Differentiation and Protective Capacity of Virus-Specific CD8+ T Cells Suggest Murine Norovirus Persistence in an Immune-Privileged Enteric Niche.

Noroviruses can establish chronic infections with active viral shedding in healthy humans but whether persistence is associated with adaptive immune dysfunction is unknown. We used genetically engineered strains of mouse norovirus (MNV) to investigate CD8+ T cell differentiation during chronic infection. We found that chronic infection drove MNV-specific tissue-resident memory (Trm) CD8+ T cells to a differentiation state resembling inflationary effector responses against latent cytomegalovirus with only limited evidence of exhaustion. These MNV-specific Trm cells remained highly functional yet appeared ignorant of ongoing viral replication. Pre-existing MNV-specific Trm cells provided partial protection against chronic infection but largely ceased to detect virus within 72 hours of challenge, demonstrating rapid sequestration of viral replication away from T cells. Our studies revealed a strategy of immune evasion by MNV via the induction of a CD8+ T cell program normally reserved for latent pathogens and persistence in an immune-privileged enteric niche.

Kinetically distinct processing pathways diversify the CD8+ T cell response to a single viral epitope.

The source proteins from which CD8+ T cell-activating peptides are derived remain enigmatic. Glycoproteins are particularly challenging in this regard owing to several potential trafficking routes within the cell. By engineering a glycoprotein-derived epitope to contain an N-linked glycosylation site, we determined that optimal CD8+ T cell expansion and function were induced by the peptides that are rapidly produced from the exceedingly minor fraction of protein mislocalized to the cytosol. In contrast, peptides derived from the much larger fraction that undergoes translocation and quality control are produced with delayed kinetics and induce suboptimal CD8+ T cell responses. This dual system of peptide generation enhances CD8+ T cell participation in diversifying both antigenicity and the kinetics of peptide display.

Differential localization of T-bet and Eomes in CD8 T cell memory populations.

In mice, two T-box transcription factors, T-box expressed in T cells (T-bet) and eomesodermin (Eomes), drive the differentiation of CD8 T cell lineages; however, little is known regarding their role in human CD8 T cell differentiation. In this study, we characterized T-bet and Eomes expression and localization within human CD8 memory T cell populations. We find that T-bet and Eomes are broadly expressed in human memory CD8 T cells, with increasing levels of T-bet and Eomes strongly correlating with differentiation from central memory to effector memory and effector subpopulations. In resting T cells, T-bet levels directly correlate to subcellular localization, with a higher propensity for nuclear expression of T-bet within T-bet(hi) cells and predominantly cytoplasmic expression in T-bet(lo) cells. In addition, Eomes is also localized to either the nucleus or the cytoplasm. Upon TCR stimulation, the percentage of T cells that express T-bet dramatically increases, whereas the percentage of cells expressing Eomes remains largely unchanged across all memory populations. Of interest, T-bet, but not Eomes, relocalizes to the nucleus in the majority of cells across all populations within 24 h post stimulation. These data indicate that T-bet and Eomes are likely regulated at the level of subcellular localization, potentially via different mechanisms. Together, these findings suggest a novel model for CD8 T cell differentiation in humans that is based on the localization of T-bet and Eomes.

Increased HIV-specific CD8+ T-cell cytotoxic potential in HIV elite controllers is associated with T-bet expression.

Recent data suggest that CD8+ T-cell effector activity is an important component in the control of HIV replication in elite controllers (ECs). One critical element of CD8+ T-cell effector function and differentiation is the T-box transcription factor T-bet. In the present study, we assessed T-bet expression, together with the effector proteins perforin, granzyme A (Grz A), granzyme B (Grz B), and granulysin, in HIV-specific CD8+ T cells from ECs (n = 20), chronically infected progressors (CPs; n = 18), and highly active antiretroviral therapy (HAART)-suppressed individuals (n = 19). Compared with the other cohort groups, HIV-specific CD8+ T cells among ECs demonstrated a superior ability to express perforin and Grz B, but with no detectable difference in the levels of Grz A or granulysin. We also observed higher levels of T-bet in HIV-specific CD8+ T cells from ECs, with an ensuing positive correlation between T-bet and levels of both perforin and Grz B. Moreover, HIV-specific CD8+ T cells in ECs up-regulated T-bet to a greater extent than CPs after in vitro expansion, with concomitant up-regulation of perforin and Grz B. These results suggest that T-bet may play an important role in driving effector function, and its modulation may lead to enhanced effector activity against HIV.

Full thickness genital burns independently increase the odds of death among pediatric burn patients.

