Spatial dysregulation of T follicular helper cells impairs vaccine responses in aging.

The magnitude and quality of the germinal center (GC) response decline with age, resulting in poor vaccine-induced immunity in older individuals. A functional GC requires the co-ordination of multiple cell types across time and space, in particular across its two functionally distinct compartments: the light and dark zones. In aged mice, there is CXCR4-mediated mislocalization of T follicular helper (TFH) cells to the dark zone and a compressed network of follicular dendritic cells (FDCs) in the light zone. Here we show that TFH cell localization is critical for the quality of the antibody response and for the expansion of the FDC network upon immunization. The smaller GC and compressed FDC network in aged mice were corrected by provision of TFH cells that colocalize with FDCs using CXCR5. This demonstrates that the age-dependent defects in the GC response are reversible and shows that TFH cells support stromal cell responses to vaccines.

Fibroblastic reticular cells provide a supportive niche for lymph node-resident macrophages.

The lymph node (LN) is home to resident macrophage populations that are essential for immune function and homeostasis, but key factors controlling this niche are undefined. Here, we show that fibroblastic reticular cells (FRCs) are an essential component of the LN macrophage niche. Genetic ablation of FRCs caused rapid loss of macrophages and monocytes from LNs across two in vivo models. Macrophages co-localized with FRCs in human LNs, and murine single-cell RNA-sequencing revealed that FRC subsets broadly expressed master macrophage regulator CSF1. Functional assays containing purified FRCs and monocytes showed that CSF1R signaling was sufficient to support macrophage development. These effects were conserved between mouse and human systems. These data indicate an important role for FRCs in maintaining the LN parenchymal macrophage niche.

Treg cells and CTLA-4: the ball and chain of the germinal center response.

The mechanism by which regulatory T cells control the germinal center response is unknown. In this issue of Immunity, Wing et al. (2014) and Sage et al. (2014) demonstrate that CTLA-4 is a critical effector molecule used by regulatory T cells to control the germinal center.

Distinct epigenetic signatures delineate transcriptional programs during virus-specific CD8(+) T cell differentiation.

The molecular mechanisms that regulate the rapid transcriptional changes that occur during cytotoxic T lymphocyte (CTL) proliferation and differentiation in response to infection are poorly understood. We have utilized ChIP-seq to assess histone H3 methylation dynamics within naive, effector, and memory virus-specific T cells isolated directly ex vivo after influenza A virus infection. Our results show that within naive T cells, codeposition of the permissive H3K4me3 and repressive H3K27me3 modifications is a signature of gene loci associated with gene transcription, replication, and cellular differentiation. Upon differentiation into effector and/or memory CTLs, the majority of these gene loci lose repressive H3K27me3 while retaining the permissive H3K4me3 modification. In contrast, immune-related effector gene promoters within naive T cells lacked the permissive H3K4me3 modification, with acquisition of this modification occurring upon differentiation into effector/memory CTLs. Thus, coordinate transcriptional regulation of CTL genes with related functions is achieved via distinct epigenetic mechanisms.

Stromal Cells in the Tumor Microenvironment.

The tumor microenvironment comprises a mass of heterogeneous cell types, including immune cells, endothelial cells, and fibroblasts, alongside cancer cells. It is increasingly becoming clear that the development of this support niche is critical to the continued uncontrolled growth of the cancer. The tumor microenvironment contributes to the maintenance of cancer stemness and also directly promotes angiogenesis, invasion, metastasis, and chronic inflammation. In this chapter, we describe on the role of fibroblasts, specifically termed cancer-associated fibroblasts (CAFs), in the promotion and maintenance of cancers. CAFs have a multitude of effects on the growth and maintenance of cancer, and here we focus on their roles in modulating immune cells and responses; CAFs both inhibit immune cell access to the tumor microenvironment and inhibit their functions within the tumor. Finally, we describe the potential modulation of CAF function as an adjunct to bolster the effectiveness of cancer immunotherapies.

