Hepatitis B virus (HBV) is the leading cause of hepatocellular carcinoma (HCC) worldwide. The prolyl hydroxylase domain (PHD)-hypoxia inducible factor (HIF) pathway is a key mammalian oxygen sensing pathway and is frequently perturbed by pathological states including infection and inflammation. We discovered a significant upregulation of hypoxia regulated gene transcripts in patients with chronic hepatitis B (CHB) in the absence of liver cirrhosis. We used state-of-the-art in vitro and in vivo HBV infection models to evaluate a role for HBV infection and the viral regulatory protein HBx to drive HIF-signalling. HBx had no significant impact on HIF expression or associated transcriptional activity under normoxic or hypoxic conditions. Furthermore, we found no evidence of hypoxia gene expression in HBV de novo infection, HBV infected human liver chimeric mice or transgenic mice with integrated HBV genome. Collectively, our data show clear evidence of hypoxia gene induction in CHB that is not recapitulated in existing models for acute HBV infection, suggesting a role for inflammatory mediators in promoting hypoxia gene expression.

Inflation vs. Exhaustion of Antiviral CD8+ T-Cell Populations in Persistent Infections: Two Sides of the Same Coin?

Persistent virus infection can drive CD8+ T-cell responses which are markedly divergent in terms of frequency, phenotype, function, and distribution. On the one hand viruses such as Lymphocytic Choriomeningitis Virus (LCMV) Clone 13 can drive T-cell "exhaustion", associated with upregulation of checkpoint molecules, loss of effector functions, and diminished control of viral replication. On the other, low-level persistence of viruses such as Cytomegalovirus and Adenoviral vaccines can drive memory "inflation," associated with sustained populations of CD8+ T-cells over time, with maintained effector functions and a distinct phenotype. Underpinning these divergent memory pools are distinct transcriptional patterns-we aimed to compare these to explore the regulation of CD8+ T-cell memory against persistent viruses at the level of molecular networks and address whether dysregulation of specific modules may account for the phenotype observed. By exploring in parallel and also merging existing datasets derived from different investigators we attempted to develop a combined model of inflation vs. exhaustion and investigate the gene expression networks that are shared in these memory pools. In such comparisons, co-ordination of a critical module of genes driven by Tbx21 is markedly different between the two memory types. These exploratory data highlight both the molecular similarities as well as the differences between inflation and exhaustion and we hypothesize that co-ordinated regulation of a key genetic module may underpin the markedly different resultant functions and phenotypes in vivo-an idea which could be tested directly in future experiments.

Single-cell transcriptome analysis of CD8 + T-cell memory inflation.

Background: Persistent viruses such as murine cytomegalovirus (MCMV) and adenovirus-based vaccines induce strong, sustained CD8 + T-cell responses, described as memory "inflation". These retain functionality, home to peripheral organs and are associated with a distinct transcriptional program. Methods: To further define the nature of the transcriptional mechanisms underpinning memory inflation at different sites we used single-cell RNA sequencing of tetramer-sorted cells from MCMV-infected mice, analyzing transcriptional networks in virus-specific populations in the spleen and gut intra-epithelial lymphocytes (IEL). Results: We provide a transcriptional map of T-cell memory and define a module of gene expression, which distinguishes memory inflation in spleen from resident memory T-cells (T RM) in the gut. Conclusions: These data indicate that CD8 + T-cell memory in the gut epithelium induced by persistent viruses and vaccines has a distinct quality from both conventional memory and "inflationary" memory which may be relevant to protection against mucosal infections.