A SHARP CONTRAST IN FUNCTIONALITY BETWEEN EBV- AND CMV-SPECIFIC CD8+ T-CELLS IN CHRONIC LYMPHOCYTIC LEUKEMIA
(Abstract release date: 05/19/16)
EHA Library. Hofland T. 06/09/16; 132582; E1033
Mr. Tom Hofland
Contributions
Contributions
Abstract
Abstract: E1033
Type: Eposter Presentation
Background
Chronic lymphocytic leukemia (CLL) is characterized by a tumor induced T-cell dysfunction, which leads to impaired cytotoxicity, which is probably caused by an inability to form immune synapses and increased expression of exhaustion markers. However, we recently found that CMV-specific CD8+ T-cells from CLL patients are functionally intact when compared to age-matched healthy controls (HC). This finding challenges the concept of a global T-cell dysfunction in CLL. Whether intact functionality of CMV-specific T-cells is a rare exception or whether T-cell functionality is indeed more heterogeneous is currently unknown. Acquired T-cell dysfunction in CLL needs to be better understood in order to improve anti-tumor immunotherapies that rely on T-cell mediated effects.
Aims
To analyze T-cell function heterogeneity in CLL, we studied the immunophenotype and functionality of CD8+ T-cells specific for Epstein-Barr-virus (EBV), another widely common chronic latent viral infection.
Methods
EBV-specific CD8+ T-cells were analyzed using EBV tetramers and 14-color flow cytometry in 45 untreated CLL patients and 23 age-matched HC. We studied T-cell differentiation, expression of exhaustion markers (PD-1, CD244 and CD160), functional markers and homing markers. To study the functionality of EBV-specific CD8+ T-cells, we determined cytokine production and polyfunctionality after stimulation with EBV-derived peptides. Furthermore, we analyzed ex vivo cytotoxicity of EBV-specific CD8+ T-cells.
Results
We found that, when compared to HC, EBV-specific T-cells in CLL patients are further differentiated with a significantly smaller percentage of “early” effector memory cells (also called EM1; CLL=39.6% vs HC=57.68%). These results are mirrored by the expression pattern of the transcription factors T-bet and Eomes; 25.79% of EBV-specific T-cells of CLL patients display a T-bethighEomeshigh phenotype vs 17.44% in HC, again pointing to an increased differentiation state. In comparison with HC, EBV-specific T-cells in CLL patients show higher expression of exhaustion markers CD244 and CD160, but not PD-1. However, there were no significant differences in granzyme B and K expression, suggesting an unaltered cytotoxic potential.On a functional level, no differences between CLL and HC were found with respect to production of the cytokines TNFα, IFNγ, IL-2 and MIP-1β and degranulation (measured as CD107a+ cells) of EBV-specific T-cells after peptide stimulation. Polyfunctionality of EBV-specific T-cells of CLL patients was comparable with HC. Surprisingly, we found that EBV-specific T-cells of CLL patients display significantly less direct cytotoxic capacity compared to HC in an ex vivo killing assay (mean specific lysis of target cells CLL=6,83% vs HC=32,4%), indicating a functional impairment despite normal differentiation and cytokine production. We are currently performing experiments to study immune synapse formation of EBV-specific T-cells (which we will be able to present during the EHA meeting) to determine involvement of the immune synapse in this acquired defect.
Conclusion
Taken together, we conclude that EBV-specific T-cells show signs of functional impairment and are, in contrast to CMV-specific T-cells, not able to evade CLL induced T-cell dysfunction. In depth studies on differences in T-cell mediated viral responses in the context of CLL will yield important clues to develop strategies to overcome T-cell dysfunction in this disease.
Session topic: E-poster
Type: Eposter Presentation
Background
Chronic lymphocytic leukemia (CLL) is characterized by a tumor induced T-cell dysfunction, which leads to impaired cytotoxicity, which is probably caused by an inability to form immune synapses and increased expression of exhaustion markers. However, we recently found that CMV-specific CD8+ T-cells from CLL patients are functionally intact when compared to age-matched healthy controls (HC). This finding challenges the concept of a global T-cell dysfunction in CLL. Whether intact functionality of CMV-specific T-cells is a rare exception or whether T-cell functionality is indeed more heterogeneous is currently unknown. Acquired T-cell dysfunction in CLL needs to be better understood in order to improve anti-tumor immunotherapies that rely on T-cell mediated effects.
