GENERATION OF EBV-SPECIFIC AND EBV-SPECIFIC, TCR-REPROGRAMMED HUMAN CD8+ CYTOTOXIC T LYMPHOCYTES WITH STEM CELL MEMORY AND CENTRAL MEMORY PROPERTIES BY MODULATING GLYCOLYTIC T CELL METABOLISM
(Abstract release date: 05/21/15)
EHA Library. Hartwig U. 06/13/15; 103117; S467
Disclosure(s): University Medical Center MainzIII. Dept. of Medicine - Hematology, Internal Oncology & Pneumology
Assoc. Prof. Udo Hartwig
Contributions
Contributions
Abstract
Abstract: S467
Type: Oral Presentation
Presentation during EHA20: From 13.06.2015 11:45 to 13.06.2015 12:00
Location: Room Stolz 2
Background
Adoptive transfer of virus-specific and TCR- or CAR-reprogrammed cytotoxic T lymphocyzes (CTL) has advanced as a valuable cellular therapy for opportunistic viral infections, Epstein-Barr Virus (EBV)-mediated lymphoma and leukemia relapse. However, durable clinical responses are often hampered by limited capability of terminally differentiated, high avidity effector T cells (TEFF) to establish sustained antileukemic immunity whereas less differentiated stem cell memory (TSCM) and central memory (TCM) T cells could be shown to elicit potent antitumor responses, prolonged survival and memory. Moreover, TSCM and TCM depend less on glucose consumption to drive oxidative phosphorylation (OXPHOS) as their primary source of adenosine triphosphate (ATP) production whereas TEFF require additional aerobic glycolysis to generate the ATP needed.
Aims
In the current study, we therefore investigated means of modulating T cell metabolism to generate EBV-specific TSCM and TCM as well as EBV-specific TSCM/CM TCR-reprogrammed T cells.
Methods
Naïve CD8+CD45RA+ T cells isolated from peripheral blood mononuclear cells (PBMC) of healthy HLA-A2+ donors using the Naive T cell isolation kit® (Milenyi Biotec, Bergisch Gladbach, Germany) and unselected (total) CD8+ T cells were primed by autologous dendritic cells loaded with EBV-peptides followed by restimulation with peptide loaded autologous PBMC in the presence of low (1 mM) glucose, glutamine, an optimized cytokine cocktail and 1 mM of the non-metabolizable glucose analogue 2-deoxy-glucose (2-DG). Moreover, either galactose or 9-oleic acid was added to glucose deprived cultures. In addition to phenotypic and functional in vitro analyses by FACS, IFN-g ELISPOT- and 51Cr-release assays, glucose uptake and lactate production was determined. OXPHOS and aerobic glycolysis were assessed by measuring oxygen-consumption rate (OCR) and extracellular acidification rate (ECAR), respectively, using a Seahorse Analyzer. Finally, the biological activity of EBV-reactive TSCM/CM was tested in vivo in NSG mice.
Results
We obtained strong expansion of EBV-reactive CTL expressing a CD8+CD45RA+CD45RO-CD95+ CD27+CD28+CD62L+CCR7+TSCM and CD8+CD45RA- CD45RO+CD95+CD27+CD28+CD62L+CCR7+ TCM phenotype upon repetitive stimulation of naïve T cells in the presence of low glucose, glutamine and 2-DG when compared to CTL stimulated without 2-DG. This effect was also seen in total CD8+ T cells although less pronounced. In contrast, supply of galactose or oleic-acid to glucose free (but glutamine containing) medium did not result in reduced TEFF differentiation, suggesting OXPHOS by galactose converted to glucose and fatty acid oxidation. Moreover, 2-DG treated TSCM and TCM as well as total CD8+ CTL showed less lactate production and ECAR as compared to untreated controls, confirming that differentiation to TEFF requires aerobic glycolytic ATP production. Functional assays in vitro did not only reveal cytolytic activity of 2-DG treated EBV-TSCM and TCM comparable to controls but elicited superior migration properties of these cells when tested to untreated naïve or total CD8+ T cells in a transwell- migration assay. We further showed that EBV-TSCM/CM can be successfully TCR-reprogrammed to recognize primary acute myeloid leukaemia (AML) blasts.