BACKGROUND: There are limited published data characterizing pediatric burn patients with genital burns (GB). OBJECTIVE: Assess prevalence of GB in pediatric burn patients and analyze clinical characteristics including predictors of mortality. STUDY DESIGN: We queried American Burn Association's National Burn Repository to identify all pediatric burn patients who presented to North American burn centers over a 10-year period. We excluded all patients aged ≥18 years and patients with unknown sex, race, and/or mortality. We also excluded subsequent encounters for patients with multiple visits. Demographic and clinical characteristics were compared between patients with and without GB. Univariable and multivariable logistic regression analyses were performed to identify predictors of mortality. RESULTS: Among 38 211 pediatric burn patients, 1244 (3.3%) suffered from second- or third-degree GB. Patients who suffered from third-degree GB (GB3) were significantly older than patients who suffered from second-degree GB (GB2) or patients without GB. Of the patients, 32.3% were aged 0-2 years. Scalding was the most common mechanism of injury for pediatric GB patients at 73.8%. Compared to non-GB patients, GB patients had significantly higher total body surface area (TBSA) burned (16.5% vs 7.0%), higher rates of associated inhalation injury (4.1% vs 2.6%), longer length of stay (LOS) (14.3 days vs 6.7 days), higher rates of urinary tract infection (UTI) (13.0% vs 2.8%) and sepsis (14.1% vs 2.3%), and higher mortality (3.5% vs 0.7%) (P < 0.0001 for all). The differences were more pronounced for the subset of patients who suffered from GB3 (TBSA 43.5%, associated inhalation injury 19.9%, LOS 42.9 days, 21.3% UTI, 33.3% sepsis, and 19.3% mortality). On multivariable analysis, the presence of GB3, TBSA, non-white ethnicity, and the presence of associated inhalation injury were significant predictors of mortality. Only 4.5% of pediatric GB patients underwent genital surgery, with the majority consisting of excision, reconstruction, or repair of the penis, vulva, or perineum. No patient required orchiectomy or suprapubic catheter placement. DISCUSSION: This is the largest study to date of pediatric GB patients. A minority of pediatric burn patients present with GB. However, when they occur, GB are associated with significantly worse clinical outcomes. Importantly, the presence of GB3 is an independent predictor of mortality in pediatric burn patients. CONCLUSION: The presence of GB appears to be a strong marker of severe burn injury. Pediatric GB patients need to be carefully assessed and aggressively managed for additional injuries, complications, surgical needs, and mortality risk.

Impact of epitope density on CD8+ T cell development and function.

Effective immune responses against intracellular pathogens and tumors frequently rely upon CD8+ cytotoxic T lymphocytes (CTLs). In turn, CTL detection of foreign material from viruses and bacteria depends on antigen presentation by the MHC class I pathway. The underpinnings of antigen processing and presentation and, subsequent T cell activation and immunological memory development, have been extensively studied, leading to a better understanding of the balance between antigen dose, context, and, the T cell activation threshold. Still, the complexity of this process leads to apparent contradictions that hinder construction of rational strategies for generating optimal CD8 + T cell responses in a variety of settings. In this review we consolidate the current knowledge around the effects of peptide MHC I complex (pMHC) density and kinetics on CD8 + T cell responses and function during the acute phase of an infection.

Corrigendum: Characterization of T-bet and Eomes in Peripheral Human Immune Cells.

[This corrects the article on p. 217 in vol. 5, PMID: 24860576.].

Targeting the mRNA-binding protein HuR impairs malignant characteristics of pancreatic ductal adenocarcinoma cells.

Post-transcriptional regulation is a powerful mediator of gene expression, and can rapidly alter the expression of numerous transcripts involved in tumorigenesis. We have previously shown that the mRNA-binding protein HuR (ELAVL1) is elevated in human pancreatic ductal adenocarcinoma (PDA) specimens compared to normal pancreatic tissues, and its cytoplasmic localization is associated with increased tumor stage. To gain a better insight into HuR's role in PDA biology and to assess it as a candidate therapeutic target, we altered HuR expression in PDA cell lines and characterized the resulting phenotype in preclinical models. HuR silencing by short hairpin and small interfering RNAs significantly decreased cell proliferation and anchorage-independent growth, as well as impaired migration and invasion. In comparison, HuR overexpression increased migration and invasion, but had no significant effects on cell proliferation and anchorage-independent growth. Importantly, two distinct targeted approaches to HuR silencing showed marked impairment in tumor growth in mouse xenografts. NanoString nCounter® analyses demonstrated that HuR regulates core biological processes, highlighting that HuR inhibition likely thwarts PDA viability through post-transcriptional regulation of diverse signaling pathways (e.g. cell cycle, apoptosis, DNA repair). Taken together, our study suggests that targeted inhibition of HuR may be a novel, promising approach to the treatment of PDA.

Characterization of T-bet and eomes in peripheral human immune cells.

The T-box transcription factors T-bet and Eomesodermin (Eomes) have been well defined as key drivers of immune cell development and cytolytic function. While the majority of studies have defined the roles of these factors in the context of murine T-cells, recent results have revealed that T-bet, and possibly Eomes, are expressed in other immune cell subsets. To date, the expression patterns of these factors in subsets of human peripheral blood mononuclear cells beyond T-cells remain relatively uncharacterized. In this study, we used multiparametric flow cytometry to characterize T-bet and Eomes expression in major human blood cell subsets, including total CD4(+) and CD8(+) T-cells, γδ T-cells, invariant NKT cells, natural killer cells, B-cells, and dendritic cells. Our studies identified novel cell subsets that express T-bet and Eomes and raise implications for their possible functions in the context of other human immune cell subsets besides their well-known roles in T-cells.