Volume 45, Issue 5 p. 1441-1451

Regular Article

Free Access

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

Sian M. Henson,

Corresponding Author

Sian M. Henson

Division of Infection and Immunity, University College London, London, UK

Full correspondence Dr. Sian M. Henson, Division of Infection and Immunity, University College London, 5 University Street, London, WC1E 6JF, UK

Fax: +44-207-679-9545 or

e-mail: [email protected]

Additional correspondence: Prof. Arne N. Akbar, Division of Infection and Immunity, University College London, 5 University Street, London, WC1E 6JF, UK.

Fax: +44-207-679-9545

e-mail: [email protected]

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Richard Macaulay,

Richard Macaulay

Division of Infection and Immunity, University College London, London, UK

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Natalie E. Riddell,

Natalie E. Riddell

Division of Infection and Immunity, University College London, London, UK

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Craig J. Nunn,

Craig J. Nunn

Division of Infection and Immunity, University College London, London, UK

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Arne N. Akbar,

Corresponding Author

Arne N. Akbar

Division of Infection and Immunity, University College London, London, UK

Full correspondence Dr. Sian M. Henson, Division of Infection and Immunity, University College London, 5 University Street, London, WC1E 6JF, UK

Fax: +44-207-679-9545 or

e-mail: [email protected]

Additional correspondence: Prof. Arne N. Akbar, Division of Infection and Immunity, University College London, 5 University Street, London, WC1E 6JF, UK.

Fax: +44-207-679-9545

e-mail: [email protected]

Search for more papers by this author

Sian M. Henson,

Corresponding Author

Sian M. Henson

Division of Infection and Immunity, University College London, London, UK

Full correspondence Dr. Sian M. Henson, Division of Infection and Immunity, University College London, 5 University Street, London, WC1E 6JF, UK

Fax: +44-207-679-9545 or

e-mail: [email protected]

Additional correspondence: Prof. Arne N. Akbar, Division of Infection and Immunity, University College London, 5 University Street, London, WC1E 6JF, UK.

Fax: +44-207-679-9545

e-mail: [email protected]

Search for more papers by this author

Richard Macaulay,

Richard Macaulay

Division of Infection and Immunity, University College London, London, UK

Search for more papers by this author

Natalie E. Riddell,

Natalie E. Riddell

Division of Infection and Immunity, University College London, London, UK

Search for more papers by this author

Craig J. Nunn,

Craig J. Nunn

Division of Infection and Immunity, University College London, London, UK

Search for more papers by this author

Arne N. Akbar,

Corresponding Author

Arne N. Akbar

Division of Infection and Immunity, University College London, London, UK

Full correspondence Dr. Sian M. Henson, Division of Infection and Immunity, University College London, 5 University Street, London, WC1E 6JF, UK

Fax: +44-207-679-9545 or

e-mail: [email protected]

Additional correspondence: Prof. Arne N. Akbar, Division of Infection and Immunity, University College London, 5 University Street, London, WC1E 6JF, UK.

Fax: +44-207-679-9545

e-mail: [email protected]

Search for more papers by this author

First published: 24 February 2015

https://doi.org/10.1002/eji.201445312

Citations: 94

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Abstract

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.

Introduction

Human T-cell memory is maintained by episodes of repeated antigenic challenge throughout life 1, 2. These antigenic encounters drive proliferation and differentiation of memory T cells, leading to the accumulation of highly differentiated T cells in older subjects 1, 3. Highly differentiated T cells also accumulate in patients with malignancy 4 and those with persistent infections 3, 5, 6. However, highly differentiated CD4⁺ and CD8⁺ T cells in humans have decreased proliferative capacity 7-9. Therefore, it is possible that the increased susceptibility to infections and malignancy during ageing may result from suboptimal maintenance of responsive pools of specific T cells due to their dysfunctional ability to divide 10, 11. This raises the question of whether it is possible to enhance T-cell proliferation in older subjects.

Human T cells at late stages of differentiation can be identified by the loss of surface markers CD27, CD28, and CCR7 and the re-expression of CD45RA 7, 9, 12. Furthermore, highly differentiated memory T cells have relatively short telomeres 13 and have changes in cell signaling pathways including defective Akt/PKB 7, 12 and increased p38 MAP kinase (MAPK) phosphorylation that are associated with cellular senescence 7, 9, 12, 14. These changes are particularly evident in highly differentiated effector memory T cells that re-express CD45RA (EMRA T cells) 7, 12, which are considered to be an end-stage or senescent population 15, 16. The EMRA CD4⁺ and CD8⁺ T cells also exhibit the loss of both proliferative capacity and telomerase activity and have increased susceptibility to apoptosis 7, 9, 12, 17 and these cells increase significantly during ageing.

Two processes have been shown to induce a loss of proliferative ability in T cells. First, repeated antigenic stimulation of T cells may induce a state of functional exhaustion where proliferative activity and cytokine production are lost 18. This immune exhaustion is initiated by external cell surface inhibitory receptors such as PD-1 and Tim3 19, 20. Second, repeated T-cell stimulation or genotoxic damage by agents such as ROS can induce replicative senescence where cells lose proliferative potential 1, 8. This process is triggered by a DNA damage response (DDR) that leads to growth arrest if the DDR is not resolved 21, 22. It is not clear if senescence and exhaustion signaling pathways are distinct processes 14 or if they act in isolation or in concert to control the proliferation of end-stage or senescent CD8⁺ T cells.

We now demonstrate that the proliferation of highly differentiated EMRA CD8⁺ T cells can be enhanced in an additive manner by blocking p38 MAPK and PD-1 signaling together, however, other functions such as telomerase activity are uniquely regulated by the p38 MAPK pathway. Furthermore, by inhibiting both pathways simultaneously, highly differentiated T cells can be induced to exhibit increased proliferative potential while also retaining their ability to secrete cytokines such as TNF-α, which cannot be achieved by blocking either pathway alone. Nevertheless in old individuals, other mechanisms controlling proliferation may exist, since the proliferative activity of EMRA CD8⁺ T cells after blockade of PD-1 and p38 MAPK signaling does not restore proliferative responses to the level observed in young subjects.

