The lifestyle of naturally occurring CD4+CD25+Foxp3+ regulatory T cells

Summary: Numerous studies over the past 10 years have demonstrated the importance of naturally occurring CD4⁺CD25⁺Foxp3⁺ regulatory T cells (nTregs) in immune regulation. We analyzed the mechanism of action of nTregs in a well‐characterized model of autoimmune gastritis and demonstrated that nTregs act at an early stage of disease progression to inhibit the differentiation of naïve T cells to pathogenic T‐helper 1 effectors. The effects of nTregs in this model are not antigen‐specific but are mediated by activation of the nTregs by ubiquitous self‐peptide major histocompatibility complex class II complexes together with cytokines released by activated effector cells. Studies in vitro confirmed that some nTregs exist in an activated state in vivo and can be activated to exert non‐specific suppressor effector function by stimulation with interleukin‐2 in the absence of engagement of their T‐cell receptor. Natural Tregs can differentiate in vitro to exhibit potent granzyme B‐dependent, partially perforin‐independent cytotoxic cells that are capable of specifically killing antigen‐presenting B cells. Natural Treg‐mediated killing of antigen‐presenting cells may represent one pathway by which they can induce long‐lasting suppression of autoimmune disease.     

Making sense of inflammation, epigenetics, and memory CD8+ T-cell differentiation in the context of infection

Summary: Recent findings suggest a new paradigm that early inflammatory cytokines promote the effector T‐cell response while inhibiting the development of CD8⁺ T‐cell memory. Although this opposing effect may appear paradoxical at first, it makes biological sense in the context of an infection, by ensuring a maximal effector response that will clear the pathogen. Once infection is controlled, the withdrawal of inflammatory cytokines allows the differentiation of effectors into long‐lived memory cells that provide protective immunity against re‐infection. Memory T cells differ from naïve T cells in their responsiveness to stimulation, which leads to the rapid expression of effector functions. The molecular basis for enhanced functionality of memory T cells remains largely unknown. Recent results indicate that certain epigenetic changes are imprinted in memory T cells that play an important role in keeping them poised to respond immediately upon antigen re‐encounter. These epigenetic modifications occur as naïve T cells become activated and are influenced by factors that regulate memory formation. Thus, epigenetic changes are an integral component of memory T‐cell differentiation, while inflammation plays an unexpected regulatory role in the process. These advances in our understanding of T‐cell memory will undoubtedly help design unconventional vaccine strategies for inducing large populations of long‐lived and functional memory CD8⁺ T cells.     

Developing and maintaining protective CD8+ memory T cells

Summary: A critical aim of vaccine‐related research is to identify the mechanisms by which memory T cells are formed and maintained over long periods of time. In recent years, we have designed experiments aimed at addressing two key questions: (i) what are the factors that maintain functionally responsive CD8⁺ memory cells over long periods of time, and (ii) what are the signals during the early stages of infection that drive the differentiation of long‐lived CD8⁺ memory T cells? We have identified a role for CD4⁺ T cells in the generation of CD8⁺ T‐cell‐mediated protection from secondary challenge. While CD4⁺ T cells appear to play a role in the programme of CD8 memory, we find that they are also required for the long‐term maintenance of CD8⁺ memory T‐cell numbers and function. This property is independent of CD40–CD40L interactions, and we propose a role for CD4⁺ T cells in maintaining the ability of CD8⁺ memory T cells to respond to interleukin‐7 (IL‐7) and IL‐15. By manipulating both the time course of infection and the timing of antigen presentation to newly recruited CD8⁺ T cells, we also demonstrate that the programming of effector and memory potential are at least partially distinct processes.     

CD4+ memory T cells: functional differentiation and homeostasis

Summary: CD4⁺ T cells are central regulators of both humoral and cellular immune responses. There are many subsets of CD4⁺ T cells, the most prominent being T‐helper 1 (Th1), Th2, Th‐17, and regulatory T cells, specialized in regulating different aspects of immunity. Without participation by these CD4⁺ T‐cell subsets, B cells cannot undergo isotype switching to generate high‐affinity antibodies, the microbicidal activity of macrophages is reduced, the efficiency of CD8⁺ T‐cell responses and CD8⁺ T‐cell memory are compromised, and downregulation of effector responses is impaired. It therefore stands to reason that memory CD4⁺ T cells are likely to fulfill an important facilitator role in the maintenance and control of protective immune responses. This review discusses some issues of importance for the generation of memory CD4⁺ T cells and focuses in particular on their heterogeneity and plasticity, with respect to both phenotypic characteristics and function. Finally, we discuss a number of factors that affect long‐term maintenance of memory CD4⁺ T cells.     

