Senescent CD8 Research Articles

Chronic Kidney Failure Provokes the Enrichment of Terminally Differentiated CD8+ T Cells, Impairing Cytotoxic Mechanisms After Kidney Transplantation.

Chronic kidney failure (KF) provokes the development of immune senescent CD8+ cytotoxic T cells, affecting the occurrence of graft rejection, viral infections, and malignancies after kidney transplantation. In this study, we analyzed the impact of KF, subsequent dialysis treatment, and kidney transplantation on the differentiation of CD8+CD31+CD45RA+CCR7+ recent thymic emigrant (CCR7+ RTE) Tregs/Tresps into CD8+CD31-CD45RA- memory (CD31- memory) Tregs/Tresps and its effect on the release of cytokines, Fas receptor, Fas ligand as well as cytotoxic mediators by naïve, central memory (CM), effector memory (EM), and terminally differentiated effector memory (TEMRA) Tresps. We found that normal age-dependent differentiation of CD8+ Tregs/Tresps generally differs in the way that TEMRA cells only arise in Tresps. Compared to healthy controls, KF patients revealed an age-independently decreased frequency of CCR7+ RTE Tregs/Tresps, but increased frequencies of CCR7+ MN Tregs/Tresps and CD31- memory Tregs/Tresps, suggesting an increased differentiation via CD31+CD45RA- memory (CD31+ memory) Tregs/Tresps into CD31- memory Tregs/Tresps. Intensified differentiation via CD31+ memory Tresps increased the emergence of apoptosis-resistant CM Tresps with strong Fas ligand-mediated cytotoxicity. CCR7+ RTE Tresp proliferation generated TEMRA Tresps, secreting high levels of cytotoxic mediators. In dialysis and transplant patients, CD31+ TEMRA Tregs/Tresps accumulated, proposing an impaired CCR7+ RTE Treg/Tresp differentiation via CD31+ memory Tregs/Tresps into CD31- memory Tregs/Tresps. Increased percentages of CD31- TEMRA Tresps, but not of CD31- TEMRA Tregs, were observed in all patient groups, indicating impaired proliferation of CCR7+ RTE Tresps, but not of CCR7+ RTE Tregs, into CD31- memory Tregs/Tresps. In transplant patients, CCR7+ RTE Tregs accumulated, while frequencies of CCR7+ RTE Tresps were decreased, suggesting that the immunosuppressive therapy only prevented excessive CCR7+ RTE Treg differentiation but not that of CCR7+ RTE Tresps. Presumably, this caused the accumulation of TEMRA Tresps with decreased release of cytotoxic mediators, such as perforin. In conclusion, we propose that chronic KF affects both the differentiation of CD8+ Tregs and CD8+ Tresps. However, the immunosuppressive therapy after transplantation may successfully prevent excessive Treg differentiation, but not as suffciently that of Tresps. Therefore, the risk for graft rejection may be reduced, while the susceptibility for infections and malignancies may be increased in these patients.

CD8+ T Cell Senescence: Lights and Shadows in Viral Infections, Autoimmune Disorders and Cancer.

CD8+ T lymphocytes are a heterogeneous class of cells that play a crucial role in the adaptive immune response against pathogens and cancer. During their lifetime, they acquire cytotoxic functions to ensure the clearance of infected or transformed cells and, in addition, they turn into memory lymphocytes, thus providing a long-term protection. During ageing, the thymic involution causes a reduction of circulating T cells and an enrichment of memory cells, partially explaining the lowering of the response towards novel antigens with implications in vaccine efficacy. Moreover, the persistent stimulation by several antigens throughout life favors the switching of CD8+ T cells towards a senescent phenotype contributing to a low-grade inflammation that is a major component of several ageing-related diseases. In genetically predisposed young people, an immunological stress caused by viral infections (e.g., HIV, CMV, SARS-CoV-2), autoimmune disorders or tumor microenvironment (TME) could mimic the ageing status with the consequent acceleration of T cell senescence. This, in turn, exacerbates the inflamed conditions with dramatic effects on the clinical progression of the disease. A better characterization of the phenotype as well as the functions of senescent CD8+ T cells can be pivotal to prevent age-related diseases, to improve vaccine strategies and, possibly, immunotherapies in autoimmune diseases and cancer.

Senescent CD4+CD28- T Lymphocytes as a Potential Driver of Th17/Treg Imbalance and Alveolar Bone Resorption during Periodontitis.

Senescent cells express a senescence-associated secretory phenotype (SASP) with a pro-inflammatory bias, which contributes to the chronicity of inflammation. During chronic inflammatory diseases, infiltrating CD4+ T lymphocytes can undergo cellular senescence and arrest the surface expression of CD28, have a response biased towards T-helper type-17 (Th17) of immunity, and show a remarkable ability to induce osteoclastogenesis. As a cellular counterpart, T regulatory lymphocytes (Tregs) can also undergo cellular senescence, and CD28− Tregs are able to express an SASP secretome, thus severely altering their immunosuppressive capacities. During periodontitis, the persistent microbial challenge and chronic inflammation favor the induction of cellular senescence. Therefore, senescence of Th17 and Treg lymphocytes could contribute to Th17/Treg imbalance and favor the tooth-supporting alveolar bone loss characteristic of the disease. In the present review, we describe the concept of cellular senescence; particularly, the one produced during chronic inflammation and persistent microbial antigen challenge. In addition, we detail the different markers used to identify senescent cells, proposing those specific to senescent T lymphocytes that can be used for periodontal research purposes. Finally, we discuss the existing literature that allows us to suggest the potential pathogenic role of senescent CD4+CD28− T lymphocytes in periodontitis.

