Dynamics of Tissue-Specific CD8+ T Cell Responses during West Nile Virus Infection.

The mouse model of West Nile virus (WNV), which is a leading cause of mosquito-borne encephalitis worldwide, has provided fundamental insights into the host and viral factors that regulate viral pathogenesis and infection outcome. In particular, CD8+ T cells are critical for controlling WNV replication and promoting protection against infection. Here, we present the characterization of a T cell receptor (TCR)-transgenic mouse with specificity for the immunodominant epitope in the WNV NS4B protein (here referred to as transgenic WNV-I mice). Using an adoptive-transfer model, we found that WNV-I CD8+ T cells behave similarly to endogenous CD8+ T cell responses, with an expansion phase in the periphery beginning around day 7 postinfection (p.i.) followed by a contraction phase through day 15 p.i. Through the use of in vivo intravascular immune cell staining, we determined the kinetics, expansion, and differentiation into effector and memory subsets of WNV-I CD8+ T cells within the spleen and brain. We found that red-pulp WNV-I CD8+ T cells were more effector-like than white-pulp WNV-I CD8+ T cells, which displayed increased differentiation into memory precursor cells. Within the central nervous system (CNS), we found that WNV-I CD8+ T cells were polyfunctional (gamma interferon [IFN-γ] and tumor necrosis factor alpha [TNF-α]), displayed tissue-resident characteristics (CD69+ and CD103+), persisted in the brain through day 15 p.i., and reduced the viral burden within the brain. The use of these TCR-transgenic WNV-I mice provides a new resource to dissect the immunological mechanisms of CD8+ T cell-mediated protection during WNV infection.IMPORTANCE West Nile Virus (WNV) is the leading cause of mosquito-borne encephalitis worldwide. There are currently no approved therapeutics or vaccines for use in humans to treat or prevent WNV infection. CD8+ T cells are critical for controlling WNV replication and protecting against infection. Here, we present a comprehensive characterization of a novel TCR-transgenic mouse with specificity for the immunodominant epitope in the WNV NS4B protein. In this study, we determine the kinetics, proliferation, differentiation into effector and memory subsets, homing, and clearance of WNV in the CNS. Our findings provide a new resource to dissect the immunological mechanisms of CD8+ T cell-mediated protection during WNV infection.

Local Inflammatory Cues Regulate Differentiation and Persistence of CD8+ Tissue-Resident Memory T Cells.

Many pathogens initiate infection at mucosal surfaces, and tissue-resident memory T (Trm) cells play an important role in protective immunity, yet the tissue-specific signals that regulate Trm differentiation are poorly defined. During Yersinia infection, CD8+ T cell recruitment to areas of inflammation within the intestine is required for differentiation of the CD103-CD69+ Trm subset. Intestinal proinflammatory microenvironments have elevated interferon (IFN)-ß and interleukin-12 (IL-12), which regulated Trm markers, including CD103. Type I interferon-receptor- or IL-12-receptor-deficient T cells functioned similarly to wild-type (WT) cells during infection; however, the inability of T cells to respond to inflammation resulted in defective differentiation of CD103-CD69+ Trm cells and reduced Trm persistence. Intestinal macrophages were the main producers of IFN-ß and IL-12 during infection, and deletion of CCR2+ IL-12-producing cells reduced the size of the CD103- Trm population. These data indicate that intestinal inflammation drives phenotypic diversity and abundance of Trm cells for optimal tissue-specific immunity.

Cutting Edge: Caspase-11 Limits the Response of CD8+ T Cells to Low-Abundance and Low-Affinity Antigens.