Summary
These studies demonstrate that modulation of T cell metabolism may be a promising approach to generate stem cell memory and central memory T cells in vitro for virus-specific and redirected immunity in adoptive cellular therapy.
Keyword(s): Adoptive immunotherapy, Antigen-specific T cells, EBV, Memory T cells
Session topic: Gene therapy, cellular immunotherapy and vaccination
Type: Oral Presentation
Presentation during EHA20: From 13.06.2015 11:45 to 13.06.2015 12:00
Location: Room Stolz 2
Background
Adoptive transfer of virus-specific and TCR- or CAR-reprogrammed cytotoxic T lymphocyzes (CTL) has advanced as a valuable cellular therapy for opportunistic viral infections, Epstein-Barr Virus (EBV)-mediated lymphoma and leukemia relapse. However, durable clinical responses are often hampered by limited capability of terminally differentiated, high avidity effector T cells (TEFF) to establish sustained antileukemic immunity whereas less differentiated stem cell memory (TSCM) and central memory (TCM) T cells could be shown to elicit potent antitumor responses, prolonged survival and memory. Moreover, TSCM and TCM depend less on glucose consumption to drive oxidative phosphorylation (OXPHOS) as their primary source of adenosine triphosphate (ATP) production whereas TEFF require additional aerobic glycolysis to generate the ATP needed.
Aims
In the current study, we therefore investigated means of modulating T cell metabolism to generate EBV-specific TSCM and TCM as well as EBV-specific TSCM/CM TCR-reprogrammed T cells.
Methods
Naïve CD8+CD45RA+ T cells isolated from peripheral blood mononuclear cells (PBMC) of healthy HLA-A2+ donors using the Naive T cell isolation kit® (Milenyi Biotec, Bergisch Gladbach, Germany) and unselected (total) CD8+ T cells were primed by autologous dendritic cells loaded with EBV-peptides followed by restimulation with peptide loaded autologous PBMC in the presence of low (1 mM) glucose, glutamine, an optimized cytokine cocktail and 1 mM of the non-metabolizable glucose analogue 2-deoxy-glucose (2-DG). Moreover, either galactose or 9-oleic acid was added to glucose deprived cultures. In addition to phenotypic and functional in vitro analyses by FACS, IFN-g ELISPOT- and 51Cr-release assays, glucose uptake and lactate production was determined. OXPHOS and aerobic glycolysis were assessed by measuring oxygen-consumption rate (OCR) and extracellular acidification rate (ECAR), respectively, using a Seahorse Analyzer. Finally, the biological activity of EBV-reactive TSCM/CM was tested in vivo in NSG mice.
Results
We obtained strong expansion of EBV-reactive CTL expressing a CD8+CD45RA+CD45RO-CD95+ CD27+CD28+CD62L+CCR7+TSCM and CD8+CD45RA- CD45RO+CD95+CD27+CD28+CD62L+CCR7+ TCM phenotype upon repetitive stimulation of naïve T cells in the presence of low glucose, glutamine and 2-DG when compared to CTL stimulated without 2-DG. This effect was also seen in total CD8+ T cells although less pronounced. In contrast, supply of galactose or oleic-acid to glucose free (but glutamine containing) medium did not result in reduced TEFF differentiation, suggesting OXPHOS by galactose converted to glucose and fatty acid oxidation. Moreover, 2-DG treated TSCM and TCM as well as total CD8+ CTL showed less lactate production and ECAR as compared to untreated controls, confirming that differentiation to TEFF requires aerobic glycolytic ATP production. Functional assays in vitro did not only reveal cytolytic activity of 2-DG treated EBV-TSCM and TCM comparable to controls but elicited superior migration properties of these cells when tested to untreated naïve or total CD8+ T cells in a transwell- migration assay. We further showed that EBV-TSCM/CM can be successfully TCR-reprogrammed to recognize primary acute myeloid leukaemia (AML) blasts.
Finally, first adoptive transfer studies into NSG mice indicated that EBV-TSCM/CM generated in low glucose can persist and mount an immune response to EBV-transformed B cells engrafted into NSG recipients.