Effect of Exercise on Acute Senescent Lymphocyte Counts: A Systematic Review and Meta-Analysis

Background: Highly differentiated, senescent lymphocytes are pro-inflammatory and contribute to age-related systemic inflammation, called inflammageing. There are several reports of acute changes in senescent lymphocyte counts post exercise, which potentially have consequences for systemic inflammation. However, there is little consensus since the studies differ with respect to participants, exercise protocols, cellular markers assessed, and the time point of assessment post exercise. Objective: We performed a systematic review and meta-analysis to assess the impact of exercise on senescent lymphocyte counts in blood immediately, 1 h and 2 h post exercise. Methods: The search was performed in PubMed (MEDLINE), Web of Science, Embase, Scopus, and Cochrane, on January 11, 2021. The 13 studies selected tested aerobic exercise effects, mainly in young men. They assessed the counts of lymphocytes (CD4 T cells, CD8 T cells, and NK cells), with the following immune cell marker combinations: KLRG1+, CD57+ (only NK cells), EMRA T cells (CD45RA+CCR7−CD28−CD27−), CD28−CD27−, KLRG1+CD28−, and CD28−. Independent extraction of articles was done by 2 researchers. Results: Standardized mean difference (SMD) and 95% confidence interval between baseline and post exercise showed significant increase (SMD >0.9, p < 0.003) in all types of senescent lymphocytes counts immediately post exercise. At 1 h post exercise, senescent CD4 T cells returned to baseline values (p = 0.74), CD8 T cells were reduced (−0.26 [−0.41; −0.11], p = 0.001), and senescent NK cells were raised (0.62 [0.14; 1.10], p = 0.01) above baseline. By 2 h post exercise, senescent CD4 T cells were reduced (−0.94 [−1.40; −0.48], p < 0.001), CD8 T cells remained below baseline (−0.53 [−1.04; −0.009], p = 0.04), and NK cells had returned to baseline values (−0.29 [−0.64; 0.07], p = 0.11). The main determinants of heterogeneity between studies were cytomegalovirus (CMV) serostatus and the characteristics of exercise protocols. CMV+ individuals had a higher immediate lymphocytosis and 1 h post lymphopenia than CMV− individuals. Exercise performed at higher intensities and shorter durations led to higher magnitude of change in senescent lymphocyte counts at all time-points. Conclusion: The differing effects of exercise on senescent NK cells and CD4 and CD8 T cells suggest differing susceptibility to factors modulating lymphocyte extravasation such as adrenaline and exercise intensity.

Increased Pro-Inflammatory T Cells, Senescent T Cells, and Immune-Check Point Molecules in the Placentas of Patients With Gestational Diabetes Mellitus.

Gestational diabetes mellitus (GDM) is the most common metabolic complication of pregnancy. To define the altered pathway in GDM placenta, we investigated the transcriptomic profiles from human placenta between GDM and controls. Clinical parameters and postpartum complications were reviewed in all participants. Differentially expressed canonical pathways were analyzed between the GDM and control groups based on transcriptomic analysis. CD4+ T, CD8+ T, and senescent T cell subsets were determined by flow cytometry based on staining for specific intracellular cytokines. Gene ontology analysis revealed that the placenta of GDM revealed upregulation of diverse mitochondria or DNA replication related pathways and downregulation of T-cell immunity related pathways. The maternal placenta of the GDM group had a higher proportion of CD4+ T and CD8+ T cells than the control group. Interestingly, senescent CD4+ T cells tended to increase and CD8+ T cells were significantly increased in GDM compared to controls, along with increased programmed cell death-1 (CD274+) expression. Programmed death-ligand 1 expression in syncytotrophoblasts was also significantly increased in patients with GDM. This study demonstrated increased proinflammatory T cells, senescent T cells and immune-check point molecules in GDM placentas, suggesting that changes in senescent T cells and immune-escape signaling might be related to the pathophysiology of GDM.

