THE JAK2V617F MUTATION IS A POTENTIAL TARGET FOR CANCER IMMUNE THERAPY
(Abstract release date: 05/19/16)
EHA Library. Holmström M. 06/11/16; 135264; S508
Mr. Morten Holmström
Contributions
Contributions
Abstract
Abstract: S508
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 16:30 - 16:45
Location: Hall C13
Background
More than 50 % of patients with Philadelphia chromosome negative chronic myeloproliferative neoplasia (MPN) harbor the JAK2V617F mutation. This is an acquired somatic mutation, and is found exclusively in myeloid malignancies, rendering it a cancer specific antigen and thus an attractive target for cancer immune therapy.
Aims
By this study we wish to clarify if the JAK2V617F mutation is recognized by the immune system, hereby proving the potential of cancer immune therapy as a whole new treatment modality for the JAK2V617F mutated MPNs.
Methods
We used the database www.syfpeithi.de to identify epitopes in the mutated JAK2 peptide with high affinity for HLA-A2. We chose the HLA-A2 restricted nonamer peptide JAK201 containing the V617F valine to phenylalanine substitution as the most promising candidate. A specific T cell culture was established from peripheral blood mononuclear cells (PBMCs) from an HLA-A2 positive healthy donor by stimulating with autologous dendritic cells, JAK201 peptide and cytokine as previously described1. Enzyme Linked ImmunoSPOT (ELISPOT), intracellular cytokine staining (ICS) and Cr51 cytotoxicity assays were used to analyze the reactivity of the specific T cells. The T cells were either stimulated with JAK201 peptide, T2 cells, HLA-A2 or HLA-A3 positive K562 cells or non-HLA K562 cells. T2 cells are unable to present endogenous peptides and thus only present exogenous peptide – in our experiments the JAK201 peptide. The HLA-A2 positive cancer cell lines UKE1 and SET2, which both harbor the JAK2V617F mutation, were used as target cells for the analyses of the T cells’ reactivity against JAK2V617F mutated cancer cells.
Results
First we analyzed the capacity of the specific T cells to release TNF-α and IFN-γ upon stimulation with JAK201 peptide. Release of both cytokines upon stimulation with JAK201 peptide was confirmed by ELISPOT (A and B) and ICS (data not shown). Next we showed, that the specific T cells were able to kill T2 cells pulsed with JAK201 peptide (data not shown). K562-A3 cells pulsed with JAK201 peptide were not killed, whereas K562-A2 cells were killed, demonstrating that killing by the specific T cells is HLA-A2 restricted (D). Next we wanted to investigate, if stimulation of T cells with cancer cells carrying the JAK2V617F mutation induces cytokine release and killing of the cancer cells. We thus stimulated T cells with the HLA-A2 and JAK2V617F positive cancer cell line UKE1. The JAK201 specific T cells released IFN-γ (C) and TNF-α (data not shown) in response to stimulation with UKE1. The target cells were stimulated with IFN-γ 48 hours before assaying to increase their antigen presentation, and the T cells showed an enhanced release of IFN-γ upon stimulation with IFN-γ treated UKE1 cells (C). Furthermore, T cells were able to kill UKE1 cells in a cytotoxicity assay, and in line with the above, we demonstrated an increased killing of the UKE1 cells after treatment with IFN-γ (E). Initially the HLA-A2 and JAK2V617F positive cancer cell line SET2 was not killed in a cytotoxicity assay, but after stimulation with IFN-γ, the SET2 cells were readily killed by the JAK201 specific T cells (data not shown). Finally, transfection with JAK2V617F siRNA into UKE1 cells abrogated T cell mediated killing, whereas mock transfected UKE1 cells were killed by the T cells (F). By this experiment we demonstrated, that killing of target cells is dependent upon the presence of the JAK2V617F mutation.
Conclusion
By establishing a T cell culture specific for the JAK2V617F mutation we have shown that the immune system is able to effectively target cells carrying the JAK2V617F mutation. Thus cancer immune therapy in the form of adoptive T cell therapy and/or vaccination could prove to be new treatment modalities for MPN in the future.References1. Munir S et al. Cancer Res. 2013;73(6):1764–76.