Results

CD8⁺ EMRA T cells from young and old donors exhibit characteristics of senescent T cells

Human CD8⁺ T cells can be subdivided into four populations on the basis of their relative surface expression of CD45RA and CD27 molecules (Fig. 1A). Four subsets can be defined, naïve (N; CD45RA⁺CD27⁺), central memory (CM; CD45RA⁻CD27⁺), effector memory (EM; CD45RA⁻CD27⁻), and effector memory T cells that re-express CD45RA (EMRA; CD45RA⁺CD27⁻). We have previously shown that the EMRA population when isolated from young individuals exhibited senescent characteristics 8, we now extend this to CD8⁺ T cells from old individuals. Using a panel of antibodies that defines senescent characteristics, KLRG1, CD57, and γH2AX (Fig. 1A), we find that upon overnight stimulation the CD8⁺ T-cell subsets isolated from old individuals express a greater array of senescent markers relative to young individuals (Fig. 1B). Furthermore, the EMRA population from old donors has more senescence characteristics than those isolated from young donors (Fig. 1C). However, what is also evident is that differential expression of senescent features between the subsets follows a similar pattern in both young and old donors, therefore the higher levels of senescent characteristics observed are dependent on age.

Details are in the caption following the image — **Figure 1**
Open in figure viewer PowerPoint

CD8⁺ EMRA cells exhibit characteri-stics of senescent T cells. (A) CD8⁺CD45RA⁺CD27⁻ T cells isolated from young and old individuals were isolated and stained for KLRG1, CD57, and γH2AX and analyzed by flow cytometry. One representative plot out of eight is shown. (B) Expression of senescence markers, KLRG1, CD57, and γH2AX, in CD8⁺ CD45RA/CD27 T-cell subsets ex vivo was analyzed by multiparameter flow cytometry T-cell. Pie charts show the average distribution of cells showing 0, 1, 2, or 3 senescent functions within each subset from eight young and eight old donors. (C) Percentage of CD8⁺ T-cell subsets (N, naïve, CD45RA⁺CD27⁺; CM, central memory, CD45RA⁻CD27⁺; EM, effector memory, CD45RA⁻CD27⁻; and EMRA, effector memory T cells that re-express CD45RA, CD45RA⁺CD27⁻) expressing three senescent features in cells isolated from young and old individuals generated using SPICE version 5.2, downloaded from http://exon.niaid.nih.gov. The graph shows the mean ± SEM for eight young (dark green) and old donors (light green) and p values were calculated using a repeated measures ANOVA with the Tukey correction used for post-hoc testing. *p < 0.05.

A lack of proliferation is a defining feature of senescence 8. We found that when using Ki67 staining as a readout for proliferation, CD8⁺ EMRAs, from both young and old individuals, exhibit a significant proliferative impairment when compared to the less differentiated subsets after stimulation (Fig. 2A). This was not thought to be caused by a lower expression of CD3 in these subsets, as we have previously published it to be elevated 23. However, the proliferative activity in all four subsets, especially in the naive and EMRA populations, was decreased in the old volunteers (Fig. 2A). In addition, we found that there was a significant reduction in telomerase in the EMRA population from young and old individuals when compared to the other three subsets (Fig. 2B). Again, the amount of telomerase activity detected in all the CD8⁺ subsets isolated from old donors was significantly less than the levels seen in young donors (Fig. 2C), which supports our previous observation that old individuals have shorter telomere lengths in all these subsets compared to the young cohort 24.

Despite the EMRA population of CD8⁺ T cells having less proliferative and telomerase activity, these cells, when isolated from both young and old donors, were significantly more multifunctional than the other subsets, defined by using a panel that measured the expression of perforin, granzyme B, IFN-γ, and TNF-α simultaneously (Fig. 3A and B). It is interesting to note that the naïve CD8⁺ T cells isolated from old donors display considerably more polyfunctionality than the CM cells (Fig. 3B), suggesting that naïve cells isolated from old donors, identified by CD45RA⁺CD27⁺ expression, may not be a truly naïve population. However, CD8⁺ EM cells from young donors were found to be more polyfunctional based on four functions relative to older donors (Fig. 3B).

We next investigated each cellular function separately in each of the subsets of young and old donors (Fig. 3C). We found that the despite their senescence characteristics, the percentage of the CD8⁺ EMRA T-cell population in both age groups expressed high levels of pro-inflammatory cytokines and cytotoxic molecules. However, while these cells from old donors expressed a higher percentage of cytokines, they exhibited lower levels of perforin and granzyme B after stimulation. Once again, a striking observation was that the naïve CD45RA⁺CD27⁺ population showed greater capacity for both cytokine secretion and cytotoxicity than their counterparts in young subjects reinforcing the possibility that these cells in old donors are not truly naïve.

Expression of PD-1 and p38 MAPK during CD8⁺ T-cell differentiation

We next investigate mechanisms that were involved in inhibiting proliferation in donors form both age groups. PD-1 is the most investigated inhibitory receptor that is expressed by exhausted CD8⁺ T cells 25. When we examined the expression of PD-1 on CD45RA/CD27 defined CD8⁺ T-cell subsets, we found the highest level of expression to be on the CM and EM subsets (Fig. 4A and B). These results together with their high functionality indicate that the CD8⁺ EMRA subset in young and old subjects is not an exhausted population.

Increased expression of p38 MAPK is associated with senescent T cells 26. We found that naïve T cells express the lowest levels and EMRA T cells express significantly higher amounts of phosphorylated p38 MAPK ex vivo (Fig. 4C and D). This supports the concept that EMRA CD8⁺ T cells are an end-stage/senescent population.

