Altered Ca2+ Homeostasis in Immune Cells during Aging: Role of Ion Channels

Aging is an unstoppable process and begins shortly after birth. Each cell of the organism is affected by the irreversible process, not only with equal density but also at varying ages and with different speed. Therefore, aging can also be understood as an adaptation to a continually changing cellular environment. One of these very prominent changes in age affects Ca2+ signaling. Especially immune cells highly rely on Ca2+-dependent processes and a strictly regulated Ca2+ homeostasis. The intricate patterns of impaired immune cell function may represent a deficit or compensatory mechanisms. Besides, altered immune function through Ca2+ signaling can profoundly affect the development of age-related disease. This review attempts to summarize changes in Ca2+ signaling due to channels and receptors in T cells and beyond in the context of aging

Heterozygous OT-I mice reveal that antigen-specific CD8+ T cells shift from apoptotic to necrotic killers in the elderly

Numerous alterations in CD8+ T cells contribute to impaired immune responses in elderly individuals. However, the discrimination between cell-intrinsic dysfunctions and microenvironmental changes is challenging. TCR transgenic OT-I mice are utilized to investigate CD8+ T-cell immunity, but their immunodeficient phenotype hampers their use especially in aging. Here, we demonstrate that using a heterozygous OT-I model minimizes the current limitations and provides a valuable tool to assess antigen-specific T-cell responses even at old age. We analyzed phenotypic and functional characteristics of CD8+ T cells from OT-I +/+ and OT-I +/− mice to prove the applicability of the heterozygous system. Our data reveal that OVA-activated CD8+ T cells from adult OT-I +/− mice proliferate, differentiate, and exert cytolytic activity equally to their homozygous counterparts. Moreover, common age-related alterations in CD8+ T cells, including naive T-cell deterioration and decreased proliferative capacity, also occur in elderly OT-I +/− mice, indicating the wide range of applications for in vivo and in vitro aging studies. We used the OT-I +/− model to investigate cell-intrinsic alterations affecting the cytotoxic behavior of aged CD8+ T cells after antigen-specific in vitro activation. Time-resolved analysis of antigen-directed target cell lysis confirmed previous observations that the cytotoxic capacity of CD8+ T cells increases with age. Surprisingly, detailed single cell analysis revealed that transcriptional upregulation of perforin in aged CD8+ T cells shifts the mode of target cell death from granzymemediated apoptosis to rapid induction of necrosis. This unexpected capability might be beneficial or detrimental for the aging host and requires detailed evaluation

Faster cytotoxicity with age : Increased perforin and granzyme levels in cytotoxic CD8+ T cells boost cancer cell elimination

A variety of intrinsic and extrinsic factors contribute to the altered efficiency of CTLs in elderly organisms. In particular, the efficacy of antiviral CD8+ T cells responses in the elderly has come back into focus since the COVID-19 pandemic outbreak. However, the exact molecular mechanisms leading to alterations in T cell function and the origin of the observed impairments have not been fully explored. Therefore, we investigated whether intrinsic changes affect the cytotoxic ability of CD8+ T cells in aging. We focused on the different subpopulations and time-resolved quantification of cytotoxicity during tumor cell elimination. We report a surprising result: Killing kinetics of CD8+ T cells from elderly mice are much faster than those of CD8+ T cells from adult mice. This is true not only in the total CD8+ T cell population but also for their effector (TEM) and central memory (TCM) T cell subpopulations. TIRF experiments reveal that CD8+ T cells from elderly mice possess comparable numbers of fusion events per cell, but significantly increased numbers of cells with granule fusion. Analysis of the cytotoxic granule (CG) content shows significantly increased perforin and granzyme levels and turns CD8+ T cells of elderly mice into very efficient killers. This highlights the importance of distinguishing between cell-intrinsic alterations and microenvironmental changes in elderly individuals. Our results also stress the importance of analyzing the dynamics of CTL cytotoxicity against cancer cells because, with a simple endpoint lysis analysis, cytotoxic differences could have easily been overlooked

High Glucose Enhances Cytotoxic T Lymphocyte-Mediated Cytotoxicity

Cytotoxic T lymphocytes (CTLs) are key players to eliminate tumorigenic or pathogen-infected cells using lytic granules (LG) and Fas ligand (FasL) pathways. Depletion of glucose leads to severely impaired cytotoxic function of CTLs. However, the impact of excessive glucose on CTL functions still remains largely unknown. Here we used primary human CD8+ T cells, which were stimulated by CD3/CD28 beads and cultured in medium either containing high glucose (HG, 25 mM) or normal glucose (NG, 5.6 mM). We found that in HG-CTLs, glucose uptake and glycolysis were enhanced, whereas proliferation remained unaltered. Furthermore, CTLs cultured in HG exhibited an enhanced CTL killing efficiency compared to their counterparts in NG. Unexpectedly, expression of cytotoxic proteins (perforin, granzyme A, granzyme B and FasL), LG release, cytokine/cytotoxic protein release and CTL migration remained unchanged in HG-cultured CTLs. Interestingly, additional extracellular Ca2+ diminished HG-enhanced CTL killing function. Our findings suggest that in an environment with excessive glucose, CTLs could eliminate target cells more efficiently, at least for a certain period of time, in a Ca2+-dependent manner

