Rafi Ahmed

Summary: Antigen‐specific T cells play a pivotal role in adaptive immune responses. However, they also contribute to the progression of a variety of diseases including autoimmune disorders, graft rejection and graft‐versus‐host disease (GVHD). Non‐specific immune‐ablation treatments compromise the ability of the host to respond to infection, whereas the selective removal of epitope‐specific T cells could theoretically ameliorate T‐cell‐mediated pathology while preserving the rest of the host immune function. In this study we investigated whether it is possible to destroy specific unwanted antigen‐specific T cells by incubating polyclonal T‐cell populations with major histocompatibility complex (MHC) tetramers that are conjugated to the ribosomal‐inactivating toxin, saporin. This strategy resulted in a dramatic reduction in the number of targeted antigen (Ag)‐specific CD8 T cells with no observable bystander toxicity in vitro. Moreover, in a model of transferable T‐cell‐dependent neurological disease induced by intracerebral (i.c.) lymphocytic choriomeningitis virus (LCMV) infection, the targeted killing of LCMV‐specific CD8 T cells extended the survival of mice or fully prevented their death, depending on the dose of cells transferred. In addition, the tetramer– saporin conjugate also reduced liver damage in a model of donor T‐cell‐mediated hepatic destruction. These data provide a proof of principle that MHC tetramers could be exploited for the elimination or clinical manipulation of T‐cell responses by linking effector molecules (a toxin in this case) to MHC tetramers. Also, the results suggest that it may be feasible to remodel T‐cell responses, especially in immunocompromised hosts who receive adoptive cell transfers with many potential alloreactive cells.

MTOR, part of the PI3K–AKT–mTOR cell-signalling cascade and a target for the antitumour drug rapamycin, is identified here as a regulator of CD8 T-cell differentiation. Despite its primarily immunosuppressive activity, rapamycin can enhance immune responses to experimental vaccines in mice and monkeys. This counterintuitive finding points to the ability to increase the functional qualities of memory T cells as a potential way of enhancing the efficacy of vaccines against pathogens and cancers. Inducing effective memory T-cell responses is a major goal of vaccines against chronic infections and tumours. Here, mTOR, the mammalian target for the immunosuppressive drug rapamycin, is shown to enhance the quantity and quality of virus-specific CD8 T cells in mouse and non-human primate models. Memory CD8 T cells are a critical component of protective immunity, and inducing effective memory T-cell responses is a major goal of vaccines against chronic infections and tumours1,2,3. Considerable effort has gone into designing vaccine regimens that will increase the magnitude of the memory response, but there has been minimal emphasis on developing strategies to improve the functional qualities of memory T cells4. Here we show that mTOR (mammalian target of rapamycin5, also known as FRAP1) is a major regulator of memory CD8 T-cell differentiation, and in contrast to what we expected, the immunosuppressive drug rapamycin has immunostimulatory effects on the generation of memory CD8 T cells. Treatment of mice with rapamycin following acute lymphocytic choriomeningitis virus infection enhanced not only the quantity but also the quality of virus-specific CD8 T cells. Similar effects were seen after immunization of mice with a vaccine based on non-replicating virus-like particles. In addition, rapamycin treatment also enhanced memory T-cell responses in non-human primates following vaccination with modified vaccinia virus Ankara. Rapamycin was effective during both the expansion and contraction phases of the T-cell response; during the expansion phase it increased the number of memory precursors, and during the contraction phase (effector to memory transition) it accelerated the memory T-cell differentiation program. Experiments using RNA interference to inhibit expression of mTOR, raptor (also known as 4932417H02Rik) or FKBP12 (also known as FKBP1A) in antigen-specific CD8 T cells showed that mTOR acts intrinsically through the mTORC1 (mTOR complex 1) pathway to regulate memory T-cell differentiation. Thus these studies identify a molecular pathway regulating memory formation and provide an effective strategy for improving the functional qualities of vaccine- or infection-induced memory T cells.