Summary
These studies demonstrate that modulation of T cell metabolism may be a promising approach to generate stem cell memory and central memory T cells in vitro for virus-specific and redirected immunity in adoptive cellular therapy.
Keyword(s): Adoptive immunotherapy, Antigen-specific T cells, EBV, Memory T cells
Session topic: Gene therapy, cellular immunotherapy and vaccination
Abstract: S467
Type: Oral Presentation
Presentation during EHA20: From 13.06.2015 11:45 to 13.06.2015 12:00
Location: Room Stolz 2
Background
Adoptive transfer of virus-specific and TCR- or CAR-reprogrammed cytotoxic T lymphocyzes (CTL) has advanced as a valuable cellular therapy for opportunistic viral infections, Epstein-Barr Virus (EBV)-mediated lymphoma and leukemia relapse. However, durable clinical responses are often hampered by limited capability of terminally differentiated, high avidity effector T cells (TEFF) to establish sustained antileukemic immunity whereas less differentiated stem cell memory (TSCM) and central memory (TCM) T cells could be shown to elicit potent antitumor responses, prolonged survival and memory. Moreover, TSCM and TCM depend less on glucose consumption to drive oxidative phosphorylation (OXPHOS) as their primary source of adenosine triphosphate (ATP) production whereas TEFF require additional aerobic glycolysis to generate the ATP needed.
Aims
In the current study, we therefore investigated means of modulating T cell metabolism to generate EBV-specific TSCM and TCM as well as EBV-specific TSCM/CM TCR-reprogrammed T cells.
Methods
Naïve CD8+CD45RA+ T cells isolated from peripheral blood mononuclear cells (PBMC) of healthy HLA-A2+ donors using the Naive T cell isolation kit® (Milenyi Biotec, Bergisch Gladbach, Germany) and unselected (total) CD8+ T cells were primed by autologous dendritic cells loaded with EBV-peptides followed by restimulation with peptide loaded autologous PBMC in the presence of low (1 mM) glucose, glutamine, an optimized cytokine cocktail and 1 mM of the non-metabolizable glucose analogue 2-deoxy-glucose (2-DG). Moreover, either galactose or 9-oleic acid was added to glucose deprived cultures. In addition to phenotypic and functional in vitro analyses by FACS, IFN-g ELISPOT- and 51Cr-release assays, glucose uptake and lactate production was determined. OXPHOS and aerobic glycolysis were assessed by measuring oxygen-consumption rate (OCR) and extracellular acidification rate (ECAR), respectively, using a Seahorse Analyzer. Finally, the biological activity of EBV-reactive TSCM/CM was tested in vivo in NSG mice.
Results
We obtained strong expansion of EBV-reactive CTL expressing a CD8+CD45RA+CD45RO-CD95+ CD27+CD28+CD62L+CCR7+TSCM and CD8+CD45RA- CD45RO+CD95+CD27+CD28+CD62L+CCR7+ TCM phenotype upon repetitive stimulation of naïve T cells in the presence of low glucose, glutamine and 2-DG when compared to CTL stimulated without 2-DG. This effect was also seen in total CD8+ T cells although less pronounced. In contrast, supply of galactose or oleic-acid to glucose free (but glutamine containing) medium did not result in reduced TEFF differentiation, suggesting OXPHOS by galactose converted to glucose and fatty acid oxidation. Moreover, 2-DG treated TSCM and TCM as well as total CD8+ CTL showed less lactate production and ECAR as compared to untreated controls, confirming that differentiation to TEFF requires aerobic glycolytic ATP production. Functional assays in vitro did not only reveal cytolytic activity of 2-DG treated EBV-TSCM and TCM comparable to controls but elicited superior migration properties of these cells when tested to untreated naïve or total CD8+ T cells in a transwell- migration assay. We further showed that EBV-TSCM/CM can be successfully TCR-reprogrammed to recognize primary acute myeloid leukaemia (AML) blasts.
Summary
These studies demonstrate that modulation of T cell metabolism may be a promising approach to generate stem cell memory and central memory T cells in vitro for virus-specific and redirected immunity in adoptive cellular therapy.