Both PD-1 and p38 MAPK signaling pathways contribute to reduced proliferation of EMRA T cells

We next investigated whether either PD-1 or p38 MAPK signaling pathway contributes to the decreased proliferative activity of EMRA CD8⁺ T cells. We isolated the four CD45RA/CD27 defined T-cell subsets from both young and old donors, which were stimulated with anti-CD3 antibody and irradiated autologous APCs in the presence of either PDL1/2 blocking antibodies, BIRB 796, or both inhibitors together. Proliferative activity was determined by staining with Ki67 antibody. Representative histograms for the EMRA subset with and without the inhibitors from young and old donors are shown in Figure 5A and C, respectively. The cumulative data from nine young and five old donors are shown in Figure 5B and D, respectively. In young donors, inhibiting PD-1 signaling increased proliferative activity in CD8⁺ T cells at all four stages of differentiation. However, the inhibition of the p38 MAPK pathway was only effective at increasing proliferation in the end-stage EMRA subset (Fig. 5A and B). There was additive enhancement of proliferation in the EMRA subset when both signaling pathways were inhibited together (Fig. 5A and B). When we investigated the old group, PD-1 blockade significantly enhanced the proliferation of EM and EMRA subsets only (Fig. 5C and D). In contrast, blockade of p38 MAPK was found to increase the proliferative capacity of both the naïve and EMRA subsets (Fig. 5D). Once again there was an additive enhancement in the EMRA subset when both signaling pathways were inhibited together (Fig. 5C and D).

We have recently published that while the EMRA population secretes high levels of cytokines in response to TCR stimulation, their response to CMV-specific stimulation is blunted 23. We therefore decided to investigate the effect of PD-1 and p38 MAPK blockade in response to antigen-specific stimulation (Fig. 6). The proliferative response, measured by Ki67 of sorted CMVpp65-specific CD8⁺ CD45RA/CD27 T-cell subsets to the peptide NLV in young (n = 5) and old (n = 4) individuals followed a similar trend to that seen when using anti-CD3 (Fig. 5). Following treatment, the most responsive subsets to blockade were the EMRAs in both the young (Fig. 6A and B) and old (Fig. 6B and D) donors. An additive enhancement was again observed when both blocks were used together (Fig. 6B and D).

Signaling through p38 MAPK but not PD-1 pathways reduces telomerase activity of EMRA T cells

We next investigated whether p38 MAPK or PD-1 signaling was involved in the decreased telomerase activity in EMRA CD8⁺ T cells isolated from young and old individuals. Our previous data indicated that p38 MAPK signaling inhibits telomerase activity in both CD4⁺ 12 and CD8⁺ 8, 27 EMRA T cells in young individuals. We stimulated CD8⁺ T-cell subsets with anti-CD3 and irradiated autologous APCs in the presence of both inhibitors separately or together as described previously. We found that incubation with BIRB 796 caused a significant increase in telomerase activity in the EMRA population isolated from young individuals, either when added alone or with anti-PDL-1/2 (Fig. 7A and B). When we examined CD8⁺ EMRA T cells from old individuals, we found that p38 MAPK inhibition also increased telomerase activity, however, the level of enzyme activity was not increased to the levels observed in young subjects (Fig. 7C). Therefore, additional mechanisms may be involved in the reduced telomerase activity in these cells during ageing.

p38 MAPK signaling inhibits TNF-α secretion; blocking PD-1 reverts this effect in CD8⁺ T cells

Previous studies have shown that p38 MAPK signaling plays an essential role in the production of TNF-α in T cells 28. The EM and EMRA CD8⁺ T cells from young donors (Fig. 8) secreted the highest levels of this cytokine after activation. As described above, both the naïve and EMRA populations from old donors secreted high levels of TNF-α . PD-1 blockade had no effect on TNF-α secretion in any of the subsets in both age groups. In contrast, the inhibition of p38 MAPK signaling significantly inhibited the secretion of TNF-α in all four subsets in both young and old individuals (Fig. 8A and B). However, when p38 MAPK and PD-1 were inhibited together, the capacity for TNF-α secretion was restored in the EMRA CD8⁺ T cells in young and old donors. We found that blocking PD-1 or p38 MAPK individually or together had no consistent effect on IFN-γ, perforin, or granzyme B. Therefore, the simultaneous blockade of p38 and PD-1 signaling pathways together restores proliferation and telomerase activity and also maintains the capacity for TNF-α production from EMRA of CD8⁺ T cells in young and old subjects. This cannot be achieved with blocking either molecule alone.

Discussion

Our main observation is that the CD8⁺ EMRA T-cell population is a potent effector subset that has its replicative potential and survival capacity that is rigidly controlled by nonoverlapping senescence- and exhaustion-related signaling pathways. The simultaneous targeting of both the p38 MAPK (senescence) and PD-1 (exhaustion) pathways enhances their proliferative responses, to both polyclonal and antigen-specific stimuli, while allowing the retention of effector capacity. This may be of benefit during vaccination where a low proliferative potential has been shown to reduce the effectiveness of therapeutic vaccination 29.

Both p38 MAPK and PD1 have been the focus of much pharmaceutical interest, with therapies targeting the PD-1/PD-L1 axis being effective in the treatment of melanoma, renal carcinoma, and nonsmall cell lung cancer 30. However, while treatment is generally well tolerated, a spectrum of immune-related adverse events have been experienced 30. Furthermore, not all patients respond to PD-1/PD-L1 therapy 31. p38 MAPK is critically involved in the induction of pro-inflammatory cytokine secretion, many pharmaceutical companies have p38 MAPK inhibitors in phase II and phase III clinical trials for the treatment of several inflammatory disorders 32-34. However, these drugs have been limited by their therapeutic index and a requirement for TNF-α to generate cytotoxic T cells. We show here that when p38 MAPK and PD-1 were inhibited together they were able to restore the capacity for TNF-α secretion in the EMRA subset, presumably owing to an unmasking of the enhancing effect on TNF-α secretion when using PD-1 blockade alone. We suggest that p38 inhibitors could also be used for immune enhancement, providing that PD-1 signaling is inhibited at the same time.