Inflammatory caspases, including caspase-11, are upregulated in CD8(+) T cells after Ag-specific activation, but little is known about their function in T cells. We report that caspase-11-deficient (Casp11(-/-)) T cells proliferated more readily in response to low-affinity and low-abundance ligands both in vitro and in vivo due to an increased ability to signal through the TCR. In addition to increased numbers, Casp11(-/-) T cells had enhanced effector function compared with wild-type cells, including increased production of IL-2 and reduced expression of CD62L. Casp11(-/-) T cells specific for endogenous Ags were more readily deleted than wild-type cells. These data indicate that caspase-11 negatively regulates TCR signaling, possibly through its ability to regulate actin polymerization, and inhibiting its activity could enhance the expansion and function of low-affinity T cells.

Proinflammatory microenvironments within the intestine regulate the differentiation of tissue-resident CD8⁺ T cells responding to infection.

We report that oral infection with Yersinia pseudotuberculosis results in the development of two distinct populations of pathogen-specific CD8(+) tissue-resident memory T cells (TRM cells) in the lamina propria. CD103(-) T cells did not require transforming growth factor-ß (TGF-ß) signaling but were true resident memory cells. Unlike CD103(+)CD8(+) T cells, which were TGF-ß dependent and were scattered in the tissue, CD103(-)CD8(+) T cells clustered with CD4(+) T cells and CX3CR1(+) macrophages and/or dendritic cells around areas of bacterial infection. CXCR3-dependent recruitment of cells to inflamed areas was critical for development of the CD103(-) population and pathogen clearance. Our studies have identified the 'preferential' development of CD103(-) TRM cells in inflammatory microenvironments within the lamina propria and suggest that this subset has a critical role in controlling infection.

Dermal-resident versus recruited γδ T cell response to cutaneous vaccinia virus infection.

The study of T cell immunity at barrier surfaces has largely focused on T cells bearing the αß TCR. However, T cells that express the γδ TCR are disproportionately represented in peripheral tissues of mice and humans, suggesting they too may play an important role responding to external stimuli. In this article, we report that, in a murine model of cutaneous infection with vaccinia virus, dermal γδ T cell numbers increased 10-fold in the infected ear and resulted in a novel γδ T cell population not found in naive skin. Circulating γδ T cells were specifically recruited to the site of inflammation and differentially contributed to dermal populations based on their CD27 expression. Recruited γδ T cells, the majority of which were CD27(+), were granzyme B(+) and made up about half of the dermal population at the peak of the response. In contrast, recruited and resident γδ T cell populations that made IL-17 were CD27(-). Using a double-chimera model that can discriminate between the resident dermal and recruited γδ T cell populations, we demonstrated their divergent functions and contributions to early stages of tissue inflammation. Specifically, the loss of the perinatal thymus-derived resident dermal population resulted in decreased cellularity and collateral damage in the tissue during viral infection. These findings have important implications for our understanding of immune coordination at barrier surfaces and the contribution of innate-like lymphocytes on the front lines of immune defense.

Approaches for the design of reduced toxicant emission cigarettes.

Cigarette smoking causes serious diseases through frequent and prolonged exposure to toxicants. Technologies are being developed to reduce smokers' toxicant exposure, including filter adsorbents, tobacco treatments and substitutes. This study examined the effect of modifications to filter ventilation, variations in cigarette circumference and active charcoal filter length and loading, as well as combinations of these features in a reduced-toxicant prototype (RTP) cigarette, on the yields of toxicants in cigarette smoke. An air-dilution mechanism, called split-tipping, was developed in which a band of porous paper in the centre of the filter tipping functions to minimise the loss of effective filter ventilation that occurs at the high flow rates encountered during human-smoking, and to facilitate the diffusional loss of volatile toxicants. As compared with conventional filter ventilation cigarettes, split-tipping reduced tar and volatile smoke constituent emissions under high flow rate machine-smoking conditions, most notably for products with a 1-mg ISO tar yield. Furthermore, mouth level exposure (MLE) to tar and nicotine was reduced among smokers of 1-mg ISO tar cigarettes in comparison to smokers of cigarettes with traditional filter ventilation. For higher ISO tar level cigarettes, however, there were no significant reductions in MLE. Smaller cigarette circumferences reduced sidestream toxicant yields and modified the balance of mainstream smoke chemistry with reduced levels of aromatic amines and benzo[a]pyrene but increased yields of formaldehyde. Smaller circumference cigarettes also had lower mainstream yields of volatile toxicants. Longer cigarette filters containing increased levels of high-activity carbon (HAC) showed reduced machine-smoking yields of volatile toxicants: with up to 97% removal for some volatile toxicants at higher HAC loadings. Split-tipping was combined with optimal filter length and cigarette circumference in an RTP cigarette that gave significantly lower mainstream (up to ~90%) and sidestream (predominately 20%-60%) smoke yields of numerous toxicants as compared with a commercial comparator cigarette under machine-smoking conditions. Significantly lower mainstream and sidestream smoke toxicant yields were observed for an RTP cigarette comprising several toxicant reducing technologies; these observations warrant further evaluation in clinical studies where real-world relevance can be tested using biomarkers of exposure and physiological effect.

