Dynamic regulation of T follicular regulatory cell responses by interleukin 2 during influenza infection.

Interleukin 2 (IL-2) promotes Foxp3+ regulatory T (Treg) cell responses, but inhibits T follicular helper (TFH) cell development. However, it is not clear how IL-2 affects T follicular regulatory (TFR) cells, a cell type with properties of both Treg and TFH cells. Using an influenza infection model, we found that high IL-2 concentrations at the peak of the infection prevented TFR cell development by a Blimp-1-dependent mechanism. However, once the immune response resolved, some Treg cells downregulated CD25, upregulated Bcl-6 and differentiated into TFR cells, which then migrated into the B cell follicles to prevent the expansion of self-reactive B cell clones. Thus, unlike its effects on conventional Treg cells, IL-2 inhibits TFR cell responses.

A stay of execution: type I interferons pardon T cells from death by natural killers.

In this issue of Immunity, Crouse et al., (2014) and Xu et al., (2014), show that by modulating the expression of natural killer (NK) cell receptor ligands, type I interferons protect responding T cells against culling by NK cells.

IL-21 and T Cell Differentiation: Consider the Context.

Accumulating studies demonstrate that IL-21 modulates the differentiation of various CD4 and CD8 T cell subsets and provide insights into the underlying cellular and molecular processes that are influenced by this cytokine. Intriguingly, the effects of IL-21 on T cells can be complex and vary depending on the experimental system used. We review our current understanding of the roles of IL-21 in the generation of phenotypically distinct CD4 and CD8 T cell populations and discuss the potential environmental cues, cellular factors, and molecular mediators that impact the actions of IL-21. We propose that IL-21 acts in a context-dependent manner to accentuate T cell subset development.

IL-10 Regulates Memory T Cell Development and the Balance between Th1 and Follicular Th Cell Responses during an Acute Viral Infection.

T cells provide protective immunity against infections by differentiating into effector cells that contribute to rapid pathogen control and by forming memory populations that survive over time and confer long-term protection. Thus, understanding the factors that regulate the development of effective T cell responses is beneficial for the design of vaccines and immune-based therapies against infectious diseases. Cytokines play important roles in shaping T cell responses, and IL-10 has been shown to modulate the differentiation of CD4 and CD8 T cells. In this study, we report that IL-10 functions in a cell-extrinsic manner early following acute lymphocytic choriomeningitis virus infection to suppress the magnitude of effector Th1 responses as well as the generation of memory CD4 and CD8 T cells. We further demonstrate that the blockade of IL-10 signaling during the priming phase refines the functional quality of memory CD4 and CD8 T cells. This inhibition strategy resulted in a lower frequency of virus-specific follicular Th (Tfh) cells and increased the Th1 to Tfh ratio. Nevertheless, neither germinal center B cells nor lymphocytic choriomeningitis virus-specific Ab levels were influenced by the blockade. Thus, our studies show that IL-10 influences the balance between Th1 and Tfh cell differentiation and negatively regulates the development of functionally mature memory T cells.

A Context-Dependent Role for IL-21 in Modulating the Differentiation, Distribution, and Abundance of Effector and Memory CD8 T Cell Subsets.

The activation of naive CD8 T cells typically results in the formation of effector cells (TE) as well as phenotypically distinct memory cells that are retained over time. Memory CD8 T cells can be further subdivided into central memory, effector memory (TEM), and tissue-resident memory (TRM) subsets, which cooperate to confer immunological protection. Using mixed bone marrow chimeras and adoptive transfer studies in which CD8 T cells either do or do not express IL-21R, we discovered that under homeostatic or lymphopenic conditions IL-21 acts directly on CD8 T cells to favor the accumulation of TE/TEM populations. The inability to perceive IL-21 signals under competitive conditions also resulted in lower levels of TRM phenotype cells and reduced expression of granzyme B in the small intestine. IL-21 differentially promoted the expression of the chemokine receptor CX3CR1 and the integrin α4ß7 on CD8 T cells primed in vitro and on circulating CD8 T cells in the mixed bone marrow chimeras. The requirement for IL-21 to establish CD8 TE/TEM and TRM subsets was overcome by acute lymphocytic choriomeningitis virus infection; nevertheless, memory virus-specific CD8 T cells remained dependent on IL-21 for optimal accumulation in lymphopenic environments. Overall, this study reveals a context-dependent role for IL-21 in sustaining effector phenotype CD8 T cells and influencing their migratory properties, accumulation, and functions.

ICAM-1-dependent tuning of memory CD8 T-cell responses following acute infection.