The greatest functional effect of simultaneous PD-1 and p38 MAPK inhibition was observed in the CD8⁺ T-cell EMRA subset, where p38 MAPK is maximally expressed but PD-1 is not. However, differing PD-1 expression levels may engage distinct intracellular targets 35. For example, PD-1 binds the SH2 domain containing protein tyrosine phosphatases SHP-1 and SHP-2 in naïve T cells, but in exhausted cells the very high levels of PD-1 expression can recruit additional signaling molecules 35. The varying levels of expression of PD-1 by CD8⁺ T cells that are at different stages of differentiation have implications for antiviral therapy. For example, hepatitis C virus (HCV), human immunodeficiency virus (HIV), and cytomegalovirus (CMV) specific CD8⁺ T-cell populations predominantly comprise cells at early, intermediate, and late differentiation stages, respectively 3, and these specific cells may therefore be differentially affected by PD-1 blockade.

The augmented levels of DNA damage observed in EMRAs may be a consequence of their highly impaired telomerase expression since telomerase can protect T cells against oxidative stress 36 in addition to telomere erosion and this enzyme is a critical regulator of the DDR 37. p38 MAPK is activated in response to DNA damage and indeed, its expression in different CD8⁺ T-cell subsets correlates with the phosphorylation of γH2AX, a component of DNA damage foci. Although p38 MAPK signaling has a central role in inducing senescence that results in apoptosis of the cells 7, it is also involved in pro-inflammatory cytokine production by CD4⁺ and CD8⁺ T cells including IFN-γ and TNF-α production 38. This suggests that p38 MAPK signaling may be a key component that regulates the development of typical effector cell characteristics such as susceptibility to death but potent functional capacity.

Also evident from this study is that naïve cells isolated on the basis of cell phenotype from old individuals are not truly naïve. Unprimed naive cells are characterized by the absence of cytotoxic factors 39, however aged “naïve cells” express significant amounts of granzyme, perforin, TNF-α, and IFN-γ, and have short telomeres compared to cells from young individuals 24. We hypothesize that these cells have undergone antigen-independent homeostatic expansion to convert to a functional state similar to that of memory T cells 7, 23, 40, which raises the question about the use of phenotypic markers to analyze functional outcomes from a heterogeneous population. We also found that CD8⁺ EMRA T cells from old donors, while expressing increased levels of TNF-α and IFN-γ, display significantly reduced levels of perforin and granzyme. This loss of cytotoxic killing apparatus by senescent T cells may contribute to their ineffective immune responses to viruses, which has been observed in HIV infection 41. We speculate that the reduced expression of cytotoxic molecules in CD8⁺ EMRA T cells from old donors may be due to a decreased production and/or responsiveness to IL-2 42-44, which has been shown to increase the expression of perforin and granzyme and enhance cytolytic function 45. Alternatively, the higher frequency of lytic molecules produced by CD8⁺ EMRA T cells of young individuals could reflect faster immunological control in the young. Epigenetic mechanisms alter chromatin structure and influence gene expression, indeed the epigenetic control of granzyme B during CD8⁺ T-cell differentiation has recently been demonstrated 46. They showed an increased susceptibility to nuclease digestion in the chromatin dense proximal region of the gzmB promoter, suggesting that gzmB transcription is mediated by manipulating the accessibility of the transcriptional machinery to the promoter 46. Given that DNA damage and inflammation are both capable of altering chromatin structure 47, it is conceivable that the epigenetic control of cytokine release is less flexible in old individuals. It is also of note that while the simultaneous blockade of PD-1 and p38 MAPK signaling in CD8⁺ EMRA T cells from old individuals has similar effects on enhancing functional responses as observed in young subjects, the reconstituted functional responses are still lower than in the young group. This suggests that there are additional defects in the functional responses in the old group that may not be regulated by PD-1 and p38 MAPK signaling.

In addition to PD-1, there is a diverse array of other cell surface inhibitory molecules that can regulate T-cell exhaustion and gene expression profiles have identified that exhausted CD8⁺ T cells co-express up to seven inhibitory receptors 25, 48. While PD-1 signaling is induced by cell surface interactions, p38 MAPK signaling is induced by intracellular DNA-damage associated signaling processes 14. An exciting prospect for the future is that it may be possible to target different receptors or downstream components of exhaustion and senescence pathways to custom design a tailored T-cell response that is appropriate for a particular situation.

It is possible that blocking PD-1 and/or p38 MAPK may enhance the function of these cells in older humans 49 or patients with skin malignancy 19, 20. However, this raises the question of whether it is safe to do so. Although senescence may have a role in preventing malignancy, exhaustion may reduce the risk of immunopathology that results from excessive immune activation 14. Therefore, the reversal of T-cell senescence by p38 MAPK blockade carries a risk of malignant transformation of these cells while reversing the process of exhaustion may lead to excessive T-cell activity that leads to tissue damage 14. However, while long-term blockade of p38 and or PD-1 signaling has associated risks, the short-term blockade of these signaling pathways may temporarily enhance immunity. This would be beneficial in older humans who have decreased immunity and in patients with cancer. These possibilities have to be investigated further to clarify whether it is feasible to use new strategies involving the blockade of both PD-1 and p38 MAPK to boost immunity in these subjects.

Materials and methods

Blood sample collection and isolation

Heparinized peripheral blood samples were collected from young and old healthy donors (young age range: 20–35, median 32 years, n = 33; old age range: 65–82, median 72 years, n = 31). Those donors were considered as healthy who had no infection or immunization within the last month, no known immunodeficiency or any history of chemotherapy or radiotherapy, and were not receiving systemic steroids within the last month or any other immunosuppressive medications within the last 6 months. All samples were obtained in accordance with the ethical committee of Royal Free and University College Medical School. PBMCs were isolated using Ficoll hypaque 50.

Flow cytometric analysis and cell sorting

Flow cytometric analysis was carried out using the following antibodies: Live/dead fixable blue dead cell stain (Invitrogen), KLRG1 PE (2F1/KLRG1), PD-1 PE (EH12.2H7), and CD45RA Brilliant Violet 605 from Biolegend. CD8 PerCP (SK1), CD8 Alexa Fluor 700 (RPA-T8), CD27 V500 (M-T271), CD27 allophycocyanin (M-T271), CD27 allophycocyanin -H7 (M-T271), CD45RA PE-Cy7 (L48), and CD57 allophycocyanin (NK-1) from BD Biosciences. For intracellular staining, the following antibodies were used: IFN-γ V450 (B27), granzyme B Alexa Fluor 700 (GB11), TNF-α PE (MAb11), perforin FITC (δG9), and p38 Alexa Fluor 488 (36/p38) all from BD Biosciences. For intranuclear staining, the following antibodies were used: γH2AX Alexa Fluor 488 (2F3, Biolegend) and Ki67 FITC (B56; BD Biosciences). Samples were processed using an LSR II (BD Biosciences) and analyzed using FlowJo software (Treestar).