JNK and p38 MAP kinases in CD4+ and CD8+ T cells

Summary: The c‐Jun aminoterminal kinase (JNK) and p38 mitogen‐activated protein (MAP) kinase signaling pathways have been associated with cell death, differentiation and proliferation. CD4⁺ and CD8⁺ T cells have different effector functions after antigen stimulation and control specific aspects of the immune response. The studies carried out in our group indicate that the role of JNK and p38 MAP kinases in CD4⁺ T cells is different from their role in CD8⁺ T cells. Moreover, these two pathways are not redundant in either T cell population. We have also shown that p38 MAP kinase regulates early stages of T cell development in the thymus. It is therefore important to consider the specific function of these kinases in each T cell population when pharmacological inhibitors of JNK and p38 MAP kinases are used for therapeutic purposes to control the immune response.     

Signals required for programming effector and memory development by CD8+ T cells

Summary: Stimulation of naïve CD8⁺ T cells with antigen and costimulation results in proliferation and weak clonal expansion, but the cells fail to develop effector functions and are tolerant long term. Initiation of the program leading to the strong expansion and development of effector functions and memory requires a third signal that can be provided by interleukin‐12 (IL‐12) or interferon‐α (IFN‐α). CD4⁺ T cells condition dendritic cells (DCs) to effectively present antigen to CD8⁺ T cells, and this conditioning involves, at least in part, CD40‐dependent upregulation of the production of these signal 3 cytokines by the DCs. Upon being fully activated, the cytotoxic T lymphocytes develop activation‐induced non‐responsiveness (AINR), a form of split anergy characterized by an inability to produce IL‐2 to support continued expansion. If antigen remains present, IL‐2 provided by CD4⁺ T cells can reverse AINR to allow further expansion of the effector population and conversion to responsive memory cells following antigen clearance. If IL‐2 or potentially other proliferative signals are not available, persistent antigen holds cells in the AINR state and prevents the development of a responsive memory population. Thus, in addition to antigen and costimulation, CD8⁺ T cells require cytokine signals at distinct stages of the response to be programmed for optimal generation of effector and memory populations.     

CD8+ T‐cell memory in tumor immunology and immunotherapy

Summary: The cellular and molecular mechanisms underlying the formation of distinct central, effector, and exhausted CD8⁺ T‐cell memory subsets were first described in the setting of acute and chronic viral diseases. The role of these T‐cell memory subsets are now being illuminated as relevant to the tumor‐bearing state. The generation and persistence of productive CD8⁺ T‐cell memory subsets is determined, in part, by antigen clearance, costimulation, responsiveness to homeostatic cytokines, and CD4⁺ T‐helper cells. By contrast, chronic exposure to antigen, negative costimulation, and immunomodulation by CD4⁺ T regulatory cells corrupt productive CD8⁺ T memory formation. It has become clear from human and mouse studies that the mere generation of CD8⁺ T‐cell memory is not a ‘surrogate marker’ for cancer vaccine efficacy. Some current cancer vaccine strategies may fail because they amplify, rather than correct or reset, the corrupted CD8⁺ memory population. Thus, much of the present effort in the development of vaccines for cancer and chronic infectious diseases is aimed at creating effective memory responses. Therapeutic vaccines for cancer and chronic infectious diseases may achieve consistent efficacy by ablation of the dysfunctional immune state and the provision of newly generated, non‐corrupted memory cells by adoptive cell transfer.     

Role of TCR specificity in CD4+CD25+ regulatory T-cell selection

Summary: CD4⁺CD25⁺ regulatory T cells play a crucial role in preventing autoimmune disease and can also modulate immune responses in settings such as transplantation and infection. We have developed a transgenic mouse system in which the role that T‐cell receptor (TCR) specificity for self‐peptides plays in the formation of CD4⁺CD25⁺ regulatory T cells can be examined. We have shown that interactions with a single self‐peptide can induce thymocytes bearing an autoreactive TCR to undergo selection to become CD4⁺CD25⁺ regulatory T cells and that thymocytes bearing TCRs with low affinity for the selecting peptide do not appear to undergo selection into this pathway. In addition, thymocytes with identical specificity for the selecting self‐peptide can undergo overt deletion versus abundant selection to become CD4⁺CD25⁺ regulatory T cells in response to variations in expression of the selecting peptide in different lineages of transgenic mice. Finally, we have shown that CD4⁺CD25⁺ T cells proliferate in response to their selecting self‐peptide in the periphery, but these cells do not proliferate in response to lymphopenia in the absence of the selecting self‐peptide. These studies are determining how the specificity of the TCR for self‐peptides directs the thymic selection and peripheral expansion of CD4⁺CD25⁺ regulatory T cells.     