Immunometabolic signatures predict recovery from thyrotoxic myopathy in patients with Graves' disease

BackgroundThyroid hormone excess induces protein energy wasting, which in turn promotes muscle weakness and bone loss in patients with Graves' disease. Although most studies have confirmed a relationship between thyrotoxicosis and muscle dysfunction, few have measured changes in plasma metabolites and immune cells during the development and recovery from thyrotoxic myopathy. The aim of this study was to identify specific plasma metabolites and T‐cell subsets that predict thyrotoxic myopathy recovery in patients with Graves' disease.MethodsOne hundred patients (mean age, 40.0 ± 14.2 years; 67.0% female), with newly diagnosed or relapsed Graves' disease were enrolled at the start of methimazole treatment. Handgrip strength and Five Times Sit to Stand Test performance time were measured at Weeks 0, 12, and 24. In an additional 35 patients (mean age, 38.9 ± 13.5 years; 65.7% female), plasma metabolites and immunophenotypes of peripheral blood were evaluated at Weeks 0 and 12, and the results of a short physical performance battery assessment were recorded at the same time.ResultsIn both patient groups, methimazole‐induced euthyroidism was associated with improved handgrip strength and lower limb muscle function at 12 weeks. Elevated plasma metabolites including acylcarnitines were restored to normal levels at Week 12 regardless of gender, body mass index, or age (P trend <0.01). Senescent CD8+CD28−CD57+ T‐cell levels in peripheral blood were positively correlated with acylcarnitine levels (P < 0.05) and decreased during thyrotoxicosis recovery (P < 0.05). High levels of senescent CD8+ T cells at Week 0 were significantly associated with small increases in handgrip strength after 12 weeks of methimazole treatment (P < 0.05), but not statistically associated with Five Times Sit to Stand Test performance.ConclusionsRestoring euthyroidism in Graves' disease patients was associated with improved skeletal muscle function and performance, while thyroid hormone‐associated changes in plasma acylcarnitines levels correlated with muscle dysfunction recovery. T‐cell senescence‐related systemic inflammation correlated with plasma acylcarnitine levels and was also associated with small increases in handgrip strength.

HPMSCs-Derived Exosomal miRNA-21 Protects Against Aging-Related Oxidative Damage of CD4+ T Cells by Targeting the PTEN/PI3K-Nrf2 Axis.

Mesenchymal stem cells (MSCs)-derived exosomes were considered a novel therapeutic approach in many aging-related diseases. This study aimed to clarify the protective effects of human placenta MSCs-derived exosomes (hPMSC-Exo) in aging-related CD4+ T cell senescence and identified the underlying mechanisms using a D-gal induced mouse aging model. Senescent T cells were detected SA-β-gal stain. The degree of DNA damage was evaluated by detecting the level of 8-OH-dG. The superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) activities were measured. The expression of aging-related proteins and senescence-associated secretory phenotype (SASP) were detected by Western blot and RT-PCR. We found that hPMSC-Exo treatment markedly decreased oxidative stress damage (ROS and 8-OH-dG), SA-β-gal positive cell number, aging-related protein expression (p53 and γ-H2AX), and SASP expression (IL-6 and OPN) in senescent CD4+ T cells. Additionally, hPMSC-Exo containing miR-21 effectively downregulated the expression of PTEN, increased p-PI3K and p-AKT expression, and Nrf2 nuclear translocation and the expression of downstream target genes (NQO1 and HO-1) in senescent CD4+ T cells. Furthermore, in vitro studies uncovered that hPMSC-Exo attenuated CD4+ T cell senescence by improving the PTEN/PI3K-Nrf2 axis by using the PTEN inhibitor bpV (HOpic). We also validated that PTEN was a target of miR-21 by using a luciferase reporter assay. Collectively, the obtained results suggested that hPMSC-Exo attenuates CD4+ T cells senescence via carrying miRNA-21 and activating PTEN/PI3K-Nrf2 axis mediated exogenous antioxidant defenses.