High Glucose Enhances Cytotoxic T Lymphocyte-Mediated Cytotoxicity.

peer reviewedCytotoxic T lymphocytes (CTLs) are key players to eliminate tumorigenic or pathogen-infected cells using lytic granules (LG) and Fas ligand (FasL) pathways. Depletion of glucose leads to severely impaired cytotoxic function of CTLs. However, the impact of excessive glucose on CTL functions still remains largely unknown. Here we used primary human CD8+ T cells, which were stimulated by CD3/CD28 beads and cultured in medium either containing high glucose (HG, 25 mM) or normal glucose (NG, 5.6 mM). We found that in HG-CTLs, glucose uptake and glycolysis were enhanced, whereas proliferation remained unaltered. Furthermore, CTLs cultured in HG exhibited an enhanced CTL killing efficiency compared to their counterparts in NG. Unexpectedly, expression of cytotoxic proteins (perforin, granzyme A, granzyme B and FasL), LG release, cytokine/cytotoxic protein release and CTL migration remained unchanged in HG-cultured CTLs. Interestingly, additional extracellular Ca2+ diminished HG-enhanced CTL killing function. Our findings suggest that in an environment with excessive glucose, CTLs could eliminate target cells more efficiently, at least for a certain period of time, in a Ca2+-dependent manner

