Alice E. Denton

Depletion of stromal cells expressing fibroblast activation protein-α from skeletal muscle and bone marrow results in cachexia and anemia

Ablation of stromal cells expressing fibroblast activation protein-α (FAP) results in cachexia and anemia, and loss of these cells is seen in transplantable tumor models.

Differentiation-dependent functional and epigenetic landscapes for cytokine genes in virus-specific CD8+ T cells

Although the simultaneous engagement of multiple effector mechanisms is thought to characterize optimal CD8+ T-cell immunity and facilitate pathogen clearance, the differentiation pathways leading to the acquisition and maintenance of such polyfunctional activity are not well understood. Division-dependent profiles of effector molecule expression for virus-specific T cells are analyzed here by using a combination of carboxyfluorescein succinimidyl ester dilution and intracellular cytokine staining subsequent to T-cell receptor ligation. The experiments show that, although the majority of naive CD8+ T-cell precursors are preprogrammed to produce TNF-α soon after stimulation and a proportion make both TNF-α and IL-2, the progressive acquisition of IFN-γ expression depends on continued lymphocyte proliferation. Furthermore, the extensive division characteristic of differentiation to peak effector activity is associated with the progressive dominance of IFN-γ and the concomitant loss of polyfunctional cytokine production, although this is not apparent for long-term CD8+ T-cell memory. Such proliferation-dependent variation in cytokine production appears tied to the epigenetic signatures within the ifnG and tnfA proximal promoters. Specifically, those cytokine gene loci that are rapidly expressed following antigen stimulation at different stages of T-cell differentiation can be shown (by ChIP) to have permissive epigenetic and RNA polymerase II docking signatures. Thus, the dynamic changes in cytokine profiles for naive, effector, and memory T cells are underpinned by specific epigenetic landscapes that regulate responsiveness following T-cell receptor ligation.

IL‐18, but not IL‐12, is required for optimal cytokine production by influenza virus‐specific CD8+ T cells

The potent innate cytokines IL‐12 and IL‐18 are considered to be important antigen‐independent mediators of IFN‐γ production by NK cells and T lymphocytes. The present analysis addresses the physiological role of IL‐12 and IL‐18 in the generation of virus‐specific CD8+ T cells. Both wt C57BL/6J (B6) mice and mice with disrupted IL‐12p40 (IL‐12p40–/–) or IL‐18 (IL‐18–/–) genes were infected with an influenza A virus and the characteristics of the resultant epitope‐specific CD8+ T cell responses were compared. While IL‐12 appeared to have no notable effect on either virus growth or on CD8+ T cell response profiles, the absence of IL‐18 was associated with delayed virus clearance from the lung and, despite normal numbers, a significantly reduced production of IFN‐γ, TNF‐α, and IL‐2 by epitope‐specific CD8+ T cells. While this cytokine phenotype was broadly maintained in IL‐12p40/IL‐18 double‐knockout mice, no evidence was seen for any additive effect. Together, our results suggest that IL‐18, but not IL‐12, induces optimal, antigen‐specific production of key cytokines by CD8+ T cells for the efficient clearance of influenza virus from the lungs of infected mice.

Fibroblastic reticular cells of the lymph node are required for retention of resting but not activated CD8+ T cells