Session topic: Myeloproliferative neoplasms - Biology
Keyword(s): Immunotherapy, Myeloproliferative disorder, T cell
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 16:30 - 16:45
Location: Hall C13
Background
More than 50 % of patients with Philadelphia chromosome negative chronic myeloproliferative neoplasia (MPN) harbor the JAK2V617F mutation. This is an acquired somatic mutation, and is found exclusively in myeloid malignancies, rendering it a cancer specific antigen and thus an attractive target for cancer immune therapy.
Aims
By this study we wish to clarify if the JAK2V617F mutation is recognized by the immune system, hereby proving the potential of cancer immune therapy as a whole new treatment modality for the JAK2V617F mutated MPNs.
Methods
We used the database www.syfpeithi.de to identify epitopes in the mutated JAK2 peptide with high affinity for HLA-A2. We chose the HLA-A2 restricted nonamer peptide JAK201 containing the V617F valine to phenylalanine substitution as the most promising candidate. A specific T cell culture was established from peripheral blood mononuclear cells (PBMCs) from an HLA-A2 positive healthy donor by stimulating with autologous dendritic cells, JAK201 peptide and cytokine as previously described1. Enzyme Linked ImmunoSPOT (ELISPOT), intracellular cytokine staining (ICS) and Cr51 cytotoxicity assays were used to analyze the reactivity of the specific T cells. The T cells were either stimulated with JAK201 peptide, T2 cells, HLA-A2 or HLA-A3 positive K562 cells or non-HLA K562 cells. T2 cells are unable to present endogenous peptides and thus only present exogenous peptide – in our experiments the JAK201 peptide. The HLA-A2 positive cancer cell lines UKE1 and SET2, which both harbor the JAK2V617F mutation, were used as target cells for the analyses of the T cells’ reactivity against JAK2V617F mutated cancer cells.
Results
First we analyzed the capacity of the specific T cells to release TNF-α and IFN-γ upon stimulation with JAK201 peptide. Release of both cytokines upon stimulation with JAK201 peptide was confirmed by ELISPOT (A and B) and ICS (data not shown). Next we showed, that the specific T cells were able to kill T2 cells pulsed with JAK201 peptide (data not shown). K562-A3 cells pulsed with JAK201 peptide were not killed, whereas K562-A2 cells were killed, demonstrating that killing by the specific T cells is HLA-A2 restricted (D). Next we wanted to investigate, if stimulation of T cells with cancer cells carrying the JAK2V617F mutation induces cytokine release and killing of the cancer cells. We thus stimulated T cells with the HLA-A2 and JAK2V617F positive cancer cell line UKE1. The JAK201 specific T cells released IFN-γ (C) and TNF-α (data not shown) in response to stimulation with UKE1. The target cells were stimulated with IFN-γ 48 hours before assaying to increase their antigen presentation, and the T cells showed an enhanced release of IFN-γ upon stimulation with IFN-γ treated UKE1 cells (C). Furthermore, T cells were able to kill UKE1 cells in a cytotoxicity assay, and in line with the above, we demonstrated an increased killing of the UKE1 cells after treatment with IFN-γ (E). Initially the HLA-A2 and JAK2V617F positive cancer cell line SET2 was not killed in a cytotoxicity assay, but after stimulation with IFN-γ, the SET2 cells were readily killed by the JAK201 specific T cells (data not shown). Finally, transfection with JAK2V617F siRNA into UKE1 cells abrogated T cell mediated killing, whereas mock transfected UKE1 cells were killed by the T cells (F). By this experiment we demonstrated, that killing of target cells is dependent upon the presence of the JAK2V617F mutation.
Conclusion
By establishing a T cell culture specific for the JAK2V617F mutation we have shown that the immune system is able to effectively target cells carrying the JAK2V617F mutation. Thus cancer immune therapy in the form of adoptive T cell therapy and/or vaccination could prove to be new treatment modalities for MPN in the future.References1. Munir S et al. Cancer Res. 2013;73(6):1764–76.