Metabolic regulator Fnip1 is crucial for iNKT lymphocyte development.

Folliculin-interacting protein 1 (Fnip1) is an adaptor protein that physically interacts with AMPK, an energy-sensing kinase that stimulates mitochondrial biogenesis and autophagy in response to low ATP, while turning off energy consumption mediated by mammalian target of rapamycin. Previous studies with Fnip1-null mice revealed that Fnip1 is essential for pre-B-cell development. Here we report a critical role of Fnip1 in invariant natural killer T (iNKT) cell development. Thymic iNKT development in Fnip1(-/-) mice was arrested at stage 2 (NK1.1(-)CD44(+)) but development of CD4, CD8, γδ T-cell, and NK cell lineages proceeded normally. Enforced expression of a Vα14Jα18 iNKT TCR transgene or loss of the proapoptotic protein Bim did not rescue iNKT cell maturation in Fnip1(-/-) mice. Whereas most known essential transcription factors for iNKT cell development were represented normally, Fnip1(-/-) iNKT cells failed to down-regulate Promyelocytic leukemia zinc finger compared with their WT counterparts. Moreover, Fnip1(-/-) iNKT cells contained hyperactive mTOR and reduced mitochondrial number despite lower ATP levels, resulting in increased sensitivity to apoptosis. These results indicate that Fnip1 is vital for iNKT cell development by maintaining metabolic homeostasis in response to metabolic stress.

A novel T-cell receptor mimic defines dendritic cells that present an immunodominant West Nile virus epitope in mice.

We used a newly generated T-cell receptor mimic monoclonal antibody (TCRm MAb) that recognizes a known nonself immunodominant peptide epitope from West Nile virus (WNV) NS4B protein to investigate epitope presentation after virus infection in C57BL/6 mice. Previous studies suggested that peptides of different length, either SSVWNATTAI (10-mer) or SSVWNATTA (9-mer) in complex with class I MHC antigen H-2D(b) , were immunodominant after WNV infection. Our data establish that both peptides are presented on the cell surface after WNV infection and that CD8(+) T cells can detect 10- and 9-mer length variants similarly. This result varies from the idea that a given T-cell receptor (TCR) prefers a single peptide length bound to its cognate class I MHC. In separate WNV infection studies with the TCRm MAb, we show that in vivo the 10-mer was presented on the surface of uninfected and infected CD8α(+) CD11c(+) dendritic cells, which suggests the use of direct and cross-presentation pathways. In contrast, CD11b(+) CD11c(-) cells bound the TCRm MAb only when they were infected. Our study demonstrates that TCR recognition of peptides is not limited to certain peptide lengths and that TCRm MAbs can be used to dissect the cell-type specific mechanisms of antigen presentation in vivo.

Memory CD8+ T cells exhibit increased antigen threshold requirements for recall proliferation.

