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The migratory patterns of virus-specific CD8 T cells during chronic viral infection are not well understood. To address this issue, we have done parabiosis experiments during chronic lymphocytic choriomeningitis virus (LCMV) infection of mice. We found that despite the high frequency of virus-specific CD8 T cells in both lymphoid and nonlymphoid tissues there was minimal migration of virus-specific CD8 T cells between the chronically infected conjoined parabiont mice. This was in contrast to parabionts between mice that had undergone an acute LCMV infection where virus-specific CD8 T cells established equilibrium demonstrating circulation of memory T cells generated after viral clearance. We have identified a population of PD-1+ TCF1+CXCR5+Tim-3- stemlike virus-specific CD8 T cells that reside in lymphoid tissues and act as resource cells for maintaining the T cell response during chronic infection. These are the cells that proliferate and give rise to the more terminally differentiated PD-1+ CXCR5-Tim-3+ CD8 T cells. Both the stemlike CD8 T cells and their terminally differentiated progeny showed minimal migration during chronic infection and the few LCMV-specific CD8 T cells that were present in circulation were the recently emerging progeny from the stemlike CD8 T cells. The PD-1+ TCF1+CXCR5+ stemlike CD8 T cells were truly resident in lymphoid tissues and did not circulate in the blood. We propose that this residency in specialized niches within lymphoid tissues is a key aspect of their biology and is essential for maintaining their quiescence and stemlike program under conditions of a chronic viral infection., Competing Interests: The authors declare no competing interest.

T cell dysfunction is a characteristic feature of chronic viral infection and cancer. Recent studies in chronic lymphocytic choriomeningitis virus (LCMV) infection have defined a PD-1 + Tcf-1 + CD8 + T cell subset capable of self-renewal and differentiation into more terminally differentiated cells that downregulate Tcf-1 and express additional inhibitory molecules such as Tim3. Here, we demonstrated that expression of the glycoprotein CD101 divides this terminally differentiated population into two subsets. Stem-like Tcf-1 + CD8 + T cells initially differentiated into a transitory population of CD101 - Tim3 + cells that later converted into CD101 + Tim3 + cells. Recently generated CD101 - Tim3 + cells proliferated in vivo, contributed to viral control, and were marked by an effector-like transcriptional signature including expression of the chemokine receptor CX3CR1, pro-inflammatory cytokines, and granzyme B. PD-1 pathway blockade increased the numbers of CD101 - Tim3 + CD8 + T cells, suggesting that these newly generated transitional cells play a critical role in PD-1-based immunotherapy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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 memory CD8 T cells remain poised to rapidly elaborate effector functions upon re-exposure to pathogens, but also have many properties in common with naive cells, including pluripotency and the ability to migrate to the lymph nodes and spleen. Thus, memory cells embody features of both naive and effector cells, fuelling a long-standing debate centred on whether memory T cells develop from effector cells or directly from naive cells. Here we show that long-lived memory CD8 T cells are derived from a subset of effector T cells through a process of dedifferentiation. To assess the developmental origin of memory CD8 T cells, we investigated changes in DNA methylation programming at naive and effector cell-associated genes in virus-specific CD8 T cells during acute lymphocytic choriomeningitis virus infection in mice. Methylation profiling of terminal effector versus memory-precursor CD8 T cell subsets showed that, rather than retaining a naive epigenetic state, the subset of cells that gives rise to memory cells acquired de novo DNA methylation programs at naive-associated genes and became demethylated at the loci of classically defined effector molecules. Conditional deletion of the de novo methyltransferase Dnmt3a at an early stage of effector differentiation resulted in reduced methylation and faster re-expression of naive-associated genes, thereby accelerating the development of memory cells. Longitudinal phenotypic and epigenetic characterization of the memory-precursor effector subset of virus-specific CD8 T cells transferred into antigen-free mice revealed that differentiation to memory cells was coupled to erasure of de novo methylation programs and re-expression of naive-associated genes. Thus, epigenetic repression of naive-associated genes in effector CD8 T cells can be reversed in cells that develop into long-lived memory CD8 T cells while key effector genes remain demethylated, demonstrating that memory T cells arise from a subset of fate-permissive effector T cells.

Translation is a critical process in protein synthesis, but translational regulation in antigen-specific T cells in vivo has not been well defined. Here we have characterized the translatome of virus-specific CD8 + effector T cells (T eff cells) during acute infection of mice with lymphocytic choriomeningitis virus (LCMV). Antigen-specific T cells exerted dynamic translational control of gene expression that correlated with cell proliferation and stimulation via the T cell antigen receptor (TCR). The translation of mRNAs that encode translation machinery, including ribosomal proteins, was upregulated during the T cell clonal-expansion phase, followed by inhibition of the translation of those transcripts when the CD8 + T eff cells stopped dividing just before the contraction phase. That translational suppression was more pronounced in terminal effector cells than in memory precursor cells and was regulated by antigenic stimulation and signals from the kinase mTOR. Our studies show that translation of transcripts encoding ribosomal proteins is regulated during the differentiation of CD8 + T eff cells and might have a role in fate 'decisions' involved in the formation of memory cells.