CD8⁺ T cells were isolated by positive selection using the VARIOMACS system (MiltenyiBiotec) according to the manufacturer's instructions. Positively selected CD8⁺ T cells were labeled with CD27 FITC (M-T271) and CD45RA allophycocyanin (HI100; BD Biosciences) and sorted using a FACSAria (BD Biosciences). Multiparameter flow cytometry was performed using SPICE version 5.2, downloaded from http://exon.niaid.nih.gov 51.

Phospho-cytometry

The level of p38 (pT180/pY182) was analyzed ex vivo using the surface markers CD45RA BV610, CD27 V500, and CD8 AF700, and PBMCs were fixed with warm Cytofix Buffer (BD Biosciences) at 37°C for 10 min. Cells were permeabilized with ice-cold Perm Buffer III (BD Biosciences) at 4°C for 30 min and incubated with the anti-p38 antibody (pT180/pY182) for 30 min at room temperature. γH2AX (pSer139) was detected in sorted subsets following activation with 0.5 μg/mL plate-coated anti-CD3 and 5 ng/mL of rhIL-2 for 4 days, after which the above method was used.

Proliferation assays

CD45RA/CD27 purified CD8⁺ T cells were stimulated with 0.5 μg/mL plate-coated anti-CD3 (OKT3) and irradiated APCs, as a source of co-stimulation, proliferation was assessed by staining for the cell cycle related nuclear antigen Ki67 after 4 days 50. Irradiated APCs consisted of the remaining PBMC fraction minus the sorted population of interest.

Proliferation in response to CMV-specific stimulation was achieved through a 4-day incubation of PBMCs with 0.2 μg/mL NLVPMVATV peptide (ProImmune), after which the cells were stained with Ki67 as detailed above.

Measurement of telomerase activity

Isolated CD8⁺ T-cell populations (2 × 10⁵ cells) were snap-frozen after stimulation. Telomerase activity was determined using the TRAPeze telomerase detection kit and the TRAPeze ELISA telomerase detection kit (Chemicon) according to the protocol provided by the manufacturer. The absolute numbers of CD8⁺ T cells were calculated using Trypan blue (Sigma). The TRAP assay was performed with samples adjusted to 500 Ki67⁺ T cells per reaction to control for the different level of proliferation in the subsets following activation 50.

PDL-1/2 blockade and p38 MAPK inhibitor

CD8⁺ and sorted subsets CD27/CD45RA were blocked by adding 10 μg/mL each of anti-PD-L1 (29E.2A3.C6) and anti-PD-L2 (24F.10C12.G12, both from Biolegend), or 10 μg/mL each of IgG2a (Mg2a-53) or IgG2b (MPC-11) isotype control antibodies (Abcam) for 4 days stimulated with anti-CD3 (purified OKT3, 0.5 μg/mL) and irradiated APCs or with 0.2 μg/mL NLVPMVATV peptide (ProImmune). The p38 inhibitor BIRB796 (Selleck Chemicals) was added to the 4-day cultures at a final concentration of 500 nM 8, 12 using 0.1% DMSO as a control.

Measurement of TNF-α following inhibition

Different CD45RA/CD27 defined CD8⁺ T-cell subsets were sorted and 2 × 10⁵ cells were cultured with 0.5 μg/mL plate-coated anti-CD3 (OKT3) and 5 ng/mL rhIL-2, with or without anti-PDL1/2 and BIRB796. Culture supernatants were collected at 48 h for the measurement of TNF-α using the Quantikine human TNF-α immunoassay (R&D Systems) according to the protocol provided by the manufacturer.

Statistical analysis

Graphpad Prism was used to perform statistical analysis. Statistical significance was evaluated using a repeated measures ANOVA with the Tukey correction used for post-hoc testing. Differences were considered significant when p was <0.05.

Acknowledgments

This work was supported by the Biotechnology and Biological Sciences Research Council (A.N.A., S.M.H., and N.E.R.).

Conflict of interest

The authors declare no financial or commercial conflicts of interest.

References

1Akbar, A. N., Beverley, P. C. and Salmon, M., Will telomere erosion lead to a loss of T-cell memory? Nat. Rev. Immunol. 2004. 4: 737–743.
10.1038/nri1440
CASPubMedWeb of Science®Google Scholar
2Nikolich-Zugich, J., Ageing and life-long maintenance of T-cell subsets in the face of latent persistent infections. Nat. Rev. Immunol. 2008. 8: 512–522.
10.1038/nri2318
CASPubMedWeb of Science®Google Scholar
3Appay, V., van Lier, R. A., Sallusto, F. and Roederer, M., Phenotype and function of human T lymphocyte subsets: consensus and issues. Cytometry A 2008. 73: 975–983.
10.1002/cyto.a.20643
PubMedWeb of Science®Google Scholar
4Valmori, D., Scheibenbogen, C., Dutoit, V., Nagorsen, D., Asemissen, A. M., Rubio-Godoy, V., Rimoldi, D. et al., Circulating tumor-reactive CD8(+) T cells in melanoma patients contain a CD45RA(+)CCR7(-) effector subset exerting ex vivo tumor-specific cytolytic activity. Cancer Res. 2002. 62: 1743–1750.