A hallmark of immunological memory is the ability of previously primed T cells to undergo rapid recall responses upon antigen reencounter. Classic work has suggested that memory T cells proliferate in response to lower doses of antigen than naive T cells and with reduced requirements for co-stimulation. In contrast to this premise, we observed that naive but not memory T cells proliferate in vivo in response to limited antigen presentation. To reconcile these observations, we tested the antigen threshold requirement for cell cycle entry in naive and central memory CD8(+) T cells. Although both naive and memory T cells detect low dose antigen, only naive T cells activate cell cycle effectors. Direct comparison of TCR signaling on a single cell basis indicated that central memory T cells do not activate Zap70, induce cMyc expression, or degrade p27 in response to antigen levels that activate these functions in naive T cells. The reduced sensitivity of memory T cells may result from both decreased surface TCR expression and increased expression of protein tyrosine phosphatases as compared with naive T cells. Our data describe a novel aspect of memory T cell antigen threshold sensitivity that may critically regulate recall expansion.

Inflammation and TCR signal strength determine the breadth of the T cell response in a bim-dependent manner.

Generating a diverse T cell memory population through vaccination is a promising strategy to overcome pathogen epitope variability and tolerance to tumor Ags. The effector and memory pool becomes broad in TCR diversity by recruiting high- and low-affinity T cells. We wanted to determine which factors dictate whether a memory T cell pool has a broad versus focused repertoire. We find that inflammation increases the magnitude of low- and high-affinity T cell responses equally well, arguing against a synergistic effect of TCR and inflammatory signals on T cell expansion. We dissect the differential effects of TCR signal strength and inflammation and demonstrate that they control effector T cell survival in a bim-dependent manner. Importantly, bim-dependent cell death is overcome with a high Ag dose in the context of an inflammatory environment. Our data define the framework for the generation of a broad T cell memory pool to inform future vaccine design.

CD8 and CD4 T cells in west nile virus immunity and pathogenesis.

CD4 and CD8 T lymphocytes are adaptive immune cells that play a key role in the immune response to pathogens. They have been extensively studied in a variety of model systems and the mechanisms by which they function are well described. However, the responses by these cell types vary widely from pathogen to pathogen. In this review, we will discuss the role of CD8 and CD4 T cells in the immune response to West Nile virus infection.

Transforming growth factor-ß signaling controls the formation and maintenance of gut-resident memory T cells by regulating migration and retention.

Tissue-resident memory T (Trm) cells represent a population of memory CD8⁺ T cells that can act as first responders to local infection. The mechanisms regulating the formation and maintenance of intestinal Trm cells remain elusive. Here we showed that transforming growth factor-ß (TGF-ß) controlled both stages of gut Trm cell differentiation through different mechanisms. During the formation phase of Trm cells, TGF-ß signaling inhibited the migration of effector CD8⁺ T cells from the spleen to the gut by dampening the expression of integrin α4ß7. During the maintenance phase, TGF-ß was required for the retention of intestinal Trm cells at least in part through the induction of integrins αEß7 and α1, as well as CD69. Thus, the cytokine acts to control cytotoxic T cell differentiation in lymphoid and peripheral organs.

A T-cell response to a liver-stage Plasmodium antigen is not boosted by repeated sporozoite immunizations.

Development of an antimalarial subunit vaccine inducing protective cytotoxic T lymphocyte (CTL)-mediated immunity could pave the way for malaria eradication. Experimental immunization with sporozoites induces this type of protective response, but the extremely large number of proteins expressed by Plasmodium parasites has so far prohibited the identification of sufficient discrete T-cell antigens to develop subunit vaccines that produce sterile immunity. Here, using mice singly immunized with Plasmodium yoelii sporozoites and high-throughput screening, we identified a unique CTL response against the parasite ribosomal L3 protein. Unlike CTL responses to the circumsporozoite protein (CSP), the population of L3-specific CTLs was not expanded by multiple sporozoite immunizations. CSP is abundant in the sporozoite itself, whereas L3 expression does not increase until the liver stage. The response induced by a single immunization with sporozoites reduces the parasite load in the liver so greatly during subsequent immunizations that L3-specific responses are only generated during the primary exposure. Functional L3-specific CTLs can, however, be expanded by heterologous prime-boost regimens. Thus, although repeat sporozoite immunization expands responses to preformed antigens like CSP that are present in the sporozoite itself, this immunization strategy may not expand CTLs targeting parasite proteins that are synthesized later. Heterologous strategies may be needed to increase CTL responses across the entire spectrum of Plasmodium liver-stage proteins.

