[Abstract unavailable

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 tumors 1-3. 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 cells 4. In this study we show that mTOR, the mammalian target of rapamycin 5, is a major regulator of memory CD8 T cell differentiation and in contrast to what we expected the mTOR specific inhibitor rapamycin, an immunosuppressive drug, had surprising immunostimulatory effects on the generation of memory CD8 T cells. Treatment of mice with rapamycin following acute lymphocytic choriomeningitis virus (LCMV) 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 non-replicating VLP based vaccine. In addition, rapamycin treatment also enhanced memory T cell responses in non-human primates following vaccination with MVA (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 RNAi to inhibit mTOR, raptor or FKBP12 expression in antigen specific CD8 T cells showed that mTOR acts intrinsically through the mTORC1 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.

Inhibitory receptors play a crucial role in regulating CD8 T-cell function during chronic viral infection. T-cell Ig-and mucin-domaincontaining molecule-3 (Tim-3) is well known to negatively regulate T-cell responses, but its role in CD8 T-cell exhaustion during chronic infection in vivo remains unclear. In this study, we document coregulation of CD8 T cell exhaustion by Tim-3 and PD-1 during chronic lymphocytic choriomeningitis virus infection. Whereas Tim-3 was only transiently expressed by CD8 T cells after acute infection, virus-specific CD8 T cells retained high Tim-3 expression throughout chronic infection. The majority (approximately 65% to 80%) of lymphocytic choriomeningitis virus-specific CD8 T cells in lymphoid and nonlymphoid organs coexpressed Tim-3 and PD-1. This coexpression of Tim-3 and PD-1 was associated with more severe CD8 T-cell exhaustion in terms of proliferation and secretion of effector cytokines such as IFN-γ, TNF-α, and IL-2. Interestingly, CD8 T cells expressing both inhibitory receptors also produced the suppressive cytokine IL-10. Most importantly, combined blockade of Tim-3 and PD-1 pathways in vivo synergistically improved CD8 T cell responses and viral control in chronically infected mice. Taken together, our study defines a parameter for determining the severity of CD8 T cell dysfunction and for identifying virus-specific CD8 T cells that produce IL-10, and shows that targeting both PD-1 and Tim-3 is an effective immune strategy for treating chronic viral infections.

Programmed cell death-1 (PD-1) targeted therapies enhance T cell responses and show efficacy in multiple cancers but the role of costimulatory molecules in this T cell rescue remains elusive. Here we demonstrate that the CD28/B7 costimulatory pathway is essential for effective PD-1 therapy during chronic viral infection of mice. Conditional gene deletion showed a cell-intrinsic requirement of CD28 for CD8 T cell proliferation after PD-1 blockade. B7-costimulation was also necessary for effective PD-1 therapy in tumor-bearing mice. In addition, we found that CD8 T cells proliferating in blood after PD-1 therapy of lung cancer patients were predominantly CD28 positive. Taken together these data demonstrate CD28-costimulation requirement for CD8 T cell rescue and suggest an important role for CD28/B7 pathway in PD-1 therapy of cancer patients.

Summary Vaccines work by eliciting an immune response and consequent immunological memory that mediates protection from infection or disease. Recently new methods have been developed to dissect the immune response in experimental animals and humans which have led to increased understanding of the molecular mechanisms that control differentiation and maintenance of memory T and B cells. In this review we will provide an overview of the cellular organization of immune memory and underline some of the outstanding questions on immunological memory and how they pertain to vaccination strategies. Finally we will discuss how we can learn about antigen design from the interrogation of our memory T and B cells – a journey from vaccines to memory and back.

Antigen-specific CD8 T cells are central to the control of chronic infections and cancer, but persistent antigen stimulation results in T cell exhaustion. Exhausted CD8 T cells have decreased effector function and proliferative capacity, partly caused by overexpression of inhibitory receptors such as programmed cell death (PD)-1. Blockade of the PD-1 pathway has opened a new therapeutic avenue for reinvigorating T cell responses, with positive outcomes especially for patients with cancer. Other strategies to restore function in exhausted CD8 T cells are currently under evaluation-many in combination with PD-1-targeted therapy. Exhausted CD8 T cells comprise heterogeneous cell populations with unique differentiation and functional states. A subset of stem cell-like PD-1 CD8 T cells responsible for the proliferative burst after PD-1 therapy has been recently described. A greater understanding of T cell exhaustion is imperative to establish rational immunotherapeutic interventions.

Memory CD8 T cells that circulate in the blood and are present in lymphoid organs are an essential component of long-lived T cell immunity. These resting memory CD8 T cells remain poised to rapidly elaborate effector functions upon re-exposure to pathogen, but also have many properties in common with naïve cells, including the ability to migrate to lymph nodes and spleen, and their pluri-potency. Thus, memory cells embody features of both naïve and effector cells, fueling a long-standing debate centered on whether memory T cells develop from effector cells or directly from naïve cells1–4. To better define the developmental path of memory CD8 T cells we investigated changes in DNA methylation programming at naïve and effector genes in virus specific CD8 T cells during acute LCMV infection of mice. Methylation profiling of effector CD8 T cell subsets at day 4 and 8 after infection showed that, rather than retaining a naïve epigenetic state, the subset of cells that gives rise to memory cells acquired de novo DNA methylation programs at naïve-associated genes and became demethylated at loci of classically defined effector molecules. Conditional deletion of the de novo methyltransferase, Dnmt3a, at an early stage of effector differentiation strikingly reduced methylation of naïve-associated genes and resulted in faster re-expression of these naïve genes, accelerating memory cell development. Longitudinal phenotypic and epigenetic characterization of virus-specific memory-precursor CD8 T cells transferred into antigen-free mice revealed that their differentiation into memory cells was coupled to cell-division independent erasure of de novo methylation programs and re-expression of naïve-associated genes. These data provide evidence that epigenetic repression of naïve-associated genes in effector CD8 T cells can be reversed in cells that develop into long-lived memory CD8 T cells supporting a differentiation model where memory T cells arise from a subset of fate-permissive effector T cells.

The importance of autophagy in memory CD8 T cell differentiation in vivo is not well defined. We show here that autophagy is dynamically regulated in virus-specific CD8 T cells during acute lymphocytic choriomeningitis virus infection. Autophagy decreased in activated proliferating T cells, and was then upregulated at the peak of the effector T cell response. Consistent with this model, deletion of the key autophagy genes Atg7 or Atg5 in virus-specific CD8 T cells had minimal effect on generating effector cells but greatly enhanced their death during the contraction phase resulting in compromised memory formation. These findings provide insight into when autophagy is needed during effector and memory T cell differentiation in vivo and also warrant a re-examination of our current concepts about the relationship between T cell activation and autophagy.