CASPubMedWeb of Science®Google Scholar
5van Lier, R. A., ten Berge, I. J. and Gamadia, L. E., Human CD8(+) T-cell differentiation in response to viruses. Nat. Rev. Immunol. 2003. 3: 931–939.
10.1038/nri1254
PubMedWeb of Science®Google Scholar
6Das, A., Hoare, M., Davies, N., Lopes, A. R., Dunn, C., Kennedy, P. T., Alexander, G. et al., Functional skewing of the global CD8 T cell population in chronic hepatitis B virus infection. J. Exp. Med. 2008. 205: 2111–2124.
10.1084/jem.20072076
CASPubMedWeb of Science®Google Scholar
7Libri, V., Azevedo, R. I., Jackson, S. E., Di Mitri, D., Lachmann, R., Fuhrmann, S., Vukmanovic-Stejic, M. et al., Cytomegalovirus infection induces the accumulation of short-lived, multifunctional CD4+CD45RA+CD27+ T cells: the potential involvement of interleukin-7 in this process. Immunology 2011. 132: 326–339.
10.1111/j.1365-2567.2010.03386.x
CASPubMedWeb of Science®Google Scholar
8Henson, S. M., Lanna, A., Riddell, N. E., Franzese, O., Macaulay, R., Griffiths, S. J., Puleston, D. J. et al., p38 signaling inhibits mTORC1-independent autophagy in senescent human CD8+ T cells. J. Clin. Invest. 2014. 124: 4004–4016.
10.1172/JCI75051
CASPubMedWeb of Science®Google Scholar
9Plunkett, F. J., Franzese, O., Finney, H. M., Fletcher, J. M., Belaramani, L. L., Salmon, M., Dokal, I. et al., The loss of telomerase activity in highly differentiated CD8+CD28-CD27- T cells is associated with decreased Akt (Ser473) phosphorylation. J. Immunol. 2007. 178: 7710–7719.
10.4049/jimmunol.178.12.7710
CASPubMedWeb of Science®Google Scholar
10Arnold, C. R., Wolf, J., Brunner, S., Herndler-Brandstetter, D. and Grubeck-Loebenstein, B., Gain and loss of T cell subsets in old age—age-related reshaping of the T cell repertoire. J. Clin. Immunol. 2011. 31: 137–146.
10.1007/s10875-010-9499-x
PubMedWeb of Science®Google Scholar
11Casado, J. G., DelaRosa, O., Pawelec, G., Peralbo, E., Duran, E., Barahona, F., Solana, R. et al., Correlation of effector function with phenotype and cell division after in vitro differentiation of naive MART-1-specific CD8+ T cells. Int. Immunol. 2009. 21: 53–62.
10.1093/intimm/dxn123
CASPubMedWeb of Science®Google Scholar
12Di Mitri, D., Azevedo, R. I., Henson, S. M., Libri, V., Riddell, N. E., Macaulay, R., Kipling, D. et al., Reversible senescence in human CD4+CD45RA+CD27- memory T cells. J. Immunol. 2011. 187: 2093–2100.
10.4049/jimmunol.1100978
CASPubMedWeb of Science®Google Scholar
13Weng, N. P., Akbar, A. N. and Goronzy, J., CD28(-) T cells: their role in the age-associated decline of immune function. Trends Immunol. 2009. 30: 306–312.
10.1016/j.it.2009.03.013
CASPubMedWeb of Science®Google Scholar
14Akbar, A. N. and Henson, S. M., Are senescence and exhaustion intertwined or unrelated processes that compromise immunity? Nat. Rev. Immunol. 2011. 11: 289–295.
10.1038/nri2959
CASPubMedWeb of Science®Google Scholar
15Koch, S., Larbi, A., Derhovanessian, E., Ozcelik, D., Naumova, E. and Pawelec, G., Multiparameter flow cytometric analysis of CD4 and CD8 T cell subsets in young and old people. Immun. Ageing 2008. 5: 6.
10.1186/1742-4933-5-6
CASPubMedWeb of Science®Google Scholar
16Monteiro, M., Evaristo, C., Legrand, A., Nicoletti, A. and Rocha, B., Cartography of gene expression in CD8 single cells: novel CCR7-subsets suggest differentiation independent of CD45RA expression. Blood 2007. 109: 2863–2870.

