Richard Macaulay

KLRG1 signaling induces defective Akt (ser473) phosphorylation and proliferative dysfunction of highly differentiated CD8+ T cells.

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.

Cytomegalovirus Infection Reduces Telomere Length of the Circulating T Cell Pool

Short telomeres of circulating leukocytes are a risk factor for age-related diseases, such as atherosclerosis, but the exact mechanisms generating variations in telomere length are unknown. We hypothesized that induction of differentiated T cells during chronic CMV infection would affect T cell telomere length. To test this, we measured the amount of differentiated T cells and telomere length of lymphocytes during primary CMV infection as well as CMV-seropositive and -seronegative healthy individuals. After primary CMV infection, we observed an increase in highly differentiated cells that coincided with a steep drop in telomere length. Moreover, we found in a cohort of 159 healthy individuals that telomere shortening was more rapid in CMV-seropositive individuals and correlated with the amount of differentiated T cells in both CD4+ T cells and CD8+ T cells. Finally, we found that telomere length measured in blood leukocytes is correlated with lymphocyte telomere length. Thus, CMV infection induces a strong decrease in T cell telomere length, which can be explained by changes in the composition of the circulating lymphocyte pool.

Reversible Senescence in Human CD4+CD45RA+CD27− Memory T Cells

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.

p38 signaling inhibits mTORC1-independent autophagy in senescent human CD8+ T cells

T cell senescence is thought to contribute to immune function decline, but the pathways that mediate senescence in these cells are not clear. Here, we evaluated T cell populations from healthy volunteers and determined that human CD8+ effector memory T cells that reexpress the naive T cell marker CD45RA have many characteristics of cellular senescence, including decreased proliferation, defective mitochondrial function, and elevated levels of both ROS and p38 MAPK. Despite their apparent senescent state, we determined that these cells secreted high levels of both TNF-α and IFN-γ and showed potent cytotoxic activity. We found that the senescent CD45RA-expressing population engaged anaerobic glycolysis to generate energy for effector functions. Furthermore, inhibition of p38 MAPK signaling in senescent CD8+ T cells increased their proliferation, telomerase activity, mitochondrial biogenesis, and fitness; however, the extra energy required for these processes did not arise from increased glucose uptake or oxidative phosphorylation. Instead, p38 MAPK blockade in these senescent cells induced an increase in autophagy through enhanced interactions between p38 interacting protein (p38IP) and autophagy protein 9 (ATG9) in an mTOR-independent manner. Together, our findings describe fundamental metabolic requirements of senescent primary human CD8+ T cells and demonstrate that p38 MAPK blockade reverses senescence via an mTOR-independent pathway.

The role of the T cell in age-related inflammation

Ageing is accompanied by alterations to T-cell immunity and also by a low-grade chronic inflammatory state termed inflammaging. The significance of these phenomena is highlighted by their being predictors of earlier mortality. We have recently published that the proinflammatory cytokine TNFα is a strong inducer of CD4+ T-cell senescence and T-cell differentiation, adding to the growing body of literature implicating proinflammatory molecules in mediating these critical age-related T-cell alterations. Moreover, the inflammatory process is also being increasingly implicated in the pathogenesis of many common and severe age-related diseases, including cancer, cardiovascular diseases and type 2 diabetes. Furthermore, major age-related risk factors for poor health, such as obesity, stress and smoking, are also associated with an upregulation in systemic inflammatory markers. We propose the idea that the ensuing inflammatory response to influenza infection propagates cardiovascular diseases and constitutes a major cause of influenza-related mortality. While inflammation is not a negative phenomenon per se, this age-related dysregulation of inflammatory responses may play crucial roles driving age-related pathologies, T-cell immunosenescence and CMV reactivation, thereby underpinning key features of the ageing process.

Report from the second cytomegalovirus and immunosenescence workshop.

The Second International Workshop on CMV & Immunosenescence was held in Cambridge, UK, 2-4th December, 2010. The presentations covered four separate sessions: cytomegalovirus and T cell phenotypes; T cell memory frequency, inflation and immunosenescence; cytomegalovirus in aging, mortality and disease states; and the immunobiology of cytomegalovirus-specific T cells and effects of the virus on vaccination. This commentary summarizes the major findings of these presentations and references subsequently published work from the presenter laboratory where appropriate and draws together major themes that were subsequently discussed along with new areas of interest that were highlighted by this discussion.