Transcriptomic Features of Immune Exhaustion and Senescence Predict Outcomes and Define Checkpoint Blockade-Unresponsive Microenvironments in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease. Reinstating immunological control of AML is highly desirable to eradicate chemotherapy-resistant clones and provide long-term disease control. We recently identified bone marrow (BM) microenvironmental transcriptomic profiles that stratify patients with newly diagnosed AML into an immune-infiltrated and an immune-depleted subtype and that refine the accuracy of survival prediction beyond that afforded by current prognosticators (Vadakekolathu J et al., 2020). We have also shown that CD8 + T cells from patients with AML exhibit features of immune exhaustion and senescence (IES), including heightened expression of killer cell lectin-like receptor subfamily G member 1 (KLRG1) and B3GAT1 (encoding CD57) (Knaus H et al., 2018). Whether deranged T-cell functions affect the likelihood of responding to antitumor therapy, including immune checkpoint blockade (ICB), is an outstanding question in AML.In the current study, we analyzed 183 BM samples collected longitudinally at time of AML onset, response assessment and disease relapse from multiple cohorts of patients with AML treated with standard-of-care induction chemotherapy, and from 33 elderly AML patients with newly diagnosed or chemotherapy-refractory/relapsed AML treated with azacitidine, and the PD-1 checkpoint inhibitor pembrolizumab (NCT02845297). Primary patient specimens and associated clinical data were obtained via informed consent in accordance with the Declaration of Helsinki on research protocols approved by the Institutional Review Boards of the participating Institutions. RNA (150-200 ng) was extracted from BM aspirates and was processed on the nCounter FLEX analysis system (NanoString Technologies, Seattle, WA) using the PanCancer Immune profiling panel, as previously published (Vadakekolathu J et al., 2020). The correlation between transcriptomic features of IES, clinical characteristics, therapeutic response and patient outcome was validated using publicly available RNA-sequencing and NanoString data from 1,698 patients with AML, including samples from the TCGA-AML (n=147 cases), Beat-AML Master Trial (n=264 cases, of which 240 with survival data and 195 with chemotherapy response data) and Children's Oncology Group (COG)-TARGET AML series (n=145 cases).We initially showed that, compared with their non-senescent CD8 +CD57 -KLRG1 - counterpart, senescent CD8 +CD57 +KLRG1 + T cells are functionally impaired in terms of their ability to effect AML-blast killing mediated by an anti-CD33/CD3 bi-specific T-cell engager antibody construct (kindly provided by Amgen, USA; effector/target [E/T] ratio = 1:5). We then used gene set enrichment analysis (GSEA) to derive a transcriptomic signature of IES encompassing natural killer (NK)-cell and stem-like CD8 + T-cell markers, and showed that IES states correlate with lymphoid infiltration, adverse-risk molecular lesions (TP53 and RUNX1 mutations), experimental gene signatures of leukemia stemness (LSC17 score; Ng et al., 2016) and poor outcome in response to standard induction chemotherapy (Fig. 1A). In independent validation cohorts of children and adults with AML, the IES score was higher at baseline in patients with primary induction failure (following a standard 2 cycles of chemotherapy) compared with complete remission, increased in post-chemotherapy BM specimens, and predicted survival with greater accuracy than the ELN cytogenetic risk classifier (Fig. 1B). In the immunotherapy setting, high IES scores at baseline defined a checkpoint blockade-unresponsive AML tumor microenvironment and correlated with significantly shorter overall survival (9.1 versus 15.56 months in patients with high and low IES scores, respectively; HR = 3.32 (95% CI = 1.19-9.25); log-rank P = 0.021; Fig. 1C). Finally, the IES-related gene set also predicted for long-term outcomes and objective responses, based on RECIST criteria, to single-agent nivolumab or pembrolizumab, or combination anti-PD-1 + anti-CTLA-4, in 106 patients with melanoma (PRJEB23709 and GSE93157 series), a tumor type known to derive durable clinical benefit from ICB (Fig. 1D).Our findings encourage the pursuit of immune senescence reversal as a strategy to functionally reinvigorate T cells and could inform the delivery of ICB and other T cell-targeting immunotherapies to patients who are likely to benefit. [Display omitted] DisclosuresRadojcic: Syndax Pharmaceuticals: Research Funding; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Allakos: Membership on an entity's Board of Directors or advisory committees. Minden: Astellas: Consultancy. Tasian: Aleta Biotherapeutics: Consultancy; Gilead Sciences: Research Funding; Kura Oncology: Consultancy; Incyte Corporation: Research Funding.

Expansion of Phenotypically Senescent CD57+ CD8 T Cells Is Associated with Impaired Immunocompetence after Allogeneic Hematopoietic Stem Cell Transplantation

Background: T cell senescence is a physiological process typically associated with aging. In addition, lymphopenia and chronic immune activation can result in T cell senescence. CD57, a member of the N-CAM family initially described as a natural killer cell marker, has been reported as a marker of human senescent CD8 T cells. Percentages of CD57+ CD8 T cells increase during aging as well as during chronic viral infections. Early studies have reported increased proportions of CD57-expressing CD8 T cells after autologous and allogeneic hematopoietic stem cell transplantation (HSCT). Whether these cells can be considered a counterpart of the senescent population found during aging is at present unknown. More importantly, whether the expansion of CD57+ CD8 T cells is associated with impaired immunocompetence after allogeneic HSCT remains to be investigated.Materials and Methods: With written informed consent, peripheral blood mononuclear cells were collected from healthy controls (HC, n=21) and patients undergoing allogeneic HSCT (n=115) at Geneva University Hospitals. Proportions of CD57+ CD8 T cells as well as phenotypic and functional characteristics of CD57+ CD8 T cells were assessed by flow cytometry. Virus-specific CD8 T cells were identified by flow cytometry based on IFNg and/or TNFa intra-cytoplasmic expression after 6h in vitro stimulation with peptides derived from CMV, EBV, HHV6, BKV and Adenovirus. Torque Teno Virus (TTV) replication was quantified by quantitative PCR as previously described (Pradier et al., Front Immunol 2020; doi: 10.3389/fimmu.2020.00998).Results: CD8 T cells recovered from allogeneic HSCT displayed significantly higher proportions of CD57+ cells compared with healthy controls (HC, median 23% [range 6%-67%]; HSCT 58% [11%-97%]; p= 8.1e−06; Figure 1A-B). Such a difference was detected at early time points after transplantation and further increased at later time points (Figure 1B). After taking into account T cell subsets heterogeneity, we observed higher proportions of CD57+ cells in CD45RA- CCR7+ CD27+ central memory (CM; p= 0.00057), CD45RA- CCR7- CD27+ transitional memory (TM; p=1.1e-05) and CD45RA- CCR7- CD27- effector memory (EM; p=2.6e−06) CD8 T cells from allogeneic HSCT recipients compared with healthy controls. We did not observe any significant differences in CD57 expression in naïve nor in TEMRA CD8 T cells. Phenotypically, CD57+ CD8 T cells from allogeneic HSCT recipients displayed a senescent immunophenotype characterized by the low surface expression of CD127 that was further downregulated in CD57+ CD8 T cells from allogeneic HSCT recipients compared with healthy controls (p=0.00067). Functionally, CD57+ CD8 T cells from allogeneic HSCT recipients displayed a similar capacity to produce IFN-g, TNF-a, granzyme B and perforin when compared to CD57+ CD8 T cells from healthy controls. Virus-specific CD8 T cells identified upon stimulation with CMV, EBV, HHV6, BKV and Adenovirus peptides mainly displayed a CD27- effector memory phenotype in HSCT recipients (Figure 1C) and expressed higher levels of CD57 in HSCT recipients compared with healthy controls for CMV (p=0.017(; EBV p=0.018; Figure 1C-D). with a trend not reaching significance was for adenovirus, HHV6 and BK-virus-specific CD8 T cells. Using the replication of the non-pathogenic anellovirus TTV as a measure of impaired immunocompetence, we observed that the proportion of CD57-expressing cells among effector memory CD8 T cells positively correlated with TTV titers in allogeneic HSCT recipients (R=0.32, p=0.019; Figure 1E).Conclusion: These results show that the proportion of phenotypically senescent CD57+ CD8 T cells increases after allogeneic HSCT as a result of an increased expression at the surface of memory CD8 T cells, including virus-specific cells. Moreover, CD57 expression at the surface of EM CD8 T cells, a highly enriched population in allogeneic HSCT recipients, correlated with higher replication of TTV, reflecting a status of impaired immunocompetence after allogeneic HSCT. Studies are ongoing to determine the utility of CD57 expression on T cells as a biomarker to predict infectious complications after allogeneic HSCT. [Display omitted] DisclosuresChalandon: Incyte, BMS, Pfizer, Abbie, MSD, Roche, Novartis, Gilead, Amgen, Jazz, Astra Zenec: Other: Travel EXpenses, Accomodation; Incyte: Speakers Bureau; Incyte, BMS, Pfizer, Abbie, MSD, Roche, Novartis, Amgen: Other: Advisory Board. Simonetta: BMS Cellgene: Other: Ad-Hoc Advisory Board.