Session topic: Myeloproliferative neoplasms - Biology
Keyword(s): Immunotherapy, Myeloproliferative disorder, T cell
Abstract: S508
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 16:30 - 16:45
Location: Hall C13
Background
More than 50 % of patients with Philadelphia chromosome negative chronic myeloproliferative neoplasia (MPN) harbor the JAK2V617F mutation. This is an acquired somatic mutation, and is found exclusively in myeloid malignancies, rendering it a cancer specific antigen and thus an attractive target for cancer immune therapy.
Aims
By this study we wish to clarify if the JAK2V617F mutation is recognized by the immune system, hereby proving the potential of cancer immune therapy as a whole new treatment modality for the JAK2V617F mutated MPNs.
Methods
We used the database www.syfpeithi.de to identify epitopes in the mutated JAK2 peptide with high affinity for HLA-A2. We chose the HLA-A2 restricted nonamer peptide JAK201 containing the V617F valine to phenylalanine substitution as the most promising candidate. A specific T cell culture was established from peripheral blood mononuclear cells (PBMCs) from an HLA-A2 positive healthy donor by stimulating with autologous dendritic cells, JAK201 peptide and cytokine as previously described1. Enzyme Linked ImmunoSPOT (ELISPOT), intracellular cytokine staining (ICS) and Cr51 cytotoxicity assays were used to analyze the reactivity of the specific T cells. The T cells were either stimulated with JAK201 peptide, T2 cells, HLA-A2 or HLA-A3 positive K562 cells or non-HLA K562 cells. T2 cells are unable to present endogenous peptides and thus only present exogenous peptide – in our experiments the JAK201 peptide. The HLA-A2 positive cancer cell lines UKE1 and SET2, which both harbor the JAK2V617F mutation, were used as target cells for the analyses of the T cells’ reactivity against JAK2V617F mutated cancer cells.
Results
First we analyzed the capacity of the specific T cells to release TNF-α and IFN-γ upon stimulation with JAK201 peptide. Release of both cytokines upon stimulation with JAK201 peptide was confirmed by ELISPOT (A and B) and ICS (data not shown). Next we showed, that the specific T cells were able to kill T2 cells pulsed with JAK201 peptide (data not shown). K562-A3 cells pulsed with JAK201 peptide were not killed, whereas K562-A2 cells were killed, demonstrating that killing by the specific T cells is HLA-A2 restricted (D). Next we wanted to investigate, if stimulation of T cells with cancer cells carrying the JAK2V617F mutation induces cytokine release and killing of the cancer cells. We thus stimulated T cells with the HLA-A2 and JAK2V617F positive cancer cell line UKE1. The JAK201 specific T cells released IFN-γ (C) and TNF-α (data not shown) in response to stimulation with UKE1. The target cells were stimulated with IFN-γ 48 hours before assaying to increase their antigen presentation, and the T cells showed an enhanced release of IFN-γ upon stimulation with IFN-γ treated UKE1 cells (C). Furthermore, T cells were able to kill UKE1 cells in a cytotoxicity assay, and in line with the above, we demonstrated an increased killing of the UKE1 cells after treatment with IFN-γ (E). Initially the HLA-A2 and JAK2V617F positive cancer cell line SET2 was not killed in a cytotoxicity assay, but after stimulation with IFN-γ, the SET2 cells were readily killed by the JAK201 specific T cells (data not shown). Finally, transfection with JAK2V617F siRNA into UKE1 cells abrogated T cell mediated killing, whereas mock transfected UKE1 cells were killed by the T cells (F). By this experiment we demonstrated, that killing of target cells is dependent upon the presence of the JAK2V617F mutation.
Conclusion
By establishing a T cell culture specific for the JAK2V617F mutation we have shown that the immune system is able to effectively target cells carrying the JAK2V617F mutation. Thus cancer immune therapy in the form of adoptive T cell therapy and/or vaccination could prove to be new treatment modalities for MPN in the future.References1. Munir S et al. Cancer Res. 2013;73(6):1764–76.