Gesteigerte Zytotoxizität muriner CD8+ T-Zellen im Alter

Das Altern geht mit einer tiefgreifenden Veränderung der Immunfunktionen einher. Ein steigendes Risiko für Krebserkrankungen und schwere Krankheitsverläufe bei Infektionen machen deutlich, welche dramatischen Auswirkungen eine Beeinträchtigung des Immunsystems auf die Lebensqualität und die Mortalität im Alter haben kann. Zytotoxische CD8+ T-Zellen besitzen die Fähigkeit zur Ausbildung eines immunologischen Gedächtnisses und spielen als Teil der adaptiven Immunantwort eine Schlüsselrolle bei der Erkennung und Abtötung virusinfizierter und entarteter Zellen. CD8+ T-Zellen nutzen für die Eliminierung ihrer Zielzellen zwei wesentliche Zytotoxizitätsmechanismen: Die über Liganden Rezeptor Interaktion induzierte Apoptose und die Exozytose vermittelte Freisetzung lytischer Granula. In der Vergangenheit wurden bereits eine Vielzahl von quantitativen und funktionalen Veränderungen von CD8+ T-Zellen im Alter identifiziert, die zu einer verminderten Immunantwort gegenüber neuen Erregern und Tumorzellen beitragen. Dennoch sind die zugrunde liegenden Mechanismen bis heute nur unvollständig verstanden. Insbesondere die Abgrenzung zelleigener Alterationen vom Einfluss der alternden Mikroumgebung bleibt eine Herausforderung, ist für die Optimierung immuntherapeutischer Ansätze jedoch von zentraler Bedeutung. Trotzt der mehrfach beschriebenen Beeinträchtigungen der CD8+ T Zell-Immunität im Alter, ist über Alterationen in der zytotoxischen Effektorfunktion der Zellen bemerkenswert wenig bekannt. Im Rahmen dieser Arbeit wurde daher untersucht welchen Einfluss zelleigene Veränderungen auf die Zytotoxizität von murinen CD8+ T-Zellen im Alter nehmen. Fluoreszenzbasierte Echtzeit Zytotoxizitätsassays auf Populations- und Einzelzellebene lieferten ein überraschendes Ergebnis: CD8+ T-Zellen aus gealterten C57BL/6J Wildtyp Mäusen zeigen nach polyklonaler in vitro Stimulation eine signifikant schnellere Kinetik der Zielzelllyse. Dies gilt nicht nur für die Gesamt CD8+ T Zellpopulation, sondern trifft ebenfalls auf die zytotoxische Aktivität zentraler Gedächtnis- und Effektor-Gedächtnis-T-Zellen zu. Diese Ergebnisse legen nahe, dass es im Alter zu einem veränderten Phänotyp beider CD8+ Gedächtnis-Populationen kommt, der mit einer erhöhten zytotoxischen Effizienz der Zellen einhergeht. Die weitere Analyse der zugrundeliegenden Zytotoxizitätsmechanismen verdeutlichte, dass die schnelle Zielzelllyse durch CD8+ T-Zellen im Alter weniger auf Alterationen in Rezeptor vermittelten Signalwegen, sondern vielmehr auf eine veränderte Granula-Exozytose-vermittelte Zytotoxizität zurückzuführen ist. Während durch die Beurteilung des Degranulationsverhaltens mittels Durchflusszytometrie und TIRF-Mikroskopie keine altersbedingte Veränderung in der Exozytose lytischer Granula nachgewiesen werden konnte, zeigte die Quantifizierung der in den lytischen Vesikeln enthaltenen Effektormoleküle eine im Alter signifikant erhöhte Expression von Perforin und Granzym B. Die Ergebnisse mit in vitro stimulierten murinen CD8+ T-Zellen lieferten somit erstmals deutliche Hinweise darauf, dass die altersbedingt unzureichende Eliminierung von Erregern und Tumorzellen in vivo, nicht durch zelleigene Alterationen in der zytotoxischen Funktion von CD8+ T-Zellen bedingt ist. Im Gegensatz zu der hier durchgeführten Antikörper-basierten polyklonalen Stimulation der CD8+ T Zellen, erfolgt die Aktivierung des T-Zell-Rezeptors unter physiologischen Bedingungen antigenspezifisch. Die Bestätigung der aus den Wildtyp-Mäusen gewonnenen Erkenntnisse erforderte daher die Untersuchung der zytotoxischen Funktion nach antigenspezifischer in vitro Aktivierung. OT I Mäuse exprimieren T-Zell-Rezeptoren auf CD8+ T-Zellen die spezifisch das über den MHC-I Komplex präsentierte Peptid OVA257-264 des Modellantigens Ovalbumin erkennen und zählen zu den am häufigsten verwendeten transgenen Mausmodellen für die Untersuchung der antigenspezifischen CD8+ T Zell Immunität. Der per se immundefiziente Phänotyp der Mäuse hat jedoch eine deutliche Reduktion der Lebenserwartung zur Folge, wodurch die üblicherweise eingesetzte homozygote OT-I Linie für Alterungsstudien weitestgehend ungeeignet ist. Um diese Limitation zu überwinden, wurde im Rahmen dieser Arbeit eine heterozygote OT-I Linie etabliert, was den Gesundheitsstatus der Mäuse deutlich verbesserte und erstmals die Charakterisierung der Effektorfunktionen antigenspezifischer OT-I CD8+ T-Zellen im Alter ermöglichte. Die kinetische Analyse der Zielzelllyse im antigenspezifischen in vitro Modell bestätigte die hohe zytotoxische Effizienz von CD8+ T-Zellen im Alter. Eine erhöhte Expression von Perforin und Granzym in CD8+ T-Zellen aus alten OT-I Mäusen unterstützt die Annahme, dass die gesteigerte Zytotoxizität der Zellen durch zellintrinsische Alterationen in der Expression lytischer Effektormoleküle bedingt ist. Die Generierung einer OVA-exprimierenden Tumorzellline mit stabiler Expression des Apoptose Reporters pCasper ermöglichte eine detailliertere Analyse des Zielzelltodes. Dabei zeigte sich, dass antigenspezifische CD8+ T-Zellen aus alten OT-I Mäusen bereits wenige Minuten nach Zielzellkontakt einen nekrotischen Zelltod verursachen. Offenbar führen die hohen Perforin Konzentrationen im Alter zu einer erheblichen Schädigung der Membranintegrität von Zielzellen, wodurch die Granzym vermittelte Apoptose durch eine schnelle Induktion von Nekrosen ersetzt wird. Die in dieser Arbeit aufgezeigten zelleigenen Veränderungen der zytotoxischen Effektorfunktion von CD8+ T-Zellen im Alter könnten sich für den alternden Organismus als vorteilhaft oder nachteilig erweisen und sollten im Rahmen immunmodulatorischer Ansätze nicht unberücksichtigt bleiben. Die Möglichkeit heterozygote OT-I Mäuse bis ins hohe Alter zu halten und die Entwicklung