Deep phenotyping of T cell populations under long-term treatment of tacrolimus and rapamycin in patients receiving renal transplantations by mass cytometry.

Tacrolimus (FK506) and rapamycin (RAPA) are widely used to maintain long‐term immunosuppression after organ transplantation. However, the impact of accumulative drug administration on the recipients’ immune systems remains unclear. We investigated the impact of 3‐year FK506 or RAPA treatment after renal transplantation on the human immune systems. A discovery cohort of 30 patients was first recruited, and we discovered two distinctive T lineage suppressive regulatory patterns induced by chronic treatment of FK506 and RAPA. The increased percentage of senescent CD8+CD57+ T lineages and less responsive T cell receptor (TCR) pathway in the FK506 group indicate better graft acceptance. Meanwhile, percentages of regulatory T cells (Tregs) and expression of CTLA‐4 were both up to two‐fold higher in the RAPA group, suggesting the inconsistent reactivation potential of the FK506 and RAPA groups when an anti‐tumour or anti‐infection immune response is concerned. Additionally, up‐regulation of phosphorylated signaling proteins in T lineages after in vitro CD3/CD28 stimulation suggested more sensitive TCR‐signaling pathways reserved in the RAPA group. An independent validation cohort of 100 renal transplantation patients was further investigated for the hypothesis that long‐term RAPA administration mitigates the development of tumours and infections during long‐term intake of immunosuppressants. Our results indicate that RAPA administration indeed results in less clinical oncogenesis and infection. The deep phenotyping of T‐cell lineages, as educated by the long‐term treatment of different immunosuppressants, provides new evidence for personalized precision medicine after renal transplantations.

T lymphocyte senescence is attenuated in Parkinson\u2019s disease

BackgroundImmune involvement is well-described in Parkinson’s disease (PD), including an adaptive T lymphocyte response. Given the increasing prevalence of Parkinson’s disease in older age, age-related dysregulation of T lymphocytes may be relevant in this disorder, and we have previously observed changes in age-associated CD8+ T cell subsets in mid-stage PD. This study aimed to further characterise T cell immunosenescence in newly diagnosed PD patients, including shifts in CD4+ and CD8+ subpopulations, and changes in markers of cellular ageing in CD8+ T lymphocytes.MethodsPeripheral blood mononuclear cells were extracted from the blood of 61 newly diagnosed PD patients and 63 age- and sex-matched controls. Flow cytometric analysis was used for immunophenotyping of CD8+ and CD4+ lymphocyte subsets, and analysis of recent thymic emigrant cells. Telomere length within CD8+ T lymphocytes was assessed, as well as the expression of the telomerase reverse transcriptase enzyme (hTERT), and the cell-ageing markers p16INK4a and p21CIP1/Waf1.ResultsThe number of CD8+ TEMRA T cells was found to be significantly reduced in PD patients compared to controls. The expression of p16INK4a in CD8+ lymphocytes was also lower in patients versus controls. Chronic latent CMV infection was associated with increased senescent CD8+ lymphocytes in healthy controls, but this shift was less apparent in PD patients.ConclusionsTaken together, our data demonstrate a reduction in CD8+ T cell replicative senescence which is present at the earliest stages of Parkinson’s disease.