Significance The balance between the retention of lymphocytes in lymph nodes and their exit is a key factor determining clonal burst size, differentiation, and the efficacy of pathogen clearance. Currently, it is understood that naïve and activated T cells regulate their migration using a similar system, that is, balancing CC chemokine receptor (CCR)7-mediated retention signals against Sphingosine-1 phosphate receptor-1 (S1PR1)-mediated exit signals. This study utilizes a mouse model to selectively deplete the cellular source of CCR7 ligands, the fibroblastic reticular cells, before or during viral infection. The study concludes that while retention of naïve lymphocytes does depend on the fibroblastic reticular cell, it may be a cell-autonomous response of activated T cells that allows them to determine in a cell-intrinsic manner the magnitude of clonal expansion. Fibroblastic reticular cells (FRCs), through their expression of CC chemokine ligand (CCL)19 and CCL21, attract and retain T cells in lymph nodes (LNs), but whether this function applies to both resting and activated T cells has not been examined. Here we describe a model for conditionally depleting FRCs from LNs based on their expression of the diphtheria toxin receptor (DTR) directed by the gene encoding fibroblast activation protein-α (FAP). As expected, depleting FAP+ FRCs causes the loss of naïve T cells, B cells, and dendritic cells from LNs, and this loss decreases the magnitude of the B- and T-cell responses to a subsequent infection with influenza A virus. In contrast, depleting FAP+ FRCs during an ongoing influenza infection does not diminish the number or continued response of activated T and B cells in the draining LNs, despite still resulting in the loss of naïve T cells. Therefore, different rules govern the LN trafficking of resting and activated T cells; once a T cell is engaged in antigen-specific clonal expansion, its retention no longer depends on FRCs or their chemokines, CCL19 and CCL21. Our findings suggest that activated T cells remain in the LN because they down-regulate the expression of the sphingosine-1 phosphate receptor-1, which mediates the exit of lymphocytes from secondary lymphoid organs. Therefore, LN retention of naïve lymphocytes and the initiation of an immune response depend on FRCs, but is an FRC independent and possibly cell-autonomous response of activated T cells, which allows the magnitude of clonal expansion to determine LN egress.

Tumor-Induced IL-6 Reprograms Host Metabolism to Suppress Anti-tumor Immunity.

Summary In patients with cancer, the wasting syndrome, cachexia, is associated with caloric deficiency. Here, we describe tumor-induced alterations of the host metabolic response to caloric deficiency that cause intratumoral immune suppression. In pre-cachectic mice with transplanted colorectal cancer or autochthonous pancreatic ductal adenocarcinoma (PDA), we find that IL-6 reduces the hepatic ketogenic potential through suppression of PPARalpha, the transcriptional master regulator of ketogenesis. When these mice are challenged with caloric deficiency, the resulting relative hypoketonemia triggers a marked rise in glucocorticoid levels. Multiple intratumoral immune pathways are suppressed by this hormonal stress response. Moreover, administering corticosterone to elevate plasma corticosterone to a level that is lower than that occurring in cachectic mice abolishes the response of mouse PDA to an immunotherapy that has advanced to clinical trials. Therefore, tumor-induced IL-6 impairs the ketogenic response to reduced caloric intake, resulting in a systemic metabolic stress response that blocks anti-cancer immunotherapy.

CD28 expression is required after T cell priming for helper T cell responses and protective immunity to infection

The co-stimulatory molecule CD28 is essential for activation of helper T cells. Despite this critical role, it is not known whether CD28 has functions in maintaining T cell responses following activation. To determine the role for CD28 after T cell priming, we generated a strain of mice where CD28 is removed from CD4+ T cells after priming. We show that continued CD28 expression is important for effector CD4+ T cells following infection; maintained CD28 is required for the expansion of T helper type 1 cells, and for the differentiation and maintenance of T follicular helper cells during viral infection. Persistent CD28 is also required for clearance of the bacterium Citrobacter rodentium from the gastrointestinal tract. Together, this study demonstrates that CD28 persistence is required for helper T cell polarization in response to infection, describing a novel function for CD28 that is distinct from its role in T cell priming. DOI: http://dx.doi.org/10.7554/eLife.03180.001

Interplay between chromatin remodeling and epigenetic changes during lineage-specific commitment to granzyme B expression

The role of chromatin remodeling and histone posttranslational modifications and how they are integrated to control gene expression during the acquisition of cell-specific functions is poorly understood. We show here that following in vitro activation of CD4+ and CD8+ T lymphocytes, both cell types show rapid histone H3 loss at the granzyme B (gzmB) proximal promoter region. However, despite the gzmB proximal promoter being remodeled in both T cell subsets, only CD8+ T cells express high levels of gzmB and display a distinct pattern of key epigenetic marks, notably differential H3 acetylation and methylation. These data suggest that for high levels of transcription to occur a distinct set of histone modifications needs to be established in addition to histone loss at the proximal promoter of gzmB.