Keyword(s): Adoptive immunotherapy, Antigen-specific T cells, EBV, Memory T cells
Session topic: Gene therapy, cellular immunotherapy and vaccination
Type: Oral Presentation
Presentation during EHA20: From 13.06.2015 11:45 to 13.06.2015 12:00
Location: Room Stolz 2
Background
Adoptive transfer of virus-specific and TCR- or CAR-reprogrammed cytotoxic T lymphocyzes (CTL) has advanced as a valuable cellular therapy for opportunistic viral infections, Epstein-Barr Virus (EBV)-mediated lymphoma and leukemia relapse. However, durable clinical responses are often hampered by limited capability of terminally differentiated, high avidity effector T cells (TEFF) to establish sustained antileukemic immunity whereas less differentiated stem cell memory (TSCM) and central memory (TCM) T cells could be shown to elicit potent antitumor responses, prolonged survival and memory. Moreover, TSCM and TCM depend less on glucose consumption to drive oxidative phosphorylation (OXPHOS) as their primary source of adenosine triphosphate (ATP) production whereas TEFF require additional aerobic glycolysis to generate the ATP needed.
Aims
In the current study, we therefore investigated means of modulating T cell metabolism to generate EBV-specific TSCM and TCM as well as EBV-specific TSCM/CM TCR-reprogrammed T cells.
Methods
Naïve CD8+CD45RA+ T cells isolated from peripheral blood mononuclear cells (PBMC) of healthy HLA-A2+ donors using the Naive T cell isolation kit® (Milenyi Biotec, Bergisch Gladbach, Germany) and unselected (total) CD8+ T cells were primed by autologous dendritic cells loaded with EBV-peptides followed by restimulation with peptide loaded autologous PBMC in the presence of low (1 mM) glucose, glutamine, an optimized cytokine cocktail and 1 mM of the non-metabolizable glucose analogue 2-deoxy-glucose (2-DG). Moreover, either galactose or 9-oleic acid was added to glucose deprived cultures. In addition to phenotypic and functional in vitro analyses by FACS, IFN-g ELISPOT- and 51Cr-release assays, glucose uptake and lactate production was determined. OXPHOS and aerobic glycolysis were assessed by measuring oxygen-consumption rate (OCR) and extracellular acidification rate (ECAR), respectively, using a Seahorse Analyzer. Finally, the biological activity of EBV-reactive TSCM/CM was tested in vivo in NSG mice.
Results
We obtained strong expansion of EBV-reactive CTL expressing a CD8+CD45RA+CD45RO-CD95+ CD27+CD28+CD62L+CCR7+TSCM and CD8+CD45RA- CD45RO+CD95+CD27+CD28+CD62L+CCR7+ TCM phenotype upon repetitive stimulation of naïve T cells in the presence of low glucose, glutamine and 2-DG when compared to CTL stimulated without 2-DG. This effect was also seen in total CD8+ T cells although less pronounced. In contrast, supply of galactose or oleic-acid to glucose free (but glutamine containing) medium did not result in reduced TEFF differentiation, suggesting OXPHOS by galactose converted to glucose and fatty acid oxidation. Moreover, 2-DG treated TSCM and TCM as well as total CD8+ CTL showed less lactate production and ECAR as compared to untreated controls, confirming that differentiation to TEFF requires aerobic glycolytic ATP production. Functional assays in vitro did not only reveal cytolytic activity of 2-DG treated EBV-TSCM and TCM comparable to controls but elicited superior migration properties of these cells when tested to untreated naïve or total CD8+ T cells in a transwell- migration assay. We further showed that EBV-TSCM/CM can be successfully TCR-reprogrammed to recognize primary acute myeloid leukaemia (AML) blasts.
Finally, first adoptive transfer studies into NSG mice indicated that EBV-TSCM/CM generated in low glucose can persist and mount an immune response to EBV-transformed B cells engrafted into NSG recipients.
Summary
These studies demonstrate that modulation of T cell metabolism may be a promising approach to generate stem cell memory and central memory T cells in vitro for virus-specific and redirected immunity in adoptive cellular therapy.
Keyword(s): Adoptive immunotherapy, Antigen-specific T cells, EBV, Memory T cells
Session topic: Gene therapy, cellular immunotherapy and vaccination
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