Altered Nutrient Uptake Causes Mitochondrial Dysfunction in Senescent CD8+ EMRA T Cells During Type 2 Diabetes.

Mitochondrial health and cellular metabolism can heavily influence the onset of senescence in T cells. CD8+ EMRA T cells exhibit mitochondrial dysfunction and alterations to oxidative phosphorylation, however, the metabolic properties of senescent CD8+ T cells from people living with type 2 diabetes (T2D) are not known. We show here that mitochondria from T2D CD8+ T cells had a higher oxidative capacity together with increased levels of mitochondrial reactive oxgen species (mtROS), compared to age-matched control cells. While fatty acid uptake was increased, fatty acid oxidation was impaired in T2D CD8+ EMRA T cells, which also showed an accumulation of lipid droplets and decreased AMPK activity. Increasing glucose and fatty acids in healthy CD8+ T cells resulted in increased p-p53 expression and a fragmented mitochondrial morphology, similar to that observed in T2D CD8+ EMRA T cells. The resulting mitochondrial changes are likely to have a profound effect on T cell function. Consequently, a better understanding of these metabolic abnormalities is crucial as metabolic manipulation of these cells may restore correct T cell function and help reduce the impact of T cell dysfunction in T2D.

Polyfunctional KLRG-1+CD57+ Senescent CD4+ T Cells Infiltrate Tumors and Are Expanded in Peripheral Blood From Breast Cancer Patients.

Senescent T cells have been described during aging, chronic infections, and cancer; however, a comprehensive study of the phenotype, function, and transcriptional program of this T cell population in breast cancer (BC) patients is missing. Compared to healthy donors (HDs), BC patients exhibit an accumulation of KLRG-1+CD57+ CD4+ and CD8+ T cells in peripheral blood. These T cells infiltrate tumors and tumor-draining lymph nodes. KLRG-1+CD57+ CD4+ and CD8+ T cells from BC patients and HDs exhibit features of senescence, and despite their inhibitory receptor expression, they produce more effector cytokines and exhibit higher expression of Perforin, Granzyme B, and CD107a than non-senescent subsets. When compared to blood counterparts, tumor-infiltrating senescent CD4+ T cells show similar surface phenotype but reduced cytokine production. Transcriptional profiling of senescent CD4+ T cells from the peripheral blood of BC patients reveals enrichment in genes associated with NK or CD8+-mediated cytotoxicity, TCR-mediated stimulation, and cell exhaustion compared to non-senescent T cells. Comparison of the transcriptional profile of senescent CD4+ T cells from peripheral blood of BC patients with those of HDs highlighted marked similarities but also relevant differences. Senescent CD4+ T cells from BC patients show enrichment in T-cell signaling, processes involved in DNA replication, p53 pathways, oncogene-induced senescence, among others compared to their counterparts in HDs. High gene expression of CD4, KLRG-1, and B3GAT1 (CD57), which correlates with increased overall survival for BC patients, underscores the usefulness of the evaluation of the frequency of senescent CD4+ T cells as a biomarker in the follow-up of patients.

Allotransplantation Is Associated With Exacerbation of CD8 T-Cell Senescence: The Particular Place of the Innate CD8 T-Cell Component.

Immunosenescence is a physiological process that is associated with changes in the immune system, particularly among CD8 T-cells. Recent studies have hypothesized that senescent CD8 T-cells are produced with chronologic age by chronic stimulation, leading to the acquisition of hallmarks of innate-like T-cells. While conventional CD8 T-cells are quite well characterized, CD8 T-cells sharing features of NK cells and memory CD8 T-cells, are a newly described immune cell population. They can be distinguished from conventional CD8 T-cells by their combined expression of panKIR/NKG2A and Eomesodermin (E), a unique phenotype closely associated with IFN-γ production in response to innate stimulation. Here, we first provided new evidence in favor of the innate character of panKIR/NKG2A(+) E(+) CD8 T-cells in normal subjects, documenting their position at an intermediate level in the innateness gradient in terms of both innate IFN-γ production and diminished mitochondrial mass. We also revealed that CD8 E(+) panKIR/NKG2A(+) T-cells, hereafter referred to as Innate E(+) CD8 T-cells, exhibit increased senescent (CD27(-) CD28(-)) phenotype, compared to their conventional memory counterparts. Surprisingly, this phenomenon was not dependent on age. Given that inflammation related to chronic viral infection is known to induce NK-like marker expression and a senescence phenotype among CD8 T-cells, we hypothesized that innate E(+) CD8 T-cells will be preferentially associated with exacerbated cellular senescence in response to chronic alloantigen exposure or CMV infection. Accordingly, in a pilot cohort of stable kidney allotransplant recipients, we observed an increased frequency of the Innate E(+) CD8 T-cell subset, together with an exacerbated senescent phenotype. Importantly, this phenotype cannot be explained by age alone, in clear contrast to their conventional memory counterparts. The senescent phenotype in CD8 T-cells was further increased in cytomegalovirus (CMV) positive serology transplant recipients, suggesting that transplantation and CMV, rather than aging by itself, may promote an exacerbated senescent phenotype of innate CD8 T-cells. In conclusion, we proposed that kidney transplantation, via the setting of inflammatory stimuli of alloantigen exposure and CMV infection, may exogenously age the CD8 T-cell compartment, especially its innate component. The physiopathological consequences of this change in the immune system remain to be elucidated.