Affinity Thresholds for Naive CD8 + CTL Activation by Peptides and Engineered Influenza A Viruses

High-avidity interactions between TCRs and peptide + class I MHC (pMHCI) epitopes drive CTL activation and expansion. Intriguing questions remain concerning the constraints determining optimal TCR/pMHCI binding. The present analysis uses the TCR transgenic OT-I model to assess how varying profiles of TCR/pMHCI avidity influence naive CTL proliferation and the acquisition of effector function following exposure to the cognate H-2Kb/OVA257–264 (SIINFEKL) epitope and to mutants provided as peptide or in engineered influenza A viruses. Stimulating naive OT-I CD8+ T cells in vitro with SIINFEKL induced full CTL proliferation and differentiation that was largely independent of any need for costimulation. By contrast, in vitro activation with the low-affinity EIINFEKL or SIIGFEKL ligands depended on the provision of IL-2 and other costimulatory signals. Importantly, although they did generate potent endogenous responses, infection of mice with influenza A viruses expressing these same OVA257 variants failed to induce the activation of adoptively transferred naive OT-I CTLps, an effect that was only partially overcome by priming with a lipopeptide vaccine. Subsequent structural and biophysical analysis of H2-KbOVA257, H2-KbE1, and H2-KbG4 established that these variations introduce small changes at the pMHCI interface and decrease epitope stability in ways that would likely impact cell surface presentation and recognition. Overall, it seems that there is an activation threshold for naive CTLps, that minimal alterations in peptide sequence can have profound effects, and that the antigenic requirements for the in vitro and in vivo induction of CTL proliferation and effector function differ substantially.

Defining the molecular blueprint that drives CD8+ T cell differentiation in response to infection

A cardinal feature of adaptive, cytotoxic T lymphocyte (CTL)-mediated immunity is the ability of naïve CTLs to undergo a program of differentiation and proliferation upon activation resulting in the acquisition of lineage-specific T cell functions and eventual establishment of immunological memory. In this review, we examine the molecular factors that shape both the acquisition and maintenance of lineage-specific effector function in virus-specific CTL during both the effector and memory phases of immunity.

Memory precursor phenotype of CD8+ T cells reflects early antigenic experience rather than memory numbers in a model of localized acute influenza infection

The mechanistic basis of memory T‐cell development is poorly defined. Phenotypic markers that define precursors at effector stages have been characterized for acute systemic infections with high antigen load. We asked whether such markers can identify memory precursors from early effectors (d6) to late memory (>d500) for two immunodominant CD8+ responses during the course of a localized low‐load influenza infection in mice. CD8+ T cells stained with the DbNP366 and DbPA224 tetramers were characterized as IL‐7Rαhi, IL‐7RαhiCD62Lhi or IL‐7RαhiKLRG1lo. While the DbNP366‐ and DbPA224‐specific responses were comparable in size, decay kinetics and memory precursor frequency, their expansion characteristics differed. This correlated with a divergence in the IL‐7Rαhi, IL‐7RαhiCD62Lhi and IL‐7RαhiKLRG1lo phenotypes on effector, but not naïve, CD8+ populations. That effect was abrogated by priming with viruses engineered to present equivalent levels of NP366 and PA224 peptides, indicating that memory phenotypes reflect early antigenic experience rather than memory potential. Thus, the IL‐7RαhiKLRG1lo phenotype had a poor predictive value in identifying memory precursors in the spleen and at the site of infection. Greater consistency in influenza‐specific IL‐7RαhiKLRG1loCD8+ T‐cell numbers was found in draining lymph nodes, suggesting that this may be the preferential site for memory establishment and maintenance following localized virus infections.