Bystander-activated memory CD8 T cells control early pathogen load in an innate-like, NKG2D-dependent manner.

During an infection the antigen-nonspecific memory CD8 T cell compartment is not simply an inert pool of cells, but becomes activated and cytotoxic. It is unknown how these cells contribute to the clearance of an infection. We measured the strength of T cell receptor (TCR) signals that bystander-activated, cytotoxic CD8 T cells (BA-CTLs) receive in vivo and found evidence of limited TCR signaling. Given this marginal contribution of the TCR, we asked how BA-CTLs identify infected target cells. We show that target cells express NKG2D ligands following bacterial infection and demonstrate that BA-CTLs directly eliminate these target cells in an innate-like, NKG2D-dependent manner. Selective inhibition of BA-CTL-mediated killing led to a significant defect in pathogen clearance. Together, these data suggest an innate role for memory CD8 T cells in the early immune response before the onset of a de novo generated, antigen-specific CD8 T cell response.

Autoreactive T cells bypass negative selection and respond to self-antigen stimulation during infection.

Central and peripheral tolerance prevent autoimmunity by deleting the most aggressive CD8(+) T cells but they spare cells that react weakly to tissue-restricted antigen (TRA). To reveal the functional characteristics of these spared cells, we generated a transgenic mouse expressing the TCR of a TRA-specific T cell that had escaped negative selection. Interestingly, the isolated TCR matches the affinity/avidity threshold for negatively selecting T cells, and when developing transgenic cells are exposed to their TRA in the thymus, only a fraction of them are eliminated but significant numbers enter the periphery. In contrast to high avidity cells, low avidity T cells persist in the antigen-positive periphery with no signs of anergy, unresponsiveness, or prior activation. Upon activation during an infection they cause autoimmunity and form memory cells. Unexpectedly, peptide ligands that are weaker in stimulating the transgenic T cells than the thymic threshold ligand also induce profound activation in the periphery. Thus, the peripheral T cell activation threshold during an infection is below that of negative selection for TRA. These results demonstrate the existence of a level of self-reactivity to TRA to which the thymus confers no protection and illustrate that organ damage can occur without genetic predisposition to autoimmunity.

The molecular signature of tissue resident memory CD8 T cells isolated from the brain.

Tissue resident memory (Trm) CD8 T cells represent a newly described memory T cell population. We have previously characterized a population of Trm cells that persists within the brain after acute virus infection. Although capable of providing marked protection against a subsequent local challenge, brain Trm cells do not undergo recall expansion after dissociation from the tissue. Furthermore, these Trm cells do not depend on the same survival factors as the circulating memory T cell pool as assessed either in vivo or in vitro. To gain greater insight into this population of cells, we compared the gene expression profiles of Trm cells isolated from the brain with those of circulating memory T cells isolated from the spleen after an acute virus infection. Trm cells displayed altered expression of genes involved in chemotaxis, expressed a distinct set of transcription factors, and overexpressed several inhibitory receptors. Cumulatively, these data indicate that Trm cells are a distinct memory T cell population disconnected from the circulating memory T cell pool and display a unique molecular signature that likely results in optimal survival and function within their local environment.

The RIG-I-like receptor LGP2 controls CD8(+) T cell survival and fitness.