Programming, demarcating, and manipulating CD8+ T-cell memory

Summary: In response to infection, antigen‐specific CD8⁺ T cells undergo massive expansion in numbers, acquire effector mechanisms, and disseminate throughout the body. The expansion phase is followed by a contraction (death) phase, where 90–95% of antigen‐specific CD8⁺ T cells are eliminated. The remaining antigen‐specific CD8⁺ T cells form the initial memory pool, which can be stably maintained for life. In this review, we discuss evidence that early events after infection ‘program’ CD8⁺ T cells to expand, contract, and generate memory in a fashion that is largely insensitive to the duration of infection or antigen display. Recent data demonstrate, despite numerical stability, that memory CD8⁺ T‐cell populations undergo phenotypic and functional changes with time after immunization. However, the early suggestion that specific markers can be used to identify memory CD8⁺ T cells has not been supported by recent studies. Thus, we argue that specific functional characteristics, such as the ability to persist and undergo vigorous secondary expansion leading to elevated memory cell numbers, remain the best markers of ‘good’ memory cells. Finally, we discuss experimental approaches to manipulate and accelerate generation of CD8⁺ T cells with memory characteristics, and how these systems can inform both basic and applied immunology.     

Non-malignant clonal expansions of CD8+ memory T cells in aged individuals

Summary: CD8⁺ T cells provide a major line of defense against intracellular pathogens. Upon encounter with antigen, CD8⁺ T cells go through three distinct phases involving proliferation, contraction, and differentiation to become eventually long‐lived CD8⁺ memory T cells. CD8⁺ memory T cells provide long‐term protection against infection by intracellular pathogens. CD8⁺ memory T‐cell proliferation and survival are regulated by many factors, including cytokines, and CD8⁺ memory T cells are stably maintained over a period of months to years. In aged humans and mice, however, there are significant alterations to the CD8⁺ memory T‐cell compartment with frequent development of monoclonal expansions of CD8⁺ memory T cells in healthy individuals. Interestingly, CD8⁺ clonal expansions are not malignant and do not progress to lymphomas, suggesting that these cells must still be under certain constraints. In this review, we discuss our current understanding of factors that contribute to and regulate these CD8⁺ clonal expansions as well as the impact of CD8⁺ clonal expansions on immune function of the aged. In addition, we discuss similarities and differences between CD8⁺ clonal expansions observed in humans and mice, and we postulate that CD8⁺ clonal expansions represent a spectrum of biological outcomes ranging from antigen‐driven to antigen‐independent phenomena.     

The effect of age on the cognate function of CD4+ T cells

Summary: With increasing age, the ability to produce protective antibodies in response to immunization declines, resulting in reduced efficacy of vaccination. We have examined how reductions in CD4⁺ T‐cell function contribute to reduced humoral responses, using a model that allows us to compare identical numbers of antigen‐specific naive T cells from young and aged T‐cell receptor transgenic mice. Naive cells from aged mice exhibit reduced responses, both in vitro and in vivo. In vitro, responses of aged T cells can be enhanced by addition of interleukin (IL)‐2. In vivo, using an adoptive transfer model with young hosts, naive cells from aged mice exhibit significant reductions in cognate helper function, leading to reduced B‐cell expansion and differentiation. These age‐related defects could be overcome by prior in vitro T helper 2 effector generation with aged T cells. This improvement in cognate function of the aged effectors may be related to the enhancement of CD154 expression, which occurs on aged T cells in the presence of exogenous IL‐2. We also found no difference in B‐cell expansion and differentiation when young cells were transferred to young or aged hosts. Our results indicate that age‐related reductions in humoral responses are mainly due to defects in the cognate helper function of naive CD4⁺ T cells from aged individuals.     

