Increased A(3)AR-dependent Vasoconstriction in Diabetic Mice Is Promoted by Myeloperoxidase

Vascular dysfunction importantly contributes to mortality and morbidity in various cardiac and metabolic diseases. Among endogenous molecules regulating vascular tone is adenosine, with the adenosine A(3) receptor (A(3)AR) exerting cardioprotective properties in ischemia and reperfusion. However, overexpression of A(3)AR is suggested to result in vascular dysfunction and inflammation. The leukocyte enzyme myeloperoxidase (MPO) is an important modulator of vascular function with nitric oxide-consuming and proinflammatory properties. Increased MPO plasma levels are observed in patients with cardiovascular disorders like heart failure, acute coronary syndromes, and arrhythmias. Given that vascular dysfunction and inflammation are also hallmarks of diabetes, the role of MPO in adenosine-dependent vasomotor function was investigated in a murine model of diabetes mellitus. Wild-type (WT) and MPO-deficient (Mpo(-/-)) mice were treated with Streptozotocin (STZ), which induced an increase of MPO plasma levels in WT mice and led to enhanced aortic superoxide generation as assessed by dihydroethidium staining in STZ-treated WT mice as compared with controls. The vasoconstriction of aortic segments in response to the A(3)AR agonist Cl-IB-MECA (2-Chloro-N6-(3-iodobenzyl)-N-methyl-5-carbamoyladenosine) as determined by isometric force measurements was augmented in diabetic WT as compared with diabetic Mpo(-/-) mice. Moreover, A(3)AR protein expression was enhanced in STZ-treated mice but was attenuated by MPO deficiency. The current data reveal an MPO-mediated increase of vascular A(3)AR expression under diabetic conditions, which leads to enhanced vasoconstriction in response to A(3)AR agonists and discloses an additional mechanism of MPO-mediated vascular dysfunction

Additional file 2 of A novel CAR-T cell product targeting CD74 is an effective therapeutic approach in preclinical mantle cell lymphoma models

Additional file 2: Figure S2. Creation of the 74bbz mutant clones. A GFP+ cells of the 74bbz mutants and parent CAR expressing Jurkat cells were sorted at the same intensity by flow cytometry. B An immunoblot of CD3ζ to show the expressing of parent, 543, 5311, 42105-74bbz clones. Endogenous CD3ζ was detected at 15 kDa while the chimeric CD3ζ on CAR was detected at 55 kDa. C CD74-ECD-Fc fusion protein was stained by Coomassie blue staining

Additional file 3 of A novel CAR-T cell product targeting CD74 is an effective therapeutic approach in preclinical mantle cell lymphoma models

Additional file 3: Figure S3. Expression of CD74 after activation on T cell and B cell. T cells and B cells isolated from PBMCs of 3 healthy blood donors were either untreated (red) or activated (blue) by CD3/CD28 soluble antibodies and IL-2 for T cells, and LPS (10 ng/mL)/ anti-IgM (10 µg/mL) for B cells. One representative of 3 healthy blood donors was shown

Additional file 5 of A novel CAR-T cell product targeting CD74 is an effective therapeutic approach in preclinical mantle cell lymphoma models

Additional file 5: Figure S5. No significant depletion of circulating immune cells pre/post peak detection of 42105-74bbz CAR-T cells in a humanized mouse model. A Absolute cell numbers of B cells, monocytes, G-MDSC, M-MDSC and NK cells in humanized NSG mice on Day 3, 11 and 23 post UTT or 74bbz CAR-T engraftment. All human cells were identified by human CD45+. B: CD33−CD19+; Monocyte: LIN−CD45+CD11b+CD33+CD14+; G-MDSC: LIN−CD11b+CD33+CD14−HLA-DR−; M-MDSC: LIN−CD11b+CD33+CD14+HLA-DR−; NK: CD33−CD3−CD56+. Mice received either UTT cells (red, n = 5) and 42105-74bbz (blue, n = 7). Bars show the median cell number. B CD74 expression (blue) of B cells, monocytes, G-MDSC, M-MDSC and NK cells on Day 18 compared to isotype control (red). Data are from one mouse from the 42105-74bbz CAR-T cell treatment group

Additional file 6 of A novel CAR-T cell product targeting CD74 is an effective therapeutic approach in preclinical mantle cell lymphoma models

Additional file 6: Figure S6. No significant change in the body weights of the 74bbz CAR-T cells-treated mice was observed. The body weights of the mice treated UTT, 19bbz CAR-T and 74bbz CAR-T cells (n = 10 per group) were monitored until the mice reached ERC and plotted over time

Additional file 1 of A novel CAR-T cell product targeting CD74 is an effective therapeutic approach in preclinical mantle cell lymphoma models

Additional file 1: Figure S1. In silico modeling of CD74-anti-CD74 scFV interaction. A Best generated models of CD74-anti-CD74 scFV interaction shown by the lowest HADDOCK score as a function of RMSD. The blue cluster was picked for further in silico mutagenesis. B Visualization of CD74-anti-CD74 scFV interaction. Red: CD74 trimer; Blue: anti-CD74 scFV

Additional file 4 of A novel CAR-T cell product targeting CD74 is an effective therapeutic approach in preclinical mantle cell lymphoma models

Additional file 4: Figure S4. Expression of CD74 on human CD34+ stem cells. Human umbilical cord blood mononuclear cells were stained with anti-CD34 and CD74 antibodies and analyzed by flow cytometry. One representative of 3 healthy cord blood donors was shown