Session topic: Myeloproliferative neoplasms - Biology
Keyword(s): Immunotherapy, Myeloproliferative disorder, T cell
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 16:30 - 16:45
Location: Hall C13
Background
More than 50 % of patients with Philadelphia chromosome negative chronic myeloproliferative neoplasia (MPN) harbor the JAK2V617F mutation. This is an acquired somatic mutation, and is found exclusively in myeloid malignancies, rendering it a cancer specific antigen and thus an attractive target for cancer immune therapy.
Aims
By this study we wish to clarify if the JAK2V617F mutation is recognized by the immune system, hereby proving the potential of cancer immune therapy as a whole new treatment modality for the JAK2V617F mutated MPNs.
Methods
We used the database www.syfpeithi.de to identify epitopes in the mutated JAK2 peptide with high affinity for HLA-A2. We chose the HLA-A2 restricted nonamer peptide JAK201 containing the V617F valine to phenylalanine substitution as the most promising candidate. A specific T cell culture was established from peripheral blood mononuclear cells (PBMCs) from an HLA-A2 positive healthy donor by stimulating with autologous dendritic cells, JAK201 peptide and cytokine as previously described1. Enzyme Linked ImmunoSPOT (ELISPOT), intracellular cytokine staining (ICS) and Cr51 cytotoxicity assays were used to analyze the reactivity of the specific T cells. The T cells were either stimulated with JAK201 peptide, T2 cells, HLA-A2 or HLA-A3 positive K562 cells or non-HLA K562 cells. T2 cells are unable to present endogenous peptides and thus only present exogenous peptide – in our experiments the JAK201 peptide. The HLA-A2 positive cancer cell lines UKE1 and SET2, which both harbor the JAK2V617F mutation, were used as target cells for the analyses of the T cells’ reactivity against JAK2V617F mutated cancer cells.
Results
First we analyzed the capacity of the specific T cells to release TNF-α and IFN-γ upon stimulation with JAK201 peptide. Release of both cytokines upon stimulation with JAK201 peptide was confirmed by ELISPOT (A and B) and ICS (data not shown). Next we showed, that the specific T cells were able to kill T2 cells pulsed with JAK201 peptide (data not shown). K562-A3 cells pulsed with JAK201 peptide were not killed, whereas K562-A2 cells were killed, demonstrating that killing by the specific T cells is HLA-A2 restricted (D). Next we wanted to investigate, if stimulation of T cells with cancer cells carrying the JAK2V617F mutation induces cytokine release and killing of the cancer cells. We thus stimulated T cells with the HLA-A2 and JAK2V617F positive cancer cell line UKE1. The JAK201 specific T cells released IFN-γ (C) and TNF-α (data not shown) in response to stimulation with UKE1. The target cells were stimulated with IFN-γ 48 hours before assaying to increase their antigen presentation, and the T cells showed an enhanced release of IFN-γ upon stimulation with IFN-γ treated UKE1 cells (C). Furthermore, T cells were able to kill UKE1 cells in a cytotoxicity assay, and in line with the above, we demonstrated an increased killing of the UKE1 cells after treatment with IFN-γ (E). Initially the HLA-A2 and JAK2V617F positive cancer cell line SET2 was not killed in a cytotoxicity assay, but after stimulation with IFN-γ, the SET2 cells were readily killed by the JAK201 specific T cells (data not shown). Finally, transfection with JAK2V617F siRNA into UKE1 cells abrogated T cell mediated killing, whereas mock transfected UKE1 cells were killed by the T cells (F). By this experiment we demonstrated, that killing of target cells is dependent upon the presence of the JAK2V617F mutation.
Conclusion
By establishing a T cell culture specific for the JAK2V617F mutation we have shown that the immune system is able to effectively target cells carrying the JAK2V617F mutation. Thus cancer immune therapy in the form of adoptive T cell therapy and/or vaccination could prove to be new treatment modalities for MPN in the future.References1. Munir S et al. Cancer Res. 2013;73(6):1764–76.
Session topic: Myeloproliferative neoplasms - Biology
Keyword(s): Immunotherapy, Myeloproliferative disorder, T cell
{{ help_message }}
{{filter}}