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

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.

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.

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-2K(b)/OVA(257-264) (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 OVA(257) 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-K(b)OVA(257), H2-K(b)E1, and H2-K(b)G4 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.

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 D(b) NP(366) and D(b) PA(224) tetramers were characterized as IL-7Rα(hi) , IL-7Rα(hi) CD62L(hi) or IL-7Rα(hi) KLRG1(lo) . While the D(b) NP(366) - and D(b) PA(224) -specific responses were comparable in size, decay kinetics and memory precursor frequency, their expansion characteristics differed. This correlated with a divergence in the IL-7Rα(hi) , IL-7Rα(hi) CD62L(hi) and IL-7Rα(hi) KLRG1(lo) phenotypes on effector, but not naïve, CD8(+) populations. That effect was abrogated by priming with viruses engineered to present equivalent levels of NP(366) and PA(224) peptides, indicating that memory phenotypes reflect early antigenic experience rather than memory potential. Thus, the IL-7Rα(hi) KLRG1(lo) 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-7Rα(hi) KLRG1(lo) CD8(+) 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.

Targeting TLR4 during vaccination boosts MAdCAM-1+ lymphoid stromal cell activation and promotes the aged germinal center response.

The failure to generate enduring humoral immunity after vaccination is a hallmark of advancing age. This can be attributed to a reduction in the germinal center (GC) response, which generates long-lived antibody-secreting cells that protect against (re)infection. Despite intensive investigation, the primary cellular defect underlying impaired GCs in aging has not been identified. Here, we used heterochronic parabiosis to demonstrate that GC formation was dictated by the age of the lymph node (LN) microenvironment rather than the age of the immune cells. Lymphoid stromal cells are a key determinant of the LN microenvironment and are also an essential component underpinning GC structure and function. Using mouse models, we demonstrated that mucosal adressin cell adhesion molecule-1 (MAdCAM-1)-expressing lymphoid stromal cells were among the first cells to respond to NP-KLH + Alum immunization, proliferating and up-regulating cell surface proteins such as podoplanin and cell adhesion molecules. This response was essentially abrogated in aged mice. By targeting TLR4 using adjuvants, we improved the MAdCAM-1+ stromal cell response to immunization. This correlated with improved GC responses in both younger adult and aged mice, suggesting a link between stromal cell responses to immunization and GC initiation. Using bone marrow chimeras, we also found that MAdCAM-1+ stromal cells could respond directly to TLR4 ligands. Thus, the age-associated defect in GC and stromal cell responses to immunization can be targeted to improve vaccines in older people.

Lymphoid stromal cells-more than just a highway to humoral immunity.

The generation of high-affinity long-lived antibody responses is dependent on the differentiation of plasma cells and memory B cells, which are themselves the product of the germinal centre (GC) response. The GC forms in secondary lymphoid organs in response to antigenic stimulation and is dependent on the coordinated interactions between many types of leucocytes. These leucocytes are brought together on an interconnected network of specialized lymphoid stromal cells, which provide physical and chemical guidance to immune cells that are essential for the GC response. In this review we will highlight recent advancements in lymphoid stromal cell immunobiology and their role in regulating the GC, and discuss the contribution of lymphoid stromal cells to age-associated immunosenescence.

B Cell Diversification Is Uncoupled from SAP-Mediated Selection Forces in Chronic Germinal Centers within Peyer's Patches.

Antibodies secreted within the intestinal tract provide protection from the invasion of microbes into the host tissues. Germinal center (GC) formation in lymph nodes and spleen strictly requires SLAM-associated protein (SAP)-mediated T cell functions; however, it is not known whether this mechanism plays a similar role in mucosal-associated lymphoid tissues. Here, we find that in Peyer's patches (PPs), SAP-mediated T cell help is required for promoting B cell selection in GCs, but not for clonal diversification. PPs of SAP-deficient mice host chronic GCs that are absent in T cell-deficient mice. GC B cells in SAP-deficient mice express AID and Bcl6 and generate plasma cells in proportion to the GC size. Single-cell IgA sequencing analysis reveals that these mice host few diversified clones that were subjected to mild selection forces. These findings demonstrate that T cell-derived help to B cells in PPs includes SAP-dependent and SAP-independent functions.