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. 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 the CD28/B7 pathway in PD-1 therapy of cancer patients., (Copyright © 2017, American Association for the Advancement of Science.)

The importance of autophagy in the generation of memory CD8(+) T cells in vivo is not well defined. We report here that autophagy was dynamically regulated in virus-specific CD8(+) T cells during acute infection of mice with lymphocytic choriomeningitis virus. In contrast to the current paradigm, autophagy decreased in activated proliferating effector CD8(+) T cells and was then upregulated when the cells stopped dividing just before the contraction phase. Consistent with those findings, deletion of the gene encoding either of the autophagy-related molecules Atg5 or Atg7 had little to no effect on the proliferation and function of effector cells, but these autophagy-deficient effector cells had survival defects that resulted in compromised formation of memory T cells. Our studies define when autophagy is needed during effector and memory differentiation and warrant reexamination of the relationship between T cell activation and autophagy.

RNA interference (RNAi) is an intracellular mechanism for silencing gene expression utilizing short fragments of double-strand RNA that are complementary to the target messenger RNA. This gene silencing technique has now become an invaluable research tool due to its specific and strong repressive effect on a target transcript. We have recently applied a retrovirus-based RNAi system to investigate the in vivo role of the mammalian target of rapamycin (mTOR) in antigen-specific CD8 T cells, and have found that mTOR regulates memory CD8 T-cell differentiation. Here, we provide a detailed protocol for knocking down mTOR and its related molecules (raptor and FKBP12) in antigen-specific CD8 T cells. In our protocol, a mouse model of lymphocytic choriomeningitis virus infection is used, but the methods can be extended to other viral and bacterial infections as well as vaccinations. Also, the similar approach can be applied to analysis of CD4 T-cell responses.

To design successful vaccines for chronic diseases, an understanding of memory CD8(+) T cell responses to persistent antigen restimulation is critical. However, most studies comparing memory and naive cell responses have been performed only in rapidly cleared acute infections. Herein, by comparing the responses of memory and naive CD8(+) T cells to acute and chronic lymphocytic choriomeningitis virus infection, we show that memory cells dominated over naive cells and were protective when present in sufficient numbers to quickly reduce infection. In contrast, when infection was not rapidly reduced, because of high antigen load or persistence, memory cells were quickly lost, unlike naive cells. This loss of memory cells was due to a block in sustaining cell proliferation, selective regulation by the inhibitory receptor 2B4, and increased reliance on CD4(+) T cell help. Thus, emphasizing the importance of designing vaccines that elicit effective CD4(+) T cell help and rapidly control infection., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Previous studies have identified the inhibitory role that the programmed death 1 (PD-1) pathway plays during chronic infection. Blockade of this pathway results in rescue of viral-specific CD8 T cells, as well as reduction of viral loads in mice chronically infected with lymphocytic choriomeningitis virus (LCMV). We tested the effect of combining PD ligand 1 (PD-L1) blockade with an agonistic regimen that induces 4-1BB costimulation during chronic LCMV infection. There is a boosting effect in the rescue of LCMV-specific CD8 T cell responses after dual treatment with PD-L1 blockade and 4-1BB agonistic Abs when the amount and timing of 4-1BB costimulation are carefully controlled. When PD-L1-blocking Abs are given together with a single low dose of anti-4-1BB agonistic Abs, there is an enhanced and stable expansion of viral-specific CD8 T cells. Conversely, when blocking Abs to PD-L1 are given with a repetitive high dose of anti-4-1BB, there is an initial synergistic expansion of viral-specific CD8 T cells by day 7, followed by dramatic apoptosis by day 14. Viral control paralleled CD8 T cell kinetics after dual treatment. By day 7 posttreatment, viral titers were lower in both of the combined regimens (compared with PD-L1 blockade alone). However, whereas the high dose of anti-4-1BB plus PD-L1 blockade resulted in rebound of viral titers to original levels, the low dose of anti-4-1BB plus PD-L1 blockade resulted in a stable reduction of viral loads. These findings demonstrate the importance of carefully manipulating the balance between activating and inhibitory signals to enhance T cell responses during chronic infection.

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.

A major unanswered question is what distinguishes the majority of activated CD8 T cells that die after an acute viral infection from the small fraction (5-10%) that survive to become long-lived memory cells. In this study we show that increased expression of the interleukin 7 receptor alpha-chain (IL-7Ralpha) identifies the effector CD8 T cells that will differentiate into memory cells. IL-7R(hi) effector cells contained increased amounts of antiapoptotic molecules, and adoptive transfer of IL-7R(hi) and IL-7R(lo) effector cells showed that IL-7R(hi) cells preferentially gave rise to memory cells that could persist and confer protective immunity. Thus, selective expression of IL-7R identifies memory cell precursors, and this marker may be useful in predicting the number of memory T cells generated after infection or immunization.