CD8 T-cell responses are critical for protection against intracellular pathogens and tumors. The induction and properties of these responses are governed by a series of integrated processes that rely heavily on cell-cell interactions. Intercellular adhesion molecule (ICAM)-1 functions to enhance the strength of antigenic stimulation, extend the duration of contact with antigen-presenting cells, and augment cytokine signals, which are all factors that influence peripheral CD8 T-cell differentiation. Although previous studies suggest that ICAM-1 is essential for establishing memory T-cell populations following peptide immunization, the roles of ICAM-1 in antiviral cellular immunity are less well understood. Here we show that, following a prototypic acute viral infection, the formation and maintenance of memory-phenotype CD127(hi), KLRG-1(lo) CD8 T cells does not require ICAM-1. Nevertheless, ICAM-1 expression on nonlymphocytes dictates the phenotypic and functional attributes of the antiviral CD8 T-cell populations that develop and promotes the gradual attrition of residual effector-like CD127(lo), KLRG-1(hi) CD8 T cells during the memory phase of the response. Although memory T cells do emerge and are maintained if ICAM-1 expression is abolished, the secondary proliferative capacity of these T cells is severely curtailed. Collectively, these studies reveal potential dual roles for ICAM-1 in both promoting the decay of effector responses and programming the sensitivity of memory CD8 T cells to secondary stimuli.

Temporal requirements for B cells in the establishment of CD4 T cell memory.

CD4 T cell memory generation is shaped by a number of factors, including the strength and duration of TCR signaling, as well as the priming environment, all of which can be modified by B cells. Studies using B cell-deficient mice indicate B cells play a critical role in generating effector and memory CD4 T cells; however, when and how B cells are acting to promote these responses has not yet been ascertained. In this study, we use anti-CD20 Ab depletion of B cells at different times following Listeria monocytogenes infection to show that B cells are necessary for the induction of optimal CD4 T cell memory, but not for the transition and maintenance of this population. Importantly, the prerequisite of B cells early postinfection is partially dependent on their expression of MHC class II. B cells are not only required during the priming phase, but also necessary for the initiation of robust secondary responses by memory CD4 T cells. Interestingly, the requirement during the recall response is independent of B cell Ag presentation. Overall, these studies demonstrate the temporally and functionally distinct roles for B cells in regulating CD4 T cell responses.

IL-17RA is essential for optimal localization of follicular Th cells in the germinal center light zone to promote autoantibody-producing B cells.

Germinal centers (GCs) provide a microenvironment that promotes and regulates the interactions of B cells with follicular Th (TFH) cells. In this study, we show that there are significantly higher frequencies of CXCR5(+)ICOS(+) TFH cells in autoimmune BXD2 mice, and these cells express both IL-21R and IL-17RA. Although IL-17 and IL-21 are both important for the formation of spontaneous GCs and development of pathogenic autoantibodies, IL-21, but not IL-17, is required for the proper development of TFH cells in BXD2 mice. The total numbers of TFH cells and their ability to induce B cell responses in vitro were not affected by a deficiency of IL-17RA in BXD2-Il17ra(-/-) mice, the majority of CXCR5(+) TFH cells from BXD2-Il17ra(-/-) mice were, however, not localized in the GC light zone (LZ). Interruption of IL-17 signaling, either acutely by AdIL-17R:Fc or chronically by Il17ra(-/-), disrupted TFH-B interactions and abrogated the generation of autoantibody-forming B cells in BXD2 mice. IL-17 upregulated the expression of regulator of G-protein signaling 16 (RGS16) to promote the ability of TFH to form conjugates with B cells, which was abolished in TFH cells from BXD2-Rgs16(-/-) mice. The results suggests that IL-17 is an extrinsic stop signal that it acts on postdifferentiated IL-17RA(+) TFH to enable its interaction with responder B cells in the LZ niche. These data suggest a novel concept that TFH differentiation and its stabilization in the LZ are two separate checkpoints and that IL-21 and IL-17 act at each checkpoint to enable pathogenic GC development.

Cytokines and the inception of CD8 T cell responses.

The activation and differentiation of CD8 T cells is a necessary first step that endows these cells with the phenotypic and functional properties required for the control of intracellular pathogens. The induction of the CD8 T cell responses typically results in the development of a massive overall population of effector cells, comprising both highly functional but short-lived terminally differentiated cells, as well as a smaller subset of precursors that are predisposed to survive and transition into the memory T cell pool. In this review, we discuss how inflammatory cytokines and IL-2 bias the initial response towards short-lived effector generation, and also highlight the potential counterbalancing role of IL-21.