Follicular Regulatory T Cells Can Access the Germinal Center Independently of CXCR5.

The germinal center (GC) response is critical for generating high-affinity humoral immunity and immunological memory, which forms the basis of successful immunization. Control of the GC response is thought to require follicular regulatory T (Tfr) cells, a subset of suppressive Foxp3+ regulatory T cells located within GCs. Relatively little is known about the exact role of Tfr cells within the GC and how they exert their suppressive function. A unique feature of Tfr cells is their reported CXCR5-dependent localization to the GC. Here, we show that the lack of CXCR5 on Foxp3+ regulatory T cells results in a reduced frequency, but not an absence, of GC-localized Tfr cells. This reduction in Tfr cells is not sufficient to alter the magnitude or output of the GC response. This demonstrates that additional, CXCR5-independent mechanisms facilitate Treg cell homing to the GC.

Embryonic FAP+ lymphoid tissue organizer cells generate the reticular network of adult lymph nodes.

The induction of adaptive immunity is dependent on the structural organization of LNs, which is in turn governed by the stromal cells that underpin LN architecture. Using a novel fate-mapping mouse model, we trace the developmental origin of mesenchymal LN stromal cells (mLNSCs) to a previously undescribed embryonic fibroblast activation protein-α (FAP)+ progenitor. FAP+ cells of the LN anlagen express lymphotoxin ß receptor (LTßR) and vascular cell adhesion molecule (VCAM), but not intercellular adhesion molecule (ICAM), suggesting they are early mesenchymal lymphoid tissue organizer (mLTo) cells. Clonal labeling shows that FAP+ progenitors locally differentiate into mLNSCs. This process is also coopted in nonlymphoid tissues in response to infection to facilitate the development of tertiary lymphoid structures, thereby mimicking the process of LN ontogeny in response to infection.

Type I interferon induces CXCL13 to support ectopic germinal center formation.

Ectopic lymphoid structures form in a wide range of inflammatory conditions, including infection, autoimmune disease, and cancer. In the context of infection, this response can be beneficial for the host: influenza A virus infection-induced pulmonary ectopic germinal centers give rise to more broadly cross-reactive antibody responses, thereby generating cross-strain protection. However, despite the ubiquity of ectopic lymphoid structures and their role in both health and disease, little is known about the mechanisms by which inflammation is able to convert a peripheral tissue into one that resembles a secondary lymphoid organ. Here, we show that type I IFN produced after viral infection can induce CXCL13 expression in a phenotypically distinct population of lung fibroblasts, driving CXCR5-dependent recruitment of B cells and initiating ectopic germinal center formation. This identifies type I IFN as a novel inducer of CXCL13, which, in combination with other stimuli, can promote lung remodeling, converting a nonlymphoid tissue into one permissive to functional tertiary lymphoid structure formation.

FcγRIIb differentially regulates pre-immune and germinal center B cell tolerance in mouse and human.

Several tolerance checkpoints exist throughout B cell development to control autoreactive B cells and prevent the generation of pathogenic autoantibodies. FcγRIIb is an Fc receptor that inhibits B cell activation and, if defective, is associated with autoimmune disease, yet its impact on specific B cell tolerance checkpoints is unknown. Here we show that reduced expression of FcγRIIb enhances the deletion and anergy of autoreactive immature B cells, but in contrast promotes autoreactive B cell expansion in the germinal center and serum autoantibody production, even in response to exogenous, non-self antigens. Our data thus show that FcγRIIb has opposing effects on pre-immune and post-immune tolerance checkpoints, and suggest that B cell tolerance requires the control of bystander germinal center B cells with low or no affinity for the immunizing antigen.

