Contributions
Abstract: EP1593
Type: Poster Sessions
Abstract Category: Therapy - Immunomodulation/Immunosuppression
Cladribine (2CdA) is deoxyadenosine antimetabolite activated by sequential deoxycytidine kinase (dCK) phosphorylation causing cell death through apoptosis and necrosis. This is reverted by cytosolic 5' deoxynucleotidase (NT5C2). Thus, high dCK/NT5C2 ratios in lymphocytes reflect 2CdA-specific lymphopenic effect. Clinical trials have shown a difference in timing of immunoreconstitution for different lymphocyte subsets in multiple sclerosis (MS) patients, suggesting that a differential susceptibility to 2CdA by these subsets and/or of their lymphoid progenitors is linked to dCK/NT5C2 ratio variability. Moreover, dCK expression and activity are enhanced during lymphopoiesis and in activated lymphocytes. On this basis, our objective is to assess the enzymes involved in 2CdA metabolism in vitro in selected immature cells, including hematopoietic stem cells, and common lymphoid (CLP) and myeloid progenitor cells (CMP) from healthy donors, as well as in activated mature lymphoid and myeloid cellular subsets isolated from the peripheral blood of healthy donors upon 2CdA stimulation.
Flow cytometry analysis using pre-validated rabbit anti-dCK and anti-NT5C2 polyclonal antibodies showed a positive dCK/NT5C2 MFI ratio in B cells, T cells and monocytes, with mean values of 5.19, 3.28 and 2.17, respectively. Within B-cell subsets, dCK and NT5C2 expression was highly heterogeneous, with plasma cells displaying the highest MFI, but no significant differences were observed in the respective B-cell dCK/NT5C2 ratios (B memory dCK MFI mean = 10580, NT5C2 MFI mean = 3206, ratio = 3.30; B mature dCK MFI mean = 7496, NT5C2 MFI mean = 2356, ratio = 3.18; B transitional dCK MFI mean = 7364, NT5C2 MFI mean = 2451, ratio = 3.00; plasma cells dCK MFI mean = 16901, NT5C2 MFI mean = 2451, ratio = 2.47).
These data support our suggestion that 2CdA affects specific immune cell subsets differentially.
Disclosure: Project funded by Merck Serono.
Federico Carlini, Federico Ivaldi, Nicole Kerlero de Rosbo and Antonio Uccelli: nothing to disclose
Ursula Boschert: Head of Neuroimmunology at EMD Serono, Inc., USA
Andrea Visconti: Medical Affairs Department, Merck Serono, Italy
Abstract: EP1593
Type: Poster Sessions
Abstract Category: Therapy - Immunomodulation/Immunosuppression
Cladribine (2CdA) is deoxyadenosine antimetabolite activated by sequential deoxycytidine kinase (dCK) phosphorylation causing cell death through apoptosis and necrosis. This is reverted by cytosolic 5' deoxynucleotidase (NT5C2). Thus, high dCK/NT5C2 ratios in lymphocytes reflect 2CdA-specific lymphopenic effect. Clinical trials have shown a difference in timing of immunoreconstitution for different lymphocyte subsets in multiple sclerosis (MS) patients, suggesting that a differential susceptibility to 2CdA by these subsets and/or of their lymphoid progenitors is linked to dCK/NT5C2 ratio variability. Moreover, dCK expression and activity are enhanced during lymphopoiesis and in activated lymphocytes. On this basis, our objective is to assess the enzymes involved in 2CdA metabolism in vitro in selected immature cells, including hematopoietic stem cells, and common lymphoid (CLP) and myeloid progenitor cells (CMP) from healthy donors, as well as in activated mature lymphoid and myeloid cellular subsets isolated from the peripheral blood of healthy donors upon 2CdA stimulation.
Flow cytometry analysis using pre-validated rabbit anti-dCK and anti-NT5C2 polyclonal antibodies showed a positive dCK/NT5C2 MFI ratio in B cells, T cells and monocytes, with mean values of 5.19, 3.28 and 2.17, respectively. Within B-cell subsets, dCK and NT5C2 expression was highly heterogeneous, with plasma cells displaying the highest MFI, but no significant differences were observed in the respective B-cell dCK/NT5C2 ratios (B memory dCK MFI mean = 10580, NT5C2 MFI mean = 3206, ratio = 3.30; B mature dCK MFI mean = 7496, NT5C2 MFI mean = 2356, ratio = 3.18; B transitional dCK MFI mean = 7364, NT5C2 MFI mean = 2451, ratio = 3.00; plasma cells dCK MFI mean = 16901, NT5C2 MFI mean = 2451, ratio = 2.47).
These data support our suggestion that 2CdA affects specific immune cell subsets differentially.
Disclosure: Project funded by Merck Serono.
Federico Carlini, Federico Ivaldi, Nicole Kerlero de Rosbo and Antonio Uccelli: nothing to disclose
Ursula Boschert: Head of Neuroimmunology at EMD Serono, Inc., USA
Andrea Visconti: Medical Affairs Department, Merck Serono, Italy