The RIG-I-like receptors (RLRs) signal innate immune defenses upon RNA virus infection, but their roles in adaptive immunity have not been clearly defined. Here, we showed that the RLR LGP2 was not essential for induction of innate immune defenses, but rather was required for controlling antigen-specific CD8(+) T cell survival and fitness during peripheral T cell-number expansion in response to virus infection. Adoptive transfer and biochemical studies demonstrated that T cell-receptor signaling induced LGP2 expression wherein LGP2 operated to regulate death-receptor signaling and imparted sensitivity to CD95-mediated cell death. Thus, LGP2 promotes an essential prosurvival signal in response to antigen stimulation to confer CD8(+) T cell-number expansion and effector functions against divergent RNA viruses, including West Nile virus and lymphocytic choriomeningitis virus.

TGF-ß signaling to T cells inhibits autoimmunity during lymphopenia-driven proliferation.

T cell-specific deletion of the receptor for transforming growth factor-ß (TGF-ß) mediated by Cre recombinase expressed early in T cell development leads to early-onset lethal autoimmune disease that cannot be controlled by regulatory T cells. However, when we deleted that receptor through the use of Cre driven by a promoter that is active much later in T cell development, adult mice in which most peripheral CD4(+) or CD8(+) T cells lacked the receptor for TGF-ß showed no signs of autoimmunity. Because of their enhanced responses to weak stimulation of the T cell antigen receptor, when transferred into lymphopenic recipients, naive TGF-ß-unresponsive T cells underwent much more proliferation and differentiation into effector cells and induced lymphoproliferative disease. We propose that TGF-ß signaling controls the self-reactivity of peripheral T cells but that in the absence of TGF-ß signals, an added trigger such as lymphopenia is needed to drive overt autoimmune disease.

TCR signaling requirements for activating T cells and for generating memory.

Over the last two decades the molecular and cellular mechanisms underlying T cell activation, expansion, differentiation, and memory formation have been intensively investigated. These studies revealed that the generation of memory T cells is critically impacted by a number of factors, including the magnitude of the inflammatory response and cytokine production, the type of dendritic cell [DC] that presents the pathogen derived antigen, their maturation status, and the concomitant provision of costimulation. Nevertheless, the primary stimulus leading to T cell activation is generated through the T cell receptor [TCR] following its engagement with a peptide MHC ligand [pMHC]. The purpose of this review is to highlight classical and recent findings on how antigen recognition, the degree of TCR stimulation, and intracellular signal transduction pathways impact the formation of effector and memory T cells.

Dissociating markers of senescence and protective ability in memory T cells.

No unique transcription factor or biomarker has been identified to reliably distinguish effector from memory T cells. Instead a set of surface markers including IL-7Rα and KLRG1 is commonly used to predict the potential of CD8 effector T cells to differentiate into memory cells. Similarly, these surface markers together with the tumor necrosis factor family member CD27 are frequently used to predict a memory T cell's ability to mount a recall response. Expression of these markers changes every time a memory cell is stimulated and repeated stimulation can lead to T cell senescence and loss of memory T cell responsiveness. This is a concern for prime-boost vaccine strategies which repeatedly stimulate T cells with the aim of increasing memory T cell frequency. The molecular cues that cause senescence are still unknown, but cell division history is likely to play a major role. We sought to dissect the roles of inflammation and cell division history in developing T cell senescence and their impact on the expression pattern of commonly used markers of senescence. We developed a system that allows priming of CD8 T cells with minimal inflammation and without acquisition of maximal effector function, such as granzyme expression, but a cell division history similar to priming with systemic inflammation. Memory cells derived from minimal effector T cells are fully functional upon rechallenge, have full access to non-lymphoid tissue and appear to be less senescent by phenotype upon rechallenge. However, we report here that these currently used biomarkers to measure senescence do not predict proliferative potential or protective ability, but merely reflect initial priming conditions.