CASPubMedWeb of Science®Google Scholar
17Fletcher, J. M., Vukmanovic-Stejic, M., Dunne, P. J., Birch, K. E., Cook, J. E., Jackson, S. E., Salmon, M. et al., Cytomegalovirus-specific CD4+ T cells in healthy carriers are continuously driven to replicative exhaustion. J. Immunol. 2005. 175: 8218–8225.
10.4049/jimmunol.175.12.8218
CASPubMedWeb of Science®Google Scholar
18Day, C. L., Kaufmann, D. E., Kiepiela, P., Brown, J. A., Moodley, E. S., Reddy, S., Mackey, E. W. et al., PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature 2006. 443: 350–354.
10.1038/nature05115
CASPubMedWeb of Science®Google Scholar
19Fourcade, J., Sun, Z., Benallaoua, M., Guillaume, P., Luescher, I. F., Sander, C., Kirkwood, J. M. et al., Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen-specific CD8+ T cell dysfunction in melanoma patients. J. Exp. Med. 2010. 207: 2175–2186.
10.1084/jem.20100637
CASPubMedWeb of Science®Google Scholar
20Sakuishi, K., Apetoh, L., Sullivan, J. M., Blazar, B. R., Kuchroo, V. K. and Anderson, A. C., Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity. J. Exp. Med. 2010. 207: 2187–2194.
10.1084/jem.20100643
CASPubMedWeb of Science®Google Scholar
21d'Adda di Fagagna, F., Reaper, P. M., Clay-Farrace, L., Fiegler, H., Carr, P., Von Zglinicki, T., Saretzki, G. et al., A DNA damage checkpoint response in telomere-initiated senescence. Nature 2003. 426: 194–198.
10.1038/nature02118
CASPubMedWeb of Science®Google Scholar
22Herbig, U., Jobling, W. A., Chen, B. P., Chen, D. J. and Sedivy, J. M., Telomere shortening triggers senescence of human cells through a pathway involving ATM, p53, and p21(CIP1), but not p16(INK4a). Mol. Cell 2004. 14: 501–513.
10.1016/S1097-2765(04)00256-4
CASPubMedWeb of Science®Google Scholar
23Griffiths, S. J., Riddell, N. E., Masters, J., Libri, V., Henson, S. M., Wertheimer, A., Wallace, D. et al., Age-associated increase of low-avidity cytomegalovirus-specific CD8+ T cells that re-express CD45RA. J. Immunol. 2013. 190: 5363–5372.
10.4049/jimmunol.1203267
CASPubMedWeb of Science®Google Scholar
24Riddell, N. E., Griffiths, S. J., Rivino, L., King, D. C., Teo, G. H., Henson, S. M., Cantisan, S. et al., Multifunctional cytomegalovirus (CMV)-specific CD8 T cells are not restricted by telomere-related senescence in young or old adults. Immunology 2014. 144: 549–560.
10.1111/imm.12409
CASWeb of Science®Google Scholar
25Blackburn, S. D., Shin, H., Haining, W. N., Zou, T., Workman, C. J., Polley, A., Betts, M. R. et al., Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection. Nat. Immunol. 2009. 10: 29–37.
10.1038/ni.1679
CASPubMedWeb of Science®Google Scholar
26Iwasa, H., Han, J. and Ishikawa, F., Mitogen-activated protein kinase p38 defines the common senescence-signalling pathway. Genes Cells 2003. 8: 131–144.
10.1046/j.1365-2443.2003.00620.x
CASPubMedWeb of Science®Google Scholar
27Lanna, A., Coutavas, E., Levati, L., Seidel, J., Rustin, M. H., Henson, S. M., Akbar, A. N. et al., IFN-alpha inhibits telomerase in human CD8(+) T cells by both hTERT downregulation and induction of p38 MAPK signaling. J. Immunol. 2013. 191: 3744–3752.
10.4049/jimmunol.1301409
CASPubMedWeb of Science®Google Scholar
28Li, C., Beavis, P., Palfreeman, A. C., Amjadi, P., Kennedy, A. and Brennan, F. M., Activation of p38 mitogen-activated protein kinase is critical step for acquisition of effector function in cytokine-activated T cells, but acts as a negative regulator in T cells activated through the T-cell receptor. Immunology 2010. 132: 104–110.
10.1111/j.1365-2567.2010.03345.x
CASPubMedWeb of Science®Google Scholar
29Wherry, E. J., Blattman, J. N., Murali-Krishna, K., van der Most, R. and Ahmed, R., Viral persistence alters CD8 T-cell immunodominance and tissue distribution and results in distinct stages of functional impairment. J. Virol. 2003. 77: 4911–4927.
10.1128/JVI.77.8.4911-4927.2003
CASPubMedWeb of Science®Google Scholar
30Merelli, B., Massi, D., Cattaneo, L. and Mandala, M., Targeting the PD1/PD-L1 axis in melanoma: biological rationale, clinical challenges and opportunities. Crit. Rev. Oncol. Hematol. 2014. 89: 140–165.
10.1016/j.critrevonc.2013.08.002
PubMedWeb of Science®Google Scholar
31Kaufmann, D. E., Kavanagh, D. G., Pereyra, F., Zaunders, J. J., Mackey, E. W., Miura, T., Palmer, S. et al., Upregulation of CTLA-4 by HIV-specific CD4+ T cells correlates with disease progression and defines a reversible immune dysfunction. Nat. Immunol. 2007. 8: 1246–1254.
10.1038/ni1515
CASPubMedWeb of Science®Google Scholar
32Zhang, X., Huang, Y., Navarro, M. T., Hisoire, G. and Caulfield, J. P., A proof-of-concept and drug-drug interaction study of pamapimod, a novel p38 MAP kinase inhibitor, with methotrexate in patients with rheumatoid arthritis. J. Clin. Pharmacol. 2010. 50: 1031–1038.
10.1177/0091270009357433
CASPubMedWeb of Science®Google Scholar
33Anand, P., Shenoy, R., Palmer, J. E., Baines, A. J., Lai, R. Y., Robertson, J., Bird, N. et al., Clinical trial of the p38 MAP kinase inhibitor dilmapimod in neuropathic pain following nerve injury. Eur. J. Pain 2011. 15: 1040–1048.
10.1016/j.ejpain.2011.04.005
CASPubMedWeb of Science®Google Scholar
34Cohen, S. B., Cheng, T. T., Chindalore, V., Damjanov, N., Burgos-Vargas, R., Delora, P., Zimany, K. et al., Evaluation of the efficacy and safety of pamapimod, a p38 MAP kinase inhibitor, in a double-blind, methotrexate-controlled study of patients with active rheumatoid arthritis. Arthritis Rheum. 2009. 60: 335–344.
10.1002/art.24266
CASPubMedWeb of Science®Google Scholar
35Riley, J. L., PD-1 signaling in primary T cells. Immunol. Rev. 2009. 229: 114–125.
10.1111/j.1600-065X.2009.00767.x
CASPubMedWeb of Science®Google Scholar
36Ahmed, S., Passos, J. F., Birket, M. J., Beckmann, T., Brings, S., Peters, H., Birch-Machin, M. A. et al., Telomerase does not counteract telomere shortening but protects mitochondrial function under oxidative stress. J. Cell Sci. 2008. 121: 1046–1053.
10.1242/jcs.019372
CASPubMedWeb of Science®Google Scholar
37Masutomi, K., Possemato, R., Wong, J. M., Currier, J. L., Tothova, Z., Manola, J. B., Ganesan, S. et al., The telomerase reverse transcriptase regulates chromatin state and DNA damage responses. Proc. Natl. Acad. Sci. USA 2005. 102: 8222–8227.
10.1073/pnas.0503095102
CASPubMedWeb of Science®Google Scholar
38Dodeller, F. and Schulze-Koops, H., The p38 mitogen-activated protein kinase signaling cascade in CD4 T cells. Arthritis Res. Ther. 2006. 8: 205.
10.1186/ar1905
CASPubMedWeb of Science®Google Scholar
39Appay, V., Dunbar, P. R., Callan, M., Klenerman, P., Gillespie, G. M., Papagno, L., Ogg, G. S. et al., Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections. Nat. Med. 2002. 8: 379–385.
10.1038/nm0402-379
CASPubMedWeb of Science®Google Scholar
40Geginat, J., Sallusto, F. and Lanzavecchia, A., Cytokine-driven proliferation and differentiation of human naive, central memory, and effector memory CD4(+) T cells. J. Exp. Med. 2001. 194: 1711–1719.
10.1084/jem.194.12.1711
CASPubMedWeb of Science®Google Scholar
41Yang, O. O., Lin, H., Dagarag, M., Ng, H. L., Effros, R. B. and Uittenbogaart, C. H., Decreased perforin and granzyme B expression in senescent HIV-1-specific cytotoxic T lymphocytes. Virology 2005. 332: 16–19.
10.1016/j.virol.2004.11.028
CASPubMedWeb of Science®Google Scholar
42Miller, R. A., Effect of aging on T lymphocyte activation. Vaccine 2000. 18: 1654–1660.
10.1016/S0264-410X(99)00502-2
CASPubMedWeb of Science®Google Scholar
43Nagel, J. E., Chopra, R. K., Powers, D. C. and Adler, W. H., Effect of age on the human high affinity interleukin 2 receptor of phytohaemagglutinin stimulated peripheral blood lymphocytes. Clin. Exp. Immunol. 1989. 75: 286–291.

