Highly differentiated CD8 ؉ CD28 ؊ CD27 ؊ T cells have short telomeres, defective telomerase activity, and reduced capacity for proliferation, indicating that they are close to replicative senescence. In addition, these cells express increased levels of the senescence-associated inhibitory receptor KLRG1 and have poor capacity for IL-2 synthesis and defective Akt (ser 473 ) phosphorylation after activation. It is not known whether signaling via KLRG1 contributes to any of the attenuated differentiation-related functional changes in CD8 ؉ T cells. To address this, we blocked KLRG1 signaling during T-cell receptor activation using antibodies against its major ligand, E-cadherin. This resulted in a significant enhancement of Akt (ser 473 ) phosphorylation and T-cell receptor-induced proliferative activity of CD8 ؉ CD28 ؊ CD27 ؊ T cells. Furthermore, the increase of proliferation was directly linked to the Akt-mediated induction of cyclin D and E and reduction in the cyclin inhibitor p27 expression. In contrast, the reduced telomerase activity in highly differentiated CD8 ؉ CD28 ؊ CD27 ؊ T cells was not altered by KLRG1 blockade, indicating the involvement of other mechanisms. This is the first demonstration of a functional role for KLRG1 in primary human CD8 ؉ T cells and highlights that certain functional defects that arise during progressive T-cell differentiation toward replicative senescence are maintained actively by inhibitory receptor signaling. (Blood. 2009;113:6619-6628)

Persistent viral infections and inflammatory syndromes induce the accumulation of T cells with characteristics of terminal differentiation or senescence. However, the mechanism that regulates the end-stage differentiation of these cells is unclear. Human CD4+ effector memory (EM) T cells (CD27−CD45RA−) and also EM T cells that re-express CD45RA (CD27−CD45RA+; EMRA) have many characteristics of end-stage differentiation. These include the expression of surface KLRG1 and CD57, reduced replicative capacity, decreased survival, and high expression of nuclear γH2AX after TCR activation. A paradoxical observation was that although CD4+ EMRA T cells exhibit defective telomerase activity after activation, they have significantly longer telomeres than central memory (CM)-like (CD27+CD45RA−) and EM (CD27−CD45RA−) CD4+ T cells. This suggested that telomerase activity was actively inhibited in this population. Because proinflammatory cytokines such as TNF-α inhibited telomerase activity in T cells via a p38 MAPK pathway, we investigated the involvement of p38 signaling in CD4+ EMRA T cells. We found that the expression of both total and phosphorylated p38 was highest in the EM and EMRA compared with that of other CD4+ T cell subsets. Furthermore, the inhibition of p38 signaling, especially in CD4+ EMRA T cells, significantly enhanced their telomerase activity and survival after TCR activation. Thus, activation of the p38 MAPK pathway is directly involved in certain senescence characteristics of highly differentiated CD4+ T cells. In particular, CD4+ EMRA T cells have features of telomere-independent senescence that are regulated by active cell signaling pathways that are reversible.

The extent of human memory T cell proliferation, differentiation, and telomere erosion that occurs after a single episode of immune challenge in vivo is unclear. To investigate this, we injected tuberculin purified protein derivative (PPD) into the skin of immune individuals and isolated responsive T cells from the site of antigenic challenge at different times. PPD-specific CD4+ T cells proliferated and differentiated extensively in the skin during this secondary response. Furthermore, significant telomere erosion occurred in specific T cells that respond in the skin, but not in those that are found in the blood from the same individuals. Tissue fluid obtained from the site of PPD challenge in the skin inhibited the induction of the enzyme telomerase in T cells in vitro. Antibody inhibition studies indicated that type I interferon (IFN), which was identified at high levels in the tissue fluid and by immunohistology, was responsible in part for the telomerase inhibition. Furthermore, the addition of IFN-α to PPD-stimulated CD4+ T cells directly inhibited telomerase activity in vitro. Therefore, these results suggest that the rate of telomere erosion in proliferating, antigen-specific CD4+ T cells may be accelerated by type I IFN during a secondary response in vivo.