typischer altersbedingter Veränderungen ihrer CD8+ T-Zellen, macht sie zu einem attraktiven Modell mit breitem Anwendungsfeld für zukünftige in vitro und in vivo Alterungsstudien.Summary Aging is accompanied by pronounced changes in immune cell functions. Higher incidences of cancer and a higher risk for severe infectious diseases underline the dramatic impact of impaired immunity on the life quality and mortality of elderly individuals. As part of the adaptive immune response, cytotoxic CD8+ T cells are key players in recognizing and killing virus-infected and tumor cells. Target cell elimination by cytotoxic CD8+ T cell can be induced by two major pathways: Induction of apoptosis by ligand-receptor interactions or exocytosis-mediated release of lytic granules. Various quantitative and functional alterations of CD8+ T cells have been identified that contribute to the impaired immune response to novel pathogens and cancer cells during aging. Nevertheless, the underlying mechanisms are still incompletely understood. In particular, separating cell-intrinsic alterations from the aged environments‘ influence remains challenging but is crucial for optimizing immunotherapeutic approaches. Despite the commonly described defects in CD8+ T cells‘ immunity in old age, remarkably little is known about alterations in the cytotoxic behavior itself. Therefore, this work addressed the impact of cell-intrinsic alterations on the cytotoxic effector function of murine CD8+ T cells during aging. Fluorescence-based real-time cytotoxicity assays at population and single-cell level revealed a surprising result: CD8+ T cells from elderly C57BL/6J wild-type mice exhibit significantly faster kinetics of target cell lysis after polyclonal in vitro stimulation. This is true for the total CD8+ T cell population and applies to the cytotoxic activity of central memory and effector memory T cells. These findings suggest a yet undescribed altered phenotype of both CD8+ memory populations, associated with an increased cytotoxic capacity with age. Further analysis of the underlying cytotoxic mechanisms revealed that alterations in the granule exocytosis pathway decisively contribute to increased cytotoxicity during aging. While evaluation of degranulation ability by flow cytometry and TIRF microscopy did not show any age-related changes in the exocytosis of lytic granules, quantification of the lytic granules’ effector molecules revealed significantly increased expression of perforin and granzyme B in CD8+ T cells from elderly mice. Together, these findings provide evidence that the age-related inadequate elimination of pathogens and tumor cells in vivo is not caused by cell-intrinsic alterations in CD8+ T cells’ cytotoxic effector function. In contrast to antibody-based polyclonal in vitro stimulation as used for CD8+ T cells from wild-type mice, physiological activation of T cell receptors requires the recognition of a specific antigen. Thus, investigating the cytotoxic behavior of aged CD8+ T cells after antigen-specific in vitro stimulation was necessary to confirm the recent findings in a more physiological context. OT-I mice express T cell receptors on CD8+ T cells specific for the OVA257-264 peptide and are among the most widely used transgenic mouse models to study antigen-specific CD8+ T cell immunity. Unfortunately, using a homozygous OT-I model in aging research is challenging due to increased mortality and general health issues of these per se immunodeficient mice. Within the scope of this work, a heterozygous OT-I model was established, which improved the health status of the mice and enabled the characterization of antigen-specific CD8+ T cells even in old age. Kinetic analysis of antigen-directed target cell lysis confirmed the high cytotoxic efficiency of CD8+ T cells with age. Increased expression of perforin and granzyme B in CD8+ T cells from elderly OT-I mice supports the notion that increased cytotoxicity is caused by cell-intrinsic alterations in the expression of lytic effector molecules. Generating an OVA-expressing tumor cell line that stably expresses the apoptosis reporter pCasper allowed a more detailed analysis of target cell death, uncovering that antigen-specific CD8+ T cells from elderly OT-I mice induce necrotic cell death within minutes after target cell contact. Age-related high perforin concentrations substantially disrupt the targets cells' membrane integrity, replacing granzyme-mediated apoptosis by rapid necrosis induction. The cell-intrinsic alterations of CD8+ T cells uncovered within the present work might prove to be beneficial or detrimental for the aging organism and are worth considering in immunomodulatory approaches. The possibility of keeping heterozygous OT-I mice into old age and the development of typical age-related changes of their CD8+ T cells opens a widely applicable tool for future in vitro and in vivo aging studies

Altered Ca2+ Homeostasis in Immune Cells during Aging: Role of Ion Channels

Aging is an unstoppable process and begins shortly after birth. Each cell of the organism is affected by the irreversible process, not only with equal density but also at varying ages and with different speed. Therefore, aging can also be understood as an adaptation to a continually changing cellular environment. One of these very prominent changes in age affects Ca2+ signaling. Especially immune cells highly rely on Ca2+-dependent processes and a strictly regulated Ca2+ homeostasis. The intricate patterns of impaired immune cell function may represent a deficit or compensatory mechanisms. Besides, altered immune function through Ca2+ signaling can profoundly affect the development of age-related disease. This review attempts to summarize changes in Ca2+ signaling due to channels and receptors in T cells and beyond in the context of aging