Abstract 510: Impact of tisagenlecleucel product attributes on clinical outcomes in pediatric and young adult patients with relapsed or refractory acute lymphoblastic leukemia (r/r ALL)

Abstract Background Tisagenlecleucel CAR-T cell therapy showed durable clinical outcomes in pediatric/young adult patients with r/r ALL in ELIANA/ENSIGN trials. We examined the effect of tisagenlecleucel product attributes on clinical outcomes (Table). Methods Key product attributes of tisagenlecleucel were examined with univariate and multivariate (lasso, random forest) approaches (N=143) to evaluate the effects on efficacy (best overall response [BOR] ≤3 months, duration of remission, event-free survival [EFS], and safety (cytokine release syndrome [CRS] and neurological events). Results Univariate: High in vitro IFNγ release positively correlated with BOR and EFS. Relative and absolute number (no.) of early differentiated (ED) CD4+ and CD8+, activated (HLA-DR+CD38+) CD8+, and absolute no. of naïve CD4+ cells positively correlated with efficacy. Total CD4+ cells with central memory markers, LAG3+, CD25+ had weak positive correlation with grade 3/4 CRS. Relative no. of effector memory CD4+ and KLRG1+CD8+ and senescent CD4+ and CD8+ cells negatively correlated with efficacy. Median T-cell transduction efficiency by flow (23%; range, 1.5%-56%) and viability did not correlate with efficacy or safety. Also, the median CD4:CD8 cell ratio did not correlate with outcomes. Multivariate: ED CD4+ and CD8+, and activated CD8+ cells positively correlated with BOR. Naïve CD4+ cells positively correlated with survival, while KLRG1+CD8+ cells and effector memory CD4+ cells negatively correlated with survival. Conclusions Less mature T cells trended toward improved efficacy, while senescent and differentiated cells led to worse outcomes. No strong correlations to safety outcomes were observed. While phenotypes do not definitively distinguish outcomes, the analyses provide insight into the potential set of parameters to be used in predictive/causal modeling. Table. Univariate analysis of tisagenlecleucel product attributes and clinical outcomesProduct Attributes (CAR+ Cells)Correlation,a +/–BORDOREFSCRS G3/4NE G3/4IFNγ++++Transduction efficiency+Total CD4++++Total CD8+++++ED CD4+ cells+++++++ED CD8+ cells++++++TIM3+ CD4+ cellsTIM3+ CD8+ cells+++Activated CD4+ cells+++Activated CD8+ cells++++++HLA-DR+ CD4+ cells+HLA-DR+ CD8+ cells++++Naïve CD4+ cells++++++Naïve CD8+ cells+LAG3+ CD4+ cells++CD25+ CD4+ cells++Effector memory CD4+ cells– –– – –KLRG1+ CD4+ cells– –KLRG1+ CD8+ cells– – –– –– –Senescent CD4+ cells– – –Senescent CD8+ cells–Key: +++/– – –, P value &amp;lt;0.01; ++/– –, P value &amp;lt;0.05; +/–, P value &amp;lt;0.1 P values refer to Cox regression or Wilcoxon tests. Note: Central memory CD4+ and CD8+, LAG3+ CD8+, CD25+ CD8+ cells and CD4:CD8 ratio were analyzed but showed no significance. aComparison of ≤median to &amp;gt;median. BOR, best overall response; CAR, chimeric antigen receptor; CD, cluster of differentiation;CRS, cytokine release syndrome; DOR, duration of remission; ED, early differentiated;EFS, event-free survival; G, grade; HLA-DR, human leukocyte antigen-DR; IFN, interferon; KLRG1, Killer cell lectin-like receptor G1; LAG3, lymphocyte-activation gene 3; NE, neurological events; TIM3, T-cell immunoglobulin and mucin domain-3. Citation Format: Irina Gershgorin, Edward R. Waldron, Stephan A. Grupp, John E. Levine, Michael A. Pulsipher, Stella M. Davies, G. Doug Myers, Margit Jeschke, Boris Engels, Chris del Corral, Andre Baruchel. Impact of tisagenlecleucel product attributes on clinical outcomes in pediatric and young adult patients with relapsed or refractory acute lymphoblastic leukemia (r/r ALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 510.

Bacterial genotoxins induce Tcell senescence.