Memory CD4 T cells emerge from effector T-cell progenitors.

A hallmark of adaptive immunity is the generation of memory T cells that confer long-lived, antigen-specific protection against repeat challenges by pathogens. Understanding the mechanisms by which memory T cells arise is important for rational vaccination strategies and improved therapeutic interventions for chronic infections and autoimmune disorders. The large clonal expansion of CD8 T cells in response to some infections has made the development of CD8 T-cell memory more amenable to study, giving rise to a model of memory cell differentiation in which a fraction of fully competent effector T cells transition into long-lived memory T cells. Delineation of CD4 T-cell memory development has proved more difficult as a result of limitations on tracking the smaller populations of CD4 effector T cells generated during a pathogenic challenge, complicating efforts to determine whether CD4 memory T cells are direct descendants of effector T cells or whether they develop by alternative pathways. Here, using two complementary cytokine reporter mouse models to identify interferon (IFN)-gamma-positive effector T cells and track their fate, we show that the lineage relationship between effector and memory CD4 T cells resembles that for CD8 T cells responding to the same pathogen. We find that, in parallel with effector CD8 T cells, IFN-gamma-positive effector CD4 T cells give rise to long-lived memory T cells capable of anamnestic responses to antigenic rechallenge.

Gfi1 controls the formation of effector CD8 T cells during chronic infection and cancer.

During chronic infections and tumor progression, CD8 T cells gradually lose their effector functions and become exhausted. These exhausted CD8 T cells are heterogeneous and comprised of different subsets, including self-renewing progenitors that give rise to Ly108 - CX3CR1 + effector-like cells. Generation of these effector-like cells is essential for the control of chronic infections and tumors, albeit limited. However, the precise cues and mechanisms directing the formation and maintenance of exhausted effector-like are incompletely understood. Using genetic mouse models challenged with LCMV Clone 13 or syngeneic tumors, we show that the expression of a transcriptional repressor, growth factor independent 1 (Gfi1) is dynamically regulated in exhausted CD8 T cells, which in turn regulates the formation of exhausted effector-like cells. Gfi1 deletion in T cells dysregulates the chromatin accessibility and transcriptomic programs associated with the differentiation of LCMV Clone 13-specific CD8 T cell exhaustion, preventing the formation of effector-like and terminally exhausted cells while maintaining progenitors and a newly identified Ly108 + CX3CR1 + state. These Ly108 + CX3CR1 + cells have a distinct chromatin profile and may represent an alternative target for therapeutic interventions to combat chronic infections and cancer. In sum, we show that Gfi1 is a critical regulator of the formation of exhausted effector-like cells.

Exhausted CD8 T cells downregulate the IL-18 receptor and become unresponsive to inflammatory cytokines and bacterial co-infections.

During many chronic infections virus-specific CD8 T cells succumb to exhaustion as they lose their ability to respond to antigenic activation. Combinations of IL-12, IL-18, and IL-21 have been shown to induce the antigen-independent production of interferon (IFN)-γ by effector and memory CD8 T cells. In this study we investigated whether exhausted CD8 T cells are sensitive to activation by these cytokines. We show that effector and memory, but not exhausted, CD8 T cells produce IFN-γ and upregulate CD25 following exposure to certain combinations of IL-12, IL-18, and IL-21. The unresponsiveness of exhausted CD8 T cells is associated with downregulation of the IL-18-receptor-α (IL-18Rα). Although IL-18Rα expression is connected with the ability of memory CD8 T cells to self-renew and efflux rhodamine 123, the IL-18Rα(lo) exhausted cells remained capable of secreting this dye. To further evaluate the consequences of IL-18Rα downregulation, we tracked the fate of IL-18Rα-deficient CD8 T cells in chronically infected mixed bone marrow chimeras and discovered that IL-18Rα affects the initial but not later phases of the response. The antigen-independent responsiveness of exhausted CD8 T cells was also investigated following co-infection with Listeria monocytogenes, which induces the expression of IL-12 and IL-18. Although IL-18Rα(hi) memory cells upregulated CD25 and produced IFN-γ, the IL-18Rα(lo) exhausted cells failed to respond. Collectively, these findings indicate that as exhausted T cells adjust to the chronically infected environment, they lose their susceptibility to antigen-independent activation by cytokines, which compromises their ability to detect bacterial co-infections.

Interleukin-21-producing HIV-1-specific CD8 T cells are preferentially seen in elite controllers.