Many persistent viral infections induce dysfunctional T cell responses. Although a negative correlation exists between viral load and T cell responses during chronic infection, it is not known whether high antigen levels are the cause or just the consequence of T cell exhaustion. Furthermore, it is unclear what role antigen presentation by bone-marrow (BM) derived versus infected parenchymal cells has on T cell exhaustion. To address these issues, we examined the influence of antigen presentation by different cell types on CD8 ⁺ T cell responses during persistent infection of mice with lymphocytic choriomeningitis virus (LCMV) clone 13. We generated BM chimeric mice, in which non-BM derived cells were MHC class I deficient. Virus-specific CD8 ⁺ T cells in lymphoid and nonlymphoid tissues were increased in both number and ability to produce cytokines in these mice soon after infection. However, viral clearance from infected MHC I ^−/− parenchyma was significantly impaired, despite increased populations of cytokine producing CTL. The CD8 ⁺ T cell response was overwhelmed by sustained antigen persistence, becoming increasingly exhausted within 4–6 weeks. Thus, we find that ( i ) sustained antigen presentation directly drives T cell exhaustion during a chronic viral infection, ( ii ) CTL require direct antigen-MHC interactions to clear virus-infected cells, and ( iii ) persistent interactions with antigen presented on both hematopoietic and nonhematopoietic cells negatively impacts virus-specific T cell responses during chronic infection.

During some persistent viral infections, virus-specific T-cell responses wane due to the antigen-specific deletion or functional inactivation (i.e., exhaustion) of responding CD8 T cells. T-cell exhaustion often correlates with high viral load and is associated with the expression of the inhibitory receptor PD-1. In other infections, functional T cells are observed despite high levels of pathogen persistence. The reasons for these different T-cell fates during chronic viral infections are not clear. Here, we tracked the fate of virus-specific CD8 T cells in lymphocytic choriomeningitis virus (LCMV)-infected mice during viral clearance, the persistence of wild-type virus, or the selection and persistence of a viral variant that abrogates the presentation of a single epitope. Viral clearance results in PD-1 ^lo functional virus-specific CD8 T cells, while the persistence of wild-type LCMV results in high PD-1 levels and T-cell exhaustion. However, following the emergence of a GP35V→A variant virus that abrogates the presentation of the GP33 epitope, GP33-specific CD8 T cells remained functional, continued to show low levels of PD-1, and reexpressed CD127, a marker of memory T-cell differentiation. In the same animals and under identical environmental conditions, CD8 T cells recognizing nonmutated viral epitopes became physically deleted or were PD-1 ^hi and nonfunctional. Thus, the upregulation of PD-1 and the functional inactivation of virus-specific T cells during chronic viral infection is dependent upon continued epitope recognition. These data suggest that optimal strategies for vaccination should induce high-magnitude broadly specific T-cell responses that prevent cytotoxic T-lymphocyte escape and highlight the need to evaluate the function of vaccine-induced T cells in the context of antigens presented during virus persistence.

Summary: The lymphoid tissues are characterized by a complex organized architecture that is supported by a network of stromal cells. These stromal cells play many important roles in addition to serving as structural support. Lymphoid stromal cells, including the specialized fibroblastic reticular cells and follicular dendritic cells, express chemokines, cytokines, adhesion molecules, as well as other factors required for the migration, homeostasis, and survival of immune cells. Studies have demonstrated the dynamic role that lymphoid stromal cells and the chemokines they produce play in directing lymphocyte migration within the spleen and lymph nodes. The stromal network may also play roles in influencing antigen presentation, via adherence of antigen‐presenting cells to the network, and cell survival, via provision of survival factors such as interleukin‐7. Recently, we have shown that dramatic changes in lymphoid chemokine expression occur during many immune responses. This can alter the trafficking and localization of immune cells in lymphoid organs and has many implications for the regulation and shut‐down of immune responses. Here we briefly summarize the roles of stromal cells and lymphoid architecture in the control of immune responses.

Summary: One potentially promising strategy to control chronic infections such as human immunodeficiency virus, hepatitis B virus, and hepatitis C virus is therapeutic vaccination, which aims to reduce persisting virus by stimulating a patient's own antiviral immune responses. However, this approach has fallen short of expectations, because antiviral T cells generated during chronic infections often become functionally exhausted and thus do not respond properly to therapeutic vaccination. Therefore, it is necessary to develop a therapeutic vaccine strategy to more effectively boost endogenous T‐cell responses to control persistent viral infections. Studies to elucidate the cause of impaired T‐cell function have pointed to sustained inhibitory receptor signaling through T‐cell expression of programmed death 1 (PD‐1). Recently, another inhibitory molecule, cytotoxic T lymphocyte antigen 4 (CTLA‐4), and also an immunosuppressive cytokine, interleukin 10 (IL‐10), have been reported to be potential factors of establishing immune suppression and viral persistence. Blocking these negative signaling pathways could restore the host immune system, enabling it to respond to further stimulation. Indeed, combining therapeutic vaccination along with the blockade of inhibitory signals could synergistically enhance functional CD8⁺ T‐cell responses and improve viral control in chronically infected mice, providing a promising strategy for the treatment of chronic viral infections. Furthermore, not only the ablation of negative signals but also the addition of stimulatory signals, such as interleukin 2 (IL‐2), might prove to be a potentially promising strategy to augment the efficacy of therapeutic vaccination against chronic viral infections.