CASPubMedWeb of Science®Google Scholar
44Weiskopf, D., Weinberger, B. and Grubeck-Loebenstein, B., The aging of the immune system. Transpl. Int. 2009. 22: 1041–1050.
10.1111/j.1432-2277.2009.00927.x
CASPubMedWeb of Science®Google Scholar
45Janas, M. L., Groves, P., Kienzle, N. and Kelso, A., IL-2 regulates perforin and granzyme gene expression in CD8+ T cells independently of its effects on survival and proliferation. J. Immunol. 2005. 175: 8003–8010.
10.4049/jimmunol.175.12.8003
CASPubMedWeb of Science®Google Scholar
46Juelich, T., Sutcliffe, E. L., Denton, A., He, Y., Doherty, P. C., Parish, C. R., Turner, S. J. et al., Interplay between chromatin remodeling and epigenetic changes during lineage-specific commitment to granzyme B expression. J. Immunol. 2009. 183: 7063–7072.
10.4049/jimmunol.0901522
CASPubMedWeb of Science®Google Scholar
47Rando, T. A. and Chang, H. Y., Aging, rejuvenation, and epigenetic reprogramming: resetting the aging clock. Cell 2012. 148: 46–57.
10.1016/j.cell.2012.01.003
CASPubMedWeb of Science®Google Scholar
48Wherry, E. J., Ha, S. J., Kaech, S. M., Haining, W. N., Sarkar, S., Kalia, V., Subramaniam, S. et al., Molecular signature of CD8+ T cell exhaustion during chronic viral infection. Immunity 2007. 27: 670–684.
10.1016/j.immuni.2007.09.006
CASPubMedWeb of Science®Google Scholar
49Henson, S. M., Macaulay, R., Franzese, O. and Akbar, A. N., Reversal of functional defects in highly differentiated young and old CD8 T cells by PDL blockade. Immunology 2012. 135: 355–363.
10.1111/j.1365-2567.2011.03550.x
CASPubMedWeb of Science®Google Scholar
50Henson, S. M., Franzese, O., Macaulay, R., Libri, V., Azevedo, R. I., Kiani-Alikhan, S., Plunkett, F. J. et al., KLRG1 signaling induces defective Akt (ser473) phosphorylation and proliferative dysfunction of highly differentiated CD8+ T cells. Blood 2009. 113: 6619–6628.
10.1182/blood-2009-01-199588
CASPubMedWeb of Science®Google Scholar
51Roederer, M., Nozzi, J. L. and Nason, M. X., SPICE: exploration and analysis of post-cytometric complex multivariate datasets. Cytometry A 2011. 79: 167–174.
10.1002/cyto.a.21015
PubMedWeb of Science®Google Scholar

Abbreviation

DDR: DNA damage response

Citing Literature

Volume45, Issue5

May 2015

Pages 1441-1451

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

Abstract

Introduction

Results

CD8⁺ EMRA T cells from young and old donors exhibit characteristics of senescent T cells

Expression of PD-1 and p38 MAPK during CD8⁺ T-cell differentiation

Both PD-1 and p38 MAPK signaling pathways contribute to reduced proliferation of EMRA T cells

Signaling through p38 MAPK but not PD-1 pathways reduces telomerase activity of EMRA T cells

p38 MAPK signaling inhibits TNF-α secretion; blocking PD-1 reverts this effect in CD8⁺ T cells

Discussion

Materials and methods

Blood sample collection and isolation

Flow cytometric analysis and cell sorting

Phospho-cytometry

Proliferation assays

Measurement of telomerase activity

PDL-1/2 blockade and p38 MAPK inhibitor

Measurement of TNF-α following inhibition

Statistical analysis

Acknowledgments

Conflict of interest

References

Abbreviation

Citing Literature

Figures

References

Information

About Wiley Online Library

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Connect with Wiley

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

Abstract

Introduction

Results

CD8+ EMRA T cells from young and old donors exhibit characteristics of senescent T cells

Expression of PD-1 and p38 MAPK during CD8+ T-cell differentiation

Both PD-1 and p38 MAPK signaling pathways contribute to reduced proliferation of EMRA T cells

Signaling through p38 MAPK but not PD-1 pathways reduces telomerase activity of EMRA T cells

p38 MAPK signaling inhibits TNF-α secretion; blocking PD-1 reverts this effect in CD8+ T cells

Discussion

Materials and methods

Blood sample collection and isolation

Flow cytometric analysis and cell sorting

Phospho-cytometry

Proliferation assays

Measurement of telomerase activity

PDL-1/2 blockade and p38 MAPK inhibitor

Measurement of TNF-α following inhibition

Statistical analysis

Acknowledgments

Conflict of interest

References

Abbreviation

Citing Literature

Figures

References

Related

Information

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

CD8⁺ EMRA T cells from young and old donors exhibit characteristics of senescent T cells

Expression of PD-1 and p38 MAPK during CD8⁺ T-cell differentiation

p38 MAPK signaling inhibits TNF-α secretion; blocking PD-1 reverts this effect in CD8⁺ T cells