Aims
To analyze T-cell function heterogeneity in CLL, we studied the immunophenotype and functionality of CD8+ T-cells specific for Epstein-Barr-virus (EBV), another widely common chronic latent viral infection.
Methods
EBV-specific CD8+ T-cells were analyzed using EBV tetramers and 14-color flow cytometry in 45 untreated CLL patients and 23 age-matched HC. We studied T-cell differentiation, expression of exhaustion markers (PD-1, CD244 and CD160), functional markers and homing markers. To study the functionality of EBV-specific CD8+ T-cells, we determined cytokine production and polyfunctionality after stimulation with EBV-derived peptides. Furthermore, we analyzed ex vivo cytotoxicity of EBV-specific CD8+ T-cells.
Results
We found that, when compared to HC, EBV-specific T-cells in CLL patients are further differentiated with a significantly smaller percentage of “early” effector memory cells (also called EM1; CLL=39.6% vs HC=57.68%). These results are mirrored by the expression pattern of the transcription factors T-bet and Eomes; 25.79% of EBV-specific T-cells of CLL patients display a T-bethighEomeshigh phenotype vs 17.44% in HC, again pointing to an increased differentiation state. In comparison with HC, EBV-specific T-cells in CLL patients show higher expression of exhaustion markers CD244 and CD160, but not PD-1. However, there were no significant differences in granzyme B and K expression, suggesting an unaltered cytotoxic potential.On a functional level, no differences between CLL and HC were found with respect to production of the cytokines TNFα, IFNγ, IL-2 and MIP-1β and degranulation (measured as CD107a+ cells) of EBV-specific T-cells after peptide stimulation. Polyfunctionality of EBV-specific T-cells of CLL patients was comparable with HC. Surprisingly, we found that EBV-specific T-cells of CLL patients display significantly less direct cytotoxic capacity compared to HC in an ex vivo killing assay (mean specific lysis of target cells CLL=6,83% vs HC=32,4%), indicating a functional impairment despite normal differentiation and cytokine production. We are currently performing experiments to study immune synapse formation of EBV-specific T-cells (which we will be able to present during the EHA meeting) to determine involvement of the immune synapse in this acquired defect.
Conclusion
Taken together, we conclude that EBV-specific T-cells show signs of functional impairment and are, in contrast to CMV-specific T-cells, not able to evade CLL induced T-cell dysfunction. In depth studies on differences in T-cell mediated viral responses in the context of CLL will yield important clues to develop strategies to overcome T-cell dysfunction in this disease.
Session topic: E-poster
Abstract: E1033
Type: Eposter Presentation
Background
Chronic lymphocytic leukemia (CLL) is characterized by a tumor induced T-cell dysfunction, which leads to impaired cytotoxicity, which is probably caused by an inability to form immune synapses and increased expression of exhaustion markers. However, we recently found that CMV-specific CD8+ T-cells from CLL patients are functionally intact when compared to age-matched healthy controls (HC). This finding challenges the concept of a global T-cell dysfunction in CLL. Whether intact functionality of CMV-specific T-cells is a rare exception or whether T-cell functionality is indeed more heterogeneous is currently unknown. Acquired T-cell dysfunction in CLL needs to be better understood in order to improve anti-tumor immunotherapies that rely on T-cell mediated effects.
Aims
To analyze T-cell function heterogeneity in CLL, we studied the immunophenotype and functionality of CD8+ T-cells specific for Epstein-Barr-virus (EBV), another widely common chronic latent viral infection.
Methods
EBV-specific CD8+ T-cells were analyzed using EBV tetramers and 14-color flow cytometry in 45 untreated CLL patients and 23 age-matched HC. We studied T-cell differentiation, expression of exhaustion markers (PD-1, CD244 and CD160), functional markers and homing markers. To study the functionality of EBV-specific CD8+ T-cells, we determined cytokine production and polyfunctionality after stimulation with EBV-derived peptides. Furthermore, we analyzed ex vivo cytotoxicity of EBV-specific CD8+ T-cells.