Blockade of PD-1 or p38 MAP kinase signaling enhances senescent human CD8(+) T-cell proliferation by distinct pathways.

Immune enhancement is desirable in situations where decreased immunity results in increased morbidity. We investigated whether blocking the surface inhibitory receptor PD‐1 and/or p38 MAP kinase could enhance the proliferation of the effector memory CD8+ T‐cell subset that re‐expresses CD45RA (EMRA) and exhibits characteristics of senescence, which include decreased proliferation and telomerase activity but increased expression of the DNA damage response related protein γH2AX. Blocking of both PD‐1 and p38 MAPK signaling in these cells enhanced proliferation and the increase was additive when both pathways were inhibited simultaneously in both young and old human subjects. In contrast, telomerase activity in EMRA CD8+ T cells was only enhanced by blocking the p38 but not the PD‐1 signaling pathway, further indicating that nonoverlapping signaling pathways were involved. Although blocking p38 MAPK inhibits TNF‐α secretion in the EMRA population, this decrease was counteracted by the simultaneous inhibition of PD‐1 signaling in these cells. Therefore, end‐stage characteristics of EMRA CD8+ T cells are stringently controlled by distinct and reversible cell signaling events. In addition, the inhibition of PD‐1 and p38 signaling pathways together may enable the enhancement of proliferation of EMRA CD8+ T cells without compromising their capacity for cytokine secretion.

Reversal of functional defects in highly differentiated young and old CD8 T cells by PDL blockade.

Highly differentiated CD8+ CD28− CD27− T cells have short telomeres, defective telomerase activity and reduced capacity for proliferation. In addition, these cells express increased levels of inhibitory receptors and display defective Akt(ser473) phosphorylation following activation. It is not known whether signalling via programmed death 1 (PD‐1) contributes to any of the attenuated differentiation‐related functional changes in CD8+ T cells. To address this we blocked PD‐1 signalling during T‐cell receptor (TCR) activation using antibodies against PD‐1 ligand 1 (PDL1) and PDL2. This resulted in a significant enhancement of Akt(ser473) phosphorylation and TCR‐induced proliferative activity of highly differentiated CD8+ CD28− CD27− T cells. In contrast, the reduced telomerase activity in these cells was not altered by blockade of PDL1/2. We also demonstrate that PD‐1 signalling can inhibit the proliferative response in primary human CD8+ T cells from both young and older humans. These data collectively highlight that some, but not all, functional changes that arise during progressive T‐cell differentiation and during ageing are maintained actively by inhibitory receptor signalling.

The Use of the Inhibitory Receptors for Modulating the Immune Responses

Inhibitory receptors of the CD28 family, CTLA-4 and PD-1 deliver negative signals that regulate the balance between T cell activation, tolerance, and immunopathology. Manipulation of these pathways has been utilized by pathogens and tumors to establish chronic infections or to promote tumor survival. In this review, we examine the role of CTLA-4 and PD-1 in regulating immune response and discuss their therapeutic potential during aging.

Differing HLA types influence inhibitory receptor signalling in CMV-specific CD8+ T cells.

The dysregulated immune response to CMV constitutes a major force driving T cell immunosenescence and growing evidence suggests that it is not a benign virus in old age. We show here that the PD-1/L pathway defines a reversible defect in CMV specific CD8(+) T cell proliferative responses in both young and old individuals. More specifically, highly differentiated CD45RA(+)CD27(-) CMV-specific CD8(+) T cells exhibit a proliferative deficit compared their central and effector memory counterparts, which is reversed following PD-L blockade. However, we also report that HLA-B(∗)07/TPR specific CD8(+) T cells express higher levels of PD-1 than HLA-A(∗)02/NLV specific cells and HLA-A(∗)02 individuals show a higher proliferative response to PD-L blockade, than HLA-B(∗)07 individuals, which we postulate may be due to the differing functional avidities for these two CMV-specific CD8(+) T cells populations. Nevertheless data presented here demonstrate that CMV-specific CD8(+) T cells can be functionally enhanced by perturbation of the PD-1/L signalling pathway, whose manipulation may provide a therapeutic modality to combat age-associated immune decline.