CD4+CD25+ T cell depletion impairs tolerance induction in a murine model of asthma

Background Regulatory T cells (Tregs) are key players in controlling the development of airway inflammation. However, their role in the mechanisms leading to tolerance in established allergic asthma is unclear. Objective To examine the role of Tregs in tolerance induction in a murine model of asthma. Methods Ovalbumin (OVA) sensitized asthmatic mice were depleted or not of CD25⁺ T cells by anti‐CD25 PC61 monoclonal antibody (mAb) before intranasal treatment (INT) with OVA, then challenged with OVA aerosol. To further evaluate the respective regulatory activity of CD4⁺CD25⁺ and CD4⁺CD25^? T cells, both T cell subsets were transferred from tolerized or non‐tolerized animals to asthmatic recipients. Bronchoalveolar lavage fluid (BALF), T cell proliferation and cytokine secretion were examined. Results Intranasal treatment with OVA led to increased levels of IL‐10, TGF‐β and IL‐17 in lung homogenates, inhibition of eosinophil recruitment into the BALF and antigen specific T cell hyporesponsiveness. CD4⁺CD25⁺Foxp3⁺ T cells were markedly upregulated in lungs and suppressed in vitro and in vivo OVA‐specific T cell responses. Depletion of CD25⁺ cells before OVA INT severely hampered tolerance induction as indicated by a strong recruitment of eosinophils into BALF and a vigorous T cell response to OVA upon challenge. However, the transfer of CD4⁺CD25^? T cells not only suppressed antigen specific T cell responsiveness but also significantly reduced eosinophil recruitment as opposed to CD4⁺CD25⁺ T cells. As compared with control mice, a significantly higher proportion of CD4⁺CD25^? T cells from OVA treated mice expressed mTGF‐β. Conclusion Both CD4⁺CD25⁺ and CD4⁺CD25^? T cells appear to be essential to tolerance induction. The relationship between both subsets and the mechanisms of their regulatory activity will have to be further analyzed.     

Expansion of circulating Foxp3+CD25bright CD4+ T cells during specific venom immunotherapy

Background Venom immunotherapy (VIT) induces long‐lasting immune tolerance to hymenoptera venom antigens, but the underlying mechanisms are not yet clarified. Regulatory T cells are thought to play an important role in allergic diseases and tolerance induction during specific immunotherapy. Aim Characterize longitudinally the impact of VIT on the pool of circulating regulatory T cells. Methods Fourteen hymenoptera venom‐allergic patients with severe reactions (grades III–IV) were studied before, 6 and 12 months after starting ultra‐rush VIT. Freshly isolated peripheral blood mononuclear cells were surface stained with a panel of markers of T cell differentiation and intracellularly for CTLA‐4 and Foxp3 and analysed by flow cytometry. foxp3 mRNA was quantified by real‐time PCR. VIT responses were assessed by measuring specific IgG4 and IgE levels. Eleven individuals with no history of insect venom allergy were studied as controls. Results VIT induces a significant progressive increase in both the proportion and the absolute numbers of regulatory T cells defined as CD25^bright and/or Foxp3⁺ CD4⁺ T cells. These changes are not related to alterations in the expression of activation markers or imbalances in the naïve/memory T cell compartments. foxp3 mRNA levels also increased significantly during VIT. Of note, the increase in circulating regulatory T cell counts significantly correlates with the venom‐specific IgG4/IgE ratio shift. Conclusion VIT is associated with a progressive expansion of circulating regulatory T cells, supporting a role for these cells in tolerance induction.     

Dendritic cells expand antigen-specific Foxp3+CD25+CD4+ regulatory T cells including suppressors of alloreactivity

Summary: Thymic derived naturally occurring CD25⁺CD4⁺ T regulatory cells (Tregs) suppress immune responses, including transplantation. Here we discuss the capacity of dendritic cells (DCs) to expand antigen‐specific Tregs, particularly polyclonal Tregs directed to alloantigens. Initial studies have shown that mature DCs are specialized antigen‐presenting cells (APCs) for expanding antigen‐specific CD25⁺ CD4⁺ Tregs from TCR transgenic mice. When triggered by specific antigen, these Tregs act back on immature DCs to block the upregulation of CD80 and CD86 costimulatory molecules. More recently, DCs have been used to expand alloantigen‐specific CD25⁺CD4⁺ Tregs from the polyclonal repertoire in the presence of interleukin‐2 (IL‐2). Allogeneic DCs are much more effective than allogeneic spleen cells for expanding CD25⁺CD4⁺ Tregs. The DC‐expanded Tregs continue to express high levels of Foxp3, even without supplemental IL‐2, whereas spleen cells poorly sustain Foxp3 expression. When suppressive activity is tested, relatively small numbers of DC‐expanded CD25⁺CD4⁺ Tregs exert antigen‐specific suppression in the mixed leukocyte reaction (MLR), blocking immune responses to the original stimulating strain 10 times more effectively than to third party stimulating cells. DC‐expanded Tregs also retard graft versus host disease (GVHD) across full major histocompatibility complex (MHC) barriers. In vitro and in vivo, the alloantigen‐specific CD25⁺CD4⁺ Tregs are much more effective suppressors of transplantation reactions than polyclonal populations. We suggest that the expansion of Tregs from a polyclonal repertoire via antigen‐presenting DCs will provide a means for antigen‐specific control of unwanted immune reactions.     