Results
We found that, when compared to HC, EBV-specific T-cells in CLL patients are further differentiated with a significantly smaller percentage of “early” effector memory cells (also called EM1; CLL=39.6% vs HC=57.68%). These results are mirrored by the expression pattern of the transcription factors T-bet and Eomes; 25.79% of EBV-specific T-cells of CLL patients display a T-bethighEomeshigh phenotype vs 17.44% in HC, again pointing to an increased differentiation state. In comparison with HC, EBV-specific T-cells in CLL patients show higher expression of exhaustion markers CD244 and CD160, but not PD-1. However, there were no significant differences in granzyme B and K expression, suggesting an unaltered cytotoxic potential.On a functional level, no differences between CLL and HC were found with respect to production of the cytokines TNFα, IFNγ, IL-2 and MIP-1β and degranulation (measured as CD107a+ cells) of EBV-specific T-cells after peptide stimulation. Polyfunctionality of EBV-specific T-cells of CLL patients was comparable with HC. Surprisingly, we found that EBV-specific T-cells of CLL patients display significantly less direct cytotoxic capacity compared to HC in an ex vivo killing assay (mean specific lysis of target cells CLL=6,83% vs HC=32,4%), indicating a functional impairment despite normal differentiation and cytokine production. We are currently performing experiments to study immune synapse formation of EBV-specific T-cells (which we will be able to present during the EHA meeting) to determine involvement of the immune synapse in this acquired defect.
Conclusion
Taken together, we conclude that EBV-specific T-cells show signs of functional impairment and are, in contrast to CMV-specific T-cells, not able to evade CLL induced T-cell dysfunction. In depth studies on differences in T-cell mediated viral responses in the context of CLL will yield important clues to develop strategies to overcome T-cell dysfunction in this disease.
Session topic: E-poster
Type: Eposter Presentation
Background
Chronic lymphocytic leukemia (CLL) is characterized by a tumor induced T-cell dysfunction, which leads to impaired cytotoxicity, which is probably caused by an inability to form immune synapses and increased expression of exhaustion markers. However, we recently found that CMV-specific CD8+ T-cells from CLL patients are functionally intact when compared to age-matched healthy controls (HC). This finding challenges the concept of a global T-cell dysfunction in CLL. Whether intact functionality of CMV-specific T-cells is a rare exception or whether T-cell functionality is indeed more heterogeneous is currently unknown. Acquired T-cell dysfunction in CLL needs to be better understood in order to improve anti-tumor immunotherapies that rely on T-cell mediated effects.
Aims
To analyze T-cell function heterogeneity in CLL, we studied the immunophenotype and functionality of CD8+ T-cells specific for Epstein-Barr-virus (EBV), another widely common chronic latent viral infection.
Methods
EBV-specific CD8+ T-cells were analyzed using EBV tetramers and 14-color flow cytometry in 45 untreated CLL patients and 23 age-matched HC. We studied T-cell differentiation, expression of exhaustion markers (PD-1, CD244 and CD160), functional markers and homing markers. To study the functionality of EBV-specific CD8+ T-cells, we determined cytokine production and polyfunctionality after stimulation with EBV-derived peptides. Furthermore, we analyzed ex vivo cytotoxicity of EBV-specific CD8+ T-cells.
Results
We found that, when compared to HC, EBV-specific T-cells in CLL patients are further differentiated with a significantly smaller percentage of “early” effector memory cells (also called EM1; CLL=39.6% vs HC=57.68%). These results are mirrored by the expression pattern of the transcription factors T-bet and Eomes; 25.79% of EBV-specific T-cells of CLL patients display a T-bethighEomeshigh phenotype vs 17.44% in HC, again pointing to an increased differentiation state. In comparison with HC, EBV-specific T-cells in CLL patients show higher expression of exhaustion markers CD244 and CD160, but not PD-1. However, there were no significant differences in granzyme B and K expression, suggesting an unaltered cytotoxic potential.On a functional level, no differences between CLL and HC were found with respect to production of the cytokines TNFα, IFNγ, IL-2 and MIP-1β and degranulation (measured as CD107a+ cells) of EBV-specific T-cells after peptide stimulation. Polyfunctionality of EBV-specific T-cells of CLL patients was comparable with HC. Surprisingly, we found that EBV-specific T-cells of CLL patients display significantly less direct cytotoxic capacity compared to HC in an ex vivo killing assay (mean specific lysis of target cells CLL=6,83% vs HC=32,4%), indicating a functional impairment despite normal differentiation and cytokine production. We are currently performing experiments to study immune synapse formation of EBV-specific T-cells (which we will be able to present during the EHA meeting) to determine involvement of the immune synapse in this acquired defect.
Conclusion
Taken together, we conclude that EBV-specific T-cells show signs of functional impairment and are, in contrast to CMV-specific T-cells, not able to evade CLL induced T-cell dysfunction. In depth studies on differences in T-cell mediated viral responses in the context of CLL will yield important clues to develop strategies to overcome T-cell dysfunction in this disease.
Session topic: E-poster
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