Summary The relative roles that ageing and lifelong cytomegalovirus (CMV) infection have in shaping naive and memory CD4+ T‐cell repertoires in healthy older people is unclear. Using multiple linear regression analysis we found that age itself is a stronger predictor than CMV seropositivity for the decrease in CD45RA+ CD27+ CD4+ T cells over time. In contrast, the increase in CD45RA− CD27− and CD45RA+ CD27− CD4+ T cells is almost exclusively the result of CMV seropositivity, with age alone having no significant effect. Furthermore, the majority of the CD45RA− CD27− and CD45RA+ CD27− CD4+ T cells in CMV‐seropositive donors are specific for this virus. CD45RA+ CD27− CD4+ T cells have significantly reduced CD28, interleukin‐7 receptor α (IL‐7Rα) and Bcl‐2 expression, Akt (ser473) phosphorylation and reduced ability to survive after T‐cell receptor activation compared with the other T‐cell subsets in the same donors. Despite this, the CD45RA+ CD27− subset is as multifunctional as the CD45RA− CD27+ and CD45RA− CD27− CD4+ T‐cell subsets, indicating that they are not an exhausted population. In addition, CD45RA+ CD27− CD4+ T cells have cytotoxic potential as they express high levels of granzyme B and perforin. CD4+ memory T cells re‐expressing CD45RA can be generated from the CD45RA− CD27+ population by the addition of IL‐7 and during this process these cells down‐regulated expression of IL‐7R and Bcl‐2 and so resemble their counterparts in vivo. Finally we showed that the proportion of CD45RA+ CD27− CD4+ T cells of multiple specificities was significantly higher in the bone marrow than the blood of the same individuals, suggesting that this may be a site where these cells are generated.

During acute infection, latent and lytic Epstein-Barr virus (EBV) epitope-specific CD8 ؉ T cells have a CD45RO ؉ CD45RA ؊ phenotype. However, after resolution of the infection, a large proportion of these cells, particularly those specific for lytic viral epitopes, re-express the CD45RA molecule. The role of CD8 ؉ CD45RA ؉ T cells in ongoing immunity to EBV and other viruses is unknown. We now demonstrate that, relative to their CD45RO ؉ counterparts, the EBV-specific CD8 ؉ T cells that revert to CD45RA expression after acute infectious mononucleosis are not in cell cycle, have longer telomeres, and are more resistant to apoptosis partly because of increased Bcl-2 expression. However, the EBV-specific CD8 ؉ CD45RA ؉ T cells have shorter telomeres than the total CD8 ؉ CD45RA ؉ T-cell pool and predominantly express low levels of the CCR7 chemokine receptor, indicating that they are not naive cells. In addition, EBV-specific CD8 ؉ CD45RA ؉ T cells can be induced to proliferate and exhibit potent cytotoxic activity against target cells loaded with specific pep- IntroductionThe central goal of this study was to identify the constraints that regulate the persistence of Epstein-Barr virus (EBV)-specific CD8 ϩ T cells after the resolution of acute infectious mononucleosis (AIM) to identify how the memory pool specific for this virus may be maintained. Primary encounter with EBV induces specific naive CD8 ϩ T cells to proliferate and express phenotypic markers of priming, such as CD45RO. 1,2 Most of this expanded population succumbs to apoptosis after the acute infection resolves. 3,4 The EBV-specific memory pool that escapes apoptosis consists of a mixture of cells specific for lytic and latent viral proteins, which are differentially expressed during early and late stages of the infection, respectively. 2,[5][6][7] It is unclear whether apoptosis remains a constraint on the persistence of EBV-specific CD8 ϩ T cells after the acute infection resolves and whether the cells that are specific for different viral epitopes are equally susceptible to death.A second limit to the maintenance of memory CD8 ϩ T cells after the initial infection may be telomere erosion, resulting from excessive proliferation of specific clones. 8,9 This may lead to the development of terminally differentiated end-stage effector cells that are unable to proliferate on restimulation. 10 It has been shown, however, that despite the considerable expansion of EBV-specific CD8 ϩ T cells, telomere loss does not occur during AIM because of the induction of the enzyme telomerase in these cells. 11,12 Telomere erosion does occur in the EBV-specific CD8 pool during persistent infection, 12 possibly because of frequent restimulation by virus, leading to progressively reduced telomerase induction that is insufficient to maintain telomere length in these cells. 9 The loss of some highly expanded clones of EBV-specific CD8 ϩ T cells during acute infection that are later replaced by other less expanded populations during chronic infection, 56 suggests indirectly...