Establishment and recall of CD8+ T-cell memory in a model of localized transient infection

Summary: The influenza A virus model of localized, transient respiratory infection provides a well‐defined experimental system for dissecting the induction and maintenance of CD8⁺ T‐cell memory. This review focuses on quantitative and qualitative aspects of the prominent D^bNP₃₆₆‐ and D^bPA₂₂₄‐specific CD8⁺ T‐cell responses in virus‐infected B6 mice. The different virus‐specific effector and memory sets are compared by phenotypic [CD62L, interleukin‐7 receptor‐α (IL‐7Rα), and IL‐15Rβ expression] and functional [interferon‐γ (IFN‐γ), tumor necrosis factor‐α (TNF‐α), and IL‐2 production] analyses. Most clonotypes [defined by T‐cell receptor (TCR) CDR3β sequence] generated during the acute phase of infection survive into memory, with those expressing the more consensus ‘canonical’ TCRs being the major contributors to the recall response. The extent of clonal expansion and the size of memory CD8⁺ T‐cell populations has been characterized for mice challenged with either wildtype or mutant viruses, where broadly equivalent D^bNP₃₆₆ and D^bPA₂₂₄ expression was achieved by disabling the peptides in their native configuration, then expressing them in the viral neuraminidase protein. Combining the clonotypic and antigen dose analyses led to a somewhat mechanistic conclusion that the magnitude of any virus‐specific CD8⁺ T‐cell response will be a direct function of antigen dose and the size of the naïve or memory CD8⁺ T‐cell precursor pool.     

Effector and memory CD8+ T cells as seen in immunity to malaria

Summary: Transgenic (Tg) mice carrying a T‐cell receptor (TCR) specific for a CD8⁺ T‐cell epitope expressed in pre‐erythrocytic stages of Plasmodium yoelii has proven to be a valuable tool to advance our understanding of this anti‐parasite T‐cell response, as it occurs in vivo. The visualization of CD8⁺ T cells in vivo and ex vivo greatly facilitated research aimed at characterizing basic features of this T‐cell response such as the kinetics of differentiation and proliferation and the in vivo antigen presentation. Importantly, this research unveiled the existence of early self‐regulatory mechanisms controlling the magnitude of the CD8⁺ T‐cell response and also identified CD4⁺ T cells as critical elements in the development of memory populations. This review discusses our recent research using Tg mice and highlights our progress in understanding the CD8⁺ T‐cell‐mediated immunity against malaria liver stages.     

Control of CD4+ T-cell memory by cytokines and costimulators

Summary: During T‐cell priming, cytokines and costimulatory molecules provide important signals that determine the magnitude and quality of the response. Although the functions of defined cytokines and costimulators in the primary T‐cell response are well characterized, much less is known about how these factors contribute to memory T‐cell development and survival. Since memory cells are thought to be long‐lived progeny of the primary response, it is conceivable that the same signals shaping initial T‐cell expansion and differentiation also contribute to memory generation. Here, we review evidence and show novel data on the role of the cytokines interleukin‐2 (IL‐2) and IL‐7 and the costimulator CD28 in CD4⁺ memory T‐cell development. We emphasize that transient IL‐2 and CD28 signals during priming imprint a long‐lasting survival advantage in primed T cells, thus contributing to the persistence of a memory population. The requirement for IL‐2 and CD28 signals is not linked to promoting T‐cell division and expansion but most likely due to their capacity to (i) promote effector cell differentiation; (ii) induce survival proteins, and, as we discuss in more detail; (iii) program expression of receptors for ‘memory survival factors’ such as IL‐7. Studies exploring the therapeutic potential of these insights are also discussed.