Magnetic Resonance Imaging Is More Sensitive Than PET for Detecting Treatment-Induced Cell Death-Dependent Changes in Glycolysis.

Metabolic imaging has been widely used to measure the early responses of tumors to treatment. Here, we assess the abilities of PET measurement of [18F]FDG uptake and MRI measurement of hyperpolarized [1-13C]pyruvate metabolism to detect early changes in glycolysis following treatment-induced cell death in human colorectal (Colo205) and breast adenocarcinoma (MDA-MB-231) xenografts in mice. A TRAIL agonist that binds to human but not mouse cells induced tumor-selective cell death. Tumor glycolysis was assessed by injecting [1,6-13C2]glucose and measuring 13C-labeled metabolites in tumor extracts. Injection of hyperpolarized [1-13C]pyruvate induced rapid reduction in lactate labeling. This decrease, which correlated with an increase in histologic markers of cell death and preceded decrease in tumor volume, reflected reduced flux from glucose to lactate and decreased lactate concentration. However, [18F]FDG uptake and phosphorylation were maintained following treatment, which has been attributed previously to increased [18F]FDG uptake by infiltrating immune cells. Quantification of [18F]FDG uptake in flow-sorted tumor and immune cells from disaggregated tumors identified CD11b+/CD45+ macrophages as the most [18F]FDG-avid cell type present, yet they represented <5% of the cells present in the tumors and could not explain the failure of [18F]FDG-PET to detect treatment response. MRI measurement of hyperpolarized [1-13C]pyruvate metabolism is therefore a more sensitive marker of the early decreases in glycolytic flux that occur following cell death than PET measurements of [18F]FDG uptake. SIGNIFICANCE: These findings demonstrate superior sensitivity of MRI measurement of hyperpolarized [1-13C]pyruvate metabolism versus PET measurement of 18F-FDG uptake for detecting early changes in glycolysis following treatment-induced tumor cell death.

Mice Deficient in Nucleoporin Nup210 Develop Peripheral T Cell Alterations.

The nucleopore is an essential structure of the eukaryotic cell, regulating passage between the nucleus and cytoplasm. While individual functions of core nucleopore proteins have been identified, the role of other components, such as Nup210, are poorly defined. Here, through the use of an unbiased ENU mutagenesis screen for mutations effecting the peripheral T cell compartment, we identified a Nup210 mutation in a mouse strain with altered CD4/CD8 T cell ratios. Through the generation of Nup210 knockout mice we identified Nup210 as having a T cell-intrinsic function in the peripheral homeostasis of T cells. Remarkably, despite the deep evolutionary conservation of this key nucleopore complex member, no other major phenotypes developed, with viable and healthy knockout mice. These results identify Nup210 as an important nucleopore complex component for peripheral T cells, and raise further questions of why this nucleopore component shows deep evolutionary conservation despite seemingly redundant functions in most cell types.

Stromal networking: cellular connections in the germinal centre.

Secondary lymphoid organs are organized into distinct zones, governed by different types of mesenchymal stromal cells. These stromal cell subsets are critical for the generation of protective humoral immunity because they direct the migration of, and interaction between, multiple immune cell types to form the germinal centre. The germinal centre response generates long-lived antibody-secreting plasma cells and memory B cells which can provide long-term protection against re-infection. Stromal cell subsets mediate this response through control of immune cell trafficking, activation, localization and antigen access within the secondary lymphoid organ. Further, distinct populations of stromal cells underpin the delicate spatial organization of immune cells within the germinal centre. Because of this, the interactions between immune cells and stromal cells in secondary lymphoid organs are fundamental to the germinal centre response. Herein we review how this unique relationship leads to effective germinal centre responses.