A hallmark of human immunodeficiency virus type 1 (HIV-1) pathogenesis is the rapid loss of CD4 T cells leading to generalized immune dysfunction, including an exhausted CD8 T cell phenotype. Understanding the necessary factors that govern the functional quality and protective potential of antiviral T cell responses would facilitate rational vaccine design and improve therapeutic strategies to combat persistent infections. Mouse models of chronic viral infection demonstrate that interleukin-21 (IL-21), produced primarily by CD4 T cells, is required for the generation and maintenance of functionally competent CD8 T cells and viral containment. We reasoned that preserved IL-21 production during HIV-1 infection would be associated with enhanced CD8 T cell function, allowing improved viral control. Here we analyzed the ability of CD4 and CD8 T cells to produce several cytokines in addition to IL-21 ex vivo following stimulation with overlapping HIV-1 peptides. Both CD4 and CD8 T cells were able to produce IL-21 in response to HIV-1 infection, with the latter cell type more closely associated with viral control. Furthermore, IL-21-producing HIV-1-specific CD4 T cells (compared to those producing other cytokines) were the best indicator of functional CD8 T cells. Our results demonstrate that HIV-1-specific IL-21-producing CD8 T cells are induced following primary infection and enriched in elite controllers, suggesting a critical role for these cells in the maintenance of viremia control.

IL-21 deficiency influences CD8 T cell quality and recall responses following an acute viral infection.

CD4 T cells are principal producers of IL-21 and are often required for optimal CD8 T cell responses. Therefore, we investigated the importance of IL-21 in determining the phenotypic attributes, functional quality, and maintenance of antiviral CD8 T cells following acute infection with the prototypic mouse pathogen lymphocytic choriomeningitis virus. Previous reports have documented an obligatory role for IL-21 in sustaining CD8 T cell responses during chronic infections. Here we show that the requirements for IL-21 are less stringent following acute infections; however, in the absence of IL-21, the capacity of CD8 T cells to attain the polyfunctional trait of IL-2 production is consistently reduced during both the effector and memory phases. This is further supported by in vitro studies showing that the addition of IL-21 promotes the differentiation of IL-2-producing CD8 T cells. Although the generation of memory CD8 T cells, which are capable of mounting protective recall responses, proceeds independently of IL-21, we demonstrate that IL-21 does function to support secondary responses, especially under competitive conditions. Collectively, these studies highlight the potential roles of IL-21 in determining the quality of CD8 T cell responses postinfection.

Pronounced virus-dependent activation drives exhaustion but sustains IFN-γ transcript levels.

During many chronic infections, the responding CD8 T cells become exhausted as they progressively lose their ability to elaborate key effector functions. Unlike prototypic memory CD8 cells, which rapidly synthesize IFN-gamma following activation, severely exhausted T cells fail to produce this effector molecule. Nevertheless, the ontogeny of exhausted CD8 T cells, as well as the underlying mechanisms that account for their functional inactivation, remains ill defined. We have used cytokine reporter mice, which mark the transcription of IFN-gamma mRNA by the expression of Thy1.1, to decipher how activation events during the early stages of a chronic infection dictate the development of exhaustion. We show that virus-specific CD8 T cells clearly respond during the early stages of chronic lymphocytic choriomeningitis virus infection, and that this early T cell response is more pronounced than that initially observed in acutely infected hosts. Thus, exhausted CD8 T cells appear to emerge from populations of potently activated precursors. Unlike acute infections, which result in massive expansion of the responding T cells, there is a rapid attenuation of further expansion during chronic infections. The exhausted T cells that subsequently emerge in chronically infected hosts are incapable of producing the IFN-gamma protein. Surprisingly, high levels of the IFN-gamma transcript are still present in exhausted cells, demonstrating that ablation of IFN-gamma production by exhausted cells is not due to transcriptional silencing. Thus, posttranscription regulatory mechanisms likely disable this effector module.

Intrinsic IL-2 production by effector CD8 T cells affects IL-2 signaling and promotes fate decisions, stemness, and protection.

Here, we show that the capacity to manufacture IL-2 identifies constituents of the expanded CD8 T cell effector pool that display stem-like features, preferentially survive, rapidly attain memory traits, resist exhaustion, and control chronic viral challenges. The cell-intrinsic synthesis of IL-2 by CD8 T cells attenuates the ability to receive IL-2-dependent STAT5 signals, thereby limiting terminal effector formation, endowing the IL-2-producing effector subset with superior protective powers. In contrast, the non-IL-2-producing effector cells respond to IL-2 signals and gain effector traits at the expense of memory formation. Despite having distinct properties during the effector phase, IL-2-producing and nonproducing CD8 T cells appear to converge transcriptionally as memory matures to form populations with equal recall abilities. Therefore, the potential to produce IL-2 during the effector, but not memory stage, is a consequential feature that dictates the protective capabilities of the response.

