Chronic viral infections are characterized by a state of CD8+ T-cell dysfunction that is associated with expression of the programmed cell death 1 (PD-1) inhibitory receptor1–4. A better understanding of the mechanisms that regulate CD8+ T cell responses during chronic infection is required to improve immunotherapies that restore function in exhausted CD8+ T cells. Here we identify a population of virus-specific CD8+ T cells that proliferate after blockade of the PD-1 inhibitory pathway in mice chronically infected with lymphocytic choriomeningitis virus (LCMV). These LCMV-specific CD8+ T cells expressed the PD-1 inhibitory receptor but also expressed several costimulatory molecules such as ICOS and CD28. This CD8+ T cell subset was characterized by a unique gene signature that was related to that of CD4+ T follicular helper (TFH) cells, CD8+ T cell memory precursors and haematopoietic stem cell progenitors, but that was distinct from that of CD4+ TH1 cells and CD8+ terminal effectors. This CD8+ T cell population was found only in lymphoid tissues and resided predominantly in the T cell zones along with naïve CD8+ T cells. These PD-1+ CD8+ T cells resembled stem cells during chronic LCMV infection, undergoing self-renewal and also differentiating into the terminally exhausted CD8+ T cells that were present in both lymphoid and non-lymphoid tissues. The proliferative burst after PD-1 blockade came almost exclusively from this CD8+ T cell subset. Notably, the transcription factor TCF1 had a cell intrinsic and essential role in the generation of this CD8+ T cell subset. These findings provide a better understanding of T cell exhaustion and have implications in the optimization of PD-1-directed immunotherapy in chronic infections and cancer.

Summary CD4+ T follicular helper (Tfh) cells provide the required signals to B cells for germinal center reactions that are necessary for long-lived antibody responses. However, it remains unclear whether there are CD4+ memory T cells committed to the Tfh cell lineage after antigen clearance. Using adoptive transfer of antigen-specific memory CD4+ T cell subpopulations in the LCMV infection model, we found that there are distinct memory CD4+ T cell populations with commitment to either Tfh- or Th1-cell lineages. Our conclusions are based on gene expression profiles, epigenetic studies, and phenotypic and functional analyses. Our findings indicate that CD4+ memory T cells “remember” their previous effector lineage after antigen clearance, being poised to reacquire their lineage-specific effector functions upon antigen reencounter. These findings have important implications for rational vaccine design, where improving the generation and engagement of memory Tfh cells could be used to enhance vaccine-induced protective immunity.

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.

Using GFP to mark recent thymic emigrants (RTEs) in mice carrying a GFP transgene driven by the recombination-activating gene 2 promoter, we demonstrate that RTEs are readily detectable even in 2-year-old mice, despite the fact that the proportion of the peripheral T cell pool comprised of RTEs declines with age. Although the number of RTEs decreases after reaching a peak at 6 weeks of age, thymic output as a function of thymic size is surprisingly age-independent. The CD4:CD8 ratio of RTEs declines with age, partly because of a striking decrease in steady-state proliferation of CD4 ؉ RTEs in older mice. RTEs in aged mice undergo phenotypic maturation in the lymphoid periphery with delayed kinetics compared with young mice. RTEs from aged mice secrete less IL-2, proliferate less well, and achieve only weak expression of earlyactivation markers compared with more mature naïve peripheral T cells from the same mice. The proportion of GFP ؊ cells in the CD4 ؉ and CD8 ؉ thymic compartments increases with age, partly as a result of leakiness in the aged thymus, allowing reentry of naïve peripheral T cells.aging ͉ recent thymic emigrants ͉ T cell development M aintenance of the peripheral T cell population throughout life depends on balancing the influx of recent thymic emigrants (RTEs) with the homeostatic regulation of mature peripheral T cells. Although T cell numbers can be sustained by homeostatic proliferation of peripheral T cells after lymphocyte depletion, the thymus is essential for maintaining a diverse antigen receptor repertoire and a substantial pool of naïve peripheral T cells. Hallmarks of the aging immune system include thymic involution, enhanced contribution of memory cells to the peripheral T cell pool, and striking clonal expansions among both CD4 and CD8 T cell populations (reviewed in refs. 1 and 2). These phenomena are interrelated, because shrinkage of the thymus limits the number of newly exported T cells, triggering the gradual decline in the naïve T cell pool, which in turn likely contributes to the expansion of select memory phenotype T cells.Understanding the contribution of thymic output to the peripheral T cell pool requires identification of RTEs as a population distinct from the bulk of naïve and previously activated peripheral T cells. Over the years, this distinction has been achieved in mice by identifying RTEs that have originated from thymocytes labeled by BrdU (3) or intrathymic injection of FITC (4, 5) by following a wave of thymocyte differentiation and egress from thymic lobes transplanted into congenic hosts (6, 7) and in mice and humans by using T cell receptor (TCR) rearrangement excision circles to identify cells that have not proliferated since rearranging antigen receptor genes (8-13). Although highly useful, these techniques suffer serious disadvantages, including the short time frame over which RTEs can be observed (4-7), the trauma inherent in the labeling technique and its potential to alter thymic output (4-7), the inherent inaccuracy of the tag itself (3,(8)(9)(10)...

Accurate dielectric function values are essential for spectroscopic ellipsometry data analysis by traditional optical model‐based analysis techniques. In this paper, we show that B‐spline basis functions offer many advantages for param‐ eterizing dielectric functions. A Kramers–Kronig consistent B‐spline formulation, based on the standard B‐spline recursion relation, is derived. B‐spline representations of typical semiconductor and metal dielectric functions are also presented. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)