Several types of pathogenic bacteria produce genotoxins that induce DNA damage in host cells. Accumulating evidence suggests that a central function of these genotoxins is to dysregulate the host's immune response, but the underlying mechanisms remain unclear. To address this issue, we investigated the effects of the most widely expressed bacterial genotoxin, the cytolethal distending toxin (CDT), on Tcells-the key mediators of adaptive immunity. We show that CDT induces premature senescence in activated CD4 Tcells invitro and provide evidence suggesting that infection with genotoxin-producing bacteria promotes Tcell senescence invivo. Moreover, we demonstrate that genotoxin-induced senescent CD4 Tcells assume a senescence-associated secretory phenotype (SASP) which, at least partly, is orchestrated by the ATM-p38 signaling axis. These findings provide insight into the immunomodulatory properties of bacterial genotoxins and uncover a putative link between bacterial infections and Tcell senescence.

Senescence-associated β-galactosidase activity and other markers of senescence are present in human peripheral blood CD8+ cells during healthy aging

Abstract Aging is associated with a decline in the immune system, termed “immunosenescence”. Cellular senescence is defined by persistent cellular growth arrest and overall loss of function. If immune cells undergo cellular senescence in vivo, and if it has a role in immunosenesence, remains controversial due to the lack of specific markers to identify these cells. Human CD8+ T cells from younger and older donors were isolated from blood and labeled with a fluorescent substrate for senescence-associated b-galactosidase (SA-βGal). CD8+ T cells were sorted based on SA-βGal and analyzed by qRT-PCR, RNA sequencing, and microscopy. We found a higher percentage of CD8+ T cells with high SA-βGal activity in the older cohort. SA-βGal high CD8+ T cells displayed increased p21, p16INK4a, and inflammatory cytokine transcripts. They also had greater DNA damage response foci and p16INK4a protein and had deficient proliferation. SA-βGal increased as T cells matured, which was consistent regardless of age. Intriguingly, a portion of effector cells did not exhibit a senescent phenotype and maintained low SA-βGal. RNA sequencing of CD8+ T cells revealed cells with low, medium, and high SA-βGal had distinct profiles. In addition, SA-βGal high CD8+ T cells had larger mitochondrial mass than cells with low SA-βGal as measured by MitoID staining. This may indicate differences in metabolism, which may contribute to senescence. The presence of p16INK4a, DNA damage, and inflammatory cytokines and deficient proliferation demonstrate that SA-βGal is a strong indicator of cellular senescence in CD8+. T cells. SA-βGal can be leveraged to further identify senescent immune cells and elucidate cellular senescence’s role in immunosenesence.

Senescence-associated β-galactosidase reveals the abundance of senescent CD8+ T cells in aging humans.

Aging leads to a progressive functional decline of the immune system, rendering the elderly increasingly susceptible to disease and infection. The degree to which immune cell senescence contributes to this decline remains unclear, however, since markers that label immune cells with classical features of cellular senescence accurately and comprehensively have not been identified. Using a second‐generation fluorogenic substrate for β‐galactosidase and multi‐parameter flow cytometry, we demonstrate here that peripheral blood mononuclear cells (PBMCs) isolated from healthy humans increasingly display cells with high senescence‐associated β‐galactosidase (SA‐βGal) activity with advancing donor age. The greatest age‐associated increases were observed in CD8+ T‐cell populations, in which the fraction of cells with high SA‐βGal activity reached average levels of 64% in donors in their 60s. CD8+ T cells with high SA‐βGal activity, but not those with low SA‐βGal activity, were found to exhibit features of telomere dysfunction‐induced senescence and p16‐mediated senescence, were impaired in their ability to proliferate, developed in various T‐cell differentiation states, and had a gene expression signature consistent with the senescence state previously observed in human fibroblasts. Based on these results, we propose that senescent CD8+ T cells with classical features of cellular senescence accumulate to levels that are significantly higher than previously reported and additionally provide a simple yet robust method for the isolation and characterization of senescent CD8+ T cells with predictive potential for biological age.

Relative increase of Th17 phenotype in senescent CD4+CD28null T cells from peripheral blood of patients with rheumatoid arthritis

Relative increase of Th17 phenotype in senescent CD4+CD28null T cells from peripheral blood of patients with rheumatoid arthritis

Traditional Thai Massage Promoted Immunity in the Elderly via Attenuation of Senescent CD4+ T Cell Subsets: A Randomized Crossover Study.

The beneficial physiological effects of traditional Thai massage (TTM) have been previously documented. However, its effect on immune status, particularly in the elderly, has not been explored. This study aimed to investigate the effects of multiple rounds of TTM on senescent CD4+ T cell subsets in the elderly. The study recruited 12 volunteers (61–75 years), with senescent CD4+ T cell subsets, who received six weekly 1-h TTM sessions or rest, using a randomized controlled crossover study with a 30-day washout period. Flow cytometry analysis of surface markers and intracellular cytokine staining was performed. TTM could attenuate the senescent CD4+ T cell subsets, especially in CD4+28null NKG2D+ T cells (n = 12; p < 0.001). The participants were allocated into two groups (low < 2.75% or high ≥ 2.75%) depending on the number of CD4+28null NKG2D+ T cells. After receiving TTM over 6 sessions, the cell population of the high group had significantly decreased (p < 0.001), but the low group had no significant changes. In conclusion, multiple rounds of TTM may promote immunity through the attenuation of aberrant CD4+ T subsets. TTM may be provided as a complementary therapy to improve the immune system in elderly populations.