T-cell exhaustion: characteristics, causes and conversion.

T-cell exhaustion is characterized by the stepwise and progressive loss of T-cell functions and can culminate in the physical deletion of the responding cells. Exhaustion is well-defined during chronic lymphocytic choriomeningitis virus infection and commonly develops under conditions of antigen-persistence, which occur following many chronic infections that are of significant public health concern including hepatitis B virus, hepatitis C virus and human immunodeficiency virus infections, as well as during tumour outgrowth. Exhaustion is not a uniformly disabled setting as a gradation of phenotypic and functional defects can manifest, and these cells are distinct from prototypic effector, memory and also anergic T cells. We are gaining insights into the extrinsic and intrinsic factors that determine the severity of exhaustion. These include the duration and magnitude of antigenic activation, availability of CD4 T-cell help, the levels of stimulatory and suppressive cytokines, as well as the expression of activatory and inhibitory receptors. More information is now becoming available regarding the molecular mechanisms that attenuate the responsiveness of exhausted T cells. As the parameters that dictate exhaustion are more thoroughly defined, this is fostering the development of methods that prevent and rejuvenate functionally inferior responses. In this article we discuss our current understanding of the properties of exhausted T cells and the mechanisms that promote and maintain this state.

Shaping successful and unsuccessful CD8 T cell responses following infection.

CD8 T cells play a vital role in the immunological protection against intracellular pathogens. Ideally, robust effector responses are induced, which eradicate the pathogen, and durable memory CD8 T cells are also established, which help confer protection against subsequent reinfection. The quality and magnitude of these responses is dictated by multiple factors, including their initial interactions with professional antigen-presenting cells, as well as the cytokine milieu and availability of CD4 T cell help. These factors set the transcriptional landscape of the responding T cells, which in turn influences their phenotypic and functional attributes as well as ultimate fate. Under certain conditions, such as during chronic infections, the development of these usually successful responses becomes subverted. Here we discuss advances in our understanding of the cellular and molecular determinants of T cell quality, and the formation of effector, memory, and exhausted CD8 T cells, during acute and chronic infections.

IL-10-producing Tfh cells accumulate with age and link inflammation with age-related immune suppression.

Aging results in profound immune dysfunction, resulting in the decline of vaccine responsiveness previously attributed to irreversible defects in the immune system. In addition to increased interleukin-6 (IL-6), we found aged mice exhibit increased systemic IL-10 that requires forkhead box P3-negative (FoxP3-), but not FoxP3+, CD4+T cells. Most IL-10-producing cells manifested a T follicular helper (Tfh) phenotype and required the Tfh cytokines IL-6 and IL-21 for their accrual, so we refer to them as Tfh10 cells. IL-21 was also required to maintain normal serum levels of IL-6 and IL-10. Notably, antigen-specific Tfh10 cells arose after immunization of aged mice, and neutralization of IL-10 receptor signaling significantly restored Tfh-dependent antibody responses, whereas depletion of FoxP3+ regulatory and follicular regulatory cells did not. Thus, these data demonstrate that immune suppression with age is reversible and implicate Tfh10 cells as an intriguing link between "inflammaging" and impaired immune responses with age.

Immune Exhaustion: Past Lessons and New Insights from Lymphocytic Choriomeningitis Virus.

Lymphocytic choriomeningitis virus (LCMV) is a paradigm-forming experimental system with a remarkable track record of contributing to the discovery of many of the fundamental concepts of modern immunology. The ability of LCMV to establish a chronic infection in immunocompetent adult mice was instrumental for identifying T cell exhaustion and this system has been invaluable for uncovering the complexity, regulators, and consequences of this state. These findings have been directly relevant for understanding why ineffective T cell responses commonly arise during many chronic infections including HIV and HCV, as well as during tumor outgrowth. The principal feature of exhausted T cells is the inability to elaborate the array of effector functions necessary to contain the underlying infection or tumor. Using LCMV to determine how to prevent and reverse T cell exhaustion has highlighted the potential of checkpoint blockade therapies, most notably PD-1 inhibition strategies, for improving cellular immunity under conditions of antigen persistence. Here, we discuss the discovery, properties, and regulators of exhausted T cells and highlight how LCMV has been at the forefront of advancing our understanding of these ineffective responses.