Therapeutic vaccination is a potentially promising strategy to enhance T cell immunity and viral control in chronically infected individuals. However, therapeutic vaccination approaches have fallen short of expectations, and effective boosting of antiviral T cell responses has not always been observed. One of the principal reasons for the limited success of therapeutic vaccination is that virus-specific T cells become functionally exhausted during chronic infections. We now provide a novel strategy for enhancing the efficacy of therapeutic vaccines. In this study, we show that blocking programmed death (PD)-1/PD-L1 inhibitory signals on exhausted CD8+ T cells, in combination with therapeutic vaccination, synergistically enhances functional CD8+ T cell responses and improves viral control in mice chronically infected with lymphocytic choriomeningitis virus. This combinatorial therapeutic vaccination was effective even in the absence of CD4+ T cell help. Thus, our study defines a potent new approach to augment the efficacy of therapeutic vaccination by blocking negative signals. Such an approach may have broad applications in developing treatment strategies for chronic infections in general, and perhaps also for tumors.

An important question in memory development is understanding the differences between effector CD8 T cells that die versus effector cells that survive and give rise to memory cells. In this study, we provide a comprehensive phenotypic, functional, and genomic profiling of terminal effectors and memory precursors. Using killer cell lectin-like receptor G1 as a marker to distinguish these effector subsets, we found that despite their diverse cell fates, both subsets possessed remarkably similar gene expression profiles and functioned as equally potent killer cells. However, only the memory precursors were capable of making interleukin (IL) 2, thus defining a novel effector cell that was cytotoxic, expressed granzyme B, and produced inflammatory cytokines in addition to IL-2. This effector population then differentiated into long-lived protective memory T cells capable of self-renewal and rapid recall responses. Experiments to understand the signals that regulate the generation of terminal effectors versus memory precursors showed that cells that continued to receive antigenic stimulation during the later stages of infection were more likely to become terminal effectors. Importantly, curtailing antigenic stimulation toward the tail end of the acute infection enhanced the generation of memory cells. These studies support the decreasing potential model of memory differentiation and show that the duration of antigenic stimulation is a critical regulator of memory formation.

The memory T cell pool serves as a relatively long‐lived heterogeneous repository of antigen‐experienced T cells that "remember" previous encounters with antigen. While heterogeneity in the memory T cell pool is now well established, signals regulating the generation of this memory T cell heterogeneity are not fully understood. Two articles in this issue of the European Journal of Immunology highlight the importance of the strength of antigenic stimulation in regulating the generation of phenotypically and functionally distinct CD4⁺ T cell memory subsets. New insights are also provided into key molecular players that likely mediate differences in homeostatic and secondary expansion between the memory subsets.See accompanying articles: http://dx.doi.org/10.1002/eji200737611 and http://dx.doi.org/10.1002/eji200737852

CD8 T cells comprise a powerful branch of the adaptive immune system, yet were not formally recognized until long after the discovery of antibody. CD8 T cells contribute to the eradication of intracellular infections and to the control of many chronic infections. There is tempered optimism that CD8 T cell memory elicited via vaccination may hold the key to manufacturing protective immunity against pathogens that cause chronic infections in humans. The specificity and destructive capabilities of CD8 T cells may also be harnessed for the eradication of tumors. However, CD8 T cells also contribute to a variety of clinical difficulties such as immune mediated pathology, rejection of organ transplants, and autoimmunity. Clearly, learning how to safely generate protective long-lived CD8 T cell memory and how to control or eliminate specific unwanted responses could deliver substantial clinical rewards, and there is a great need for continued research on the biology and therapeutic potential of these cells. Herein, we recount the historical developments leading to the discovery of CD8 T cells, highlight a few of the important discoveries that have followed, and discuss some of the critical issues on the horizon.