1. Hydroxychloroquine inhibits calcium signals in T cells: a new mechanism to explain its immunomodulatory properties
by Frederick D. Goldman, Andrew L. Gilman, Clay Hollenback, Roberta M. Kato, Brett A. Premack, and David J. Rawlings
Blood
Volume 95(11):
June 1, 2000
©2000 by American Society of Hematology

2. HCQ down-regulates TCR-induced CD69 expression
HCQ down-regulates TCR-induced CD69 expression.Jurkat T cells were cultured for 24 hours with varying concentrations of HCQ, then incubated an additional 12 hours in 24-well plates (1 × 106/mL) in medium alone (dotted lines), PMA (50 ng/mL), or anti-TCR mAb...
HCQ down-regulates TCR-induced CD69 expression.Jurkat T cells were cultured for 24 hours with varying concentrations of HCQ, then incubated an additional 12 hours in 24-well plates (1 × 106/mL) in medium alone (dotted lines), PMA (50 ng/mL), or anti-TCR mAb C305 (ascites, 1:2000). Cells were then harvested and stained with FITC-conjugated anti-CD69, and fluorescence intensity was measured on a FACScan. Cell counts and viability were indistinguishable in untreated and HCQ-treated cell populations. Representative data from more than 5 similar experiments are shown.
Frederick D. Goldman et al. Blood 2000;95:
©2000 by American Society of Hematology

3. HCQ does not inhibit TCR-mediated inductive protein tyrosine phosphorylation.Jurkat T cells (1 × 106) were treated with varying concentrations of HCQ for 24 hours, stimulated with C305 for 5 minutes, and then lysed in NP-40 detergent.
HCQ does not inhibit TCR-mediated inductive protein tyrosine phosphorylation.Jurkat T cells (1 × 106) were treated with varying concentrations of HCQ for 24 hours, stimulated with C305 for 5 minutes, and then lysed in NP-40 detergent. Proteins from whole-cell lysates were separated by 10% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antiphosphotyrosine antibody (4G10). Immunoreactive bands were visualized by enhanced chemiluminescence. Representative data from more than 3 similar experiments are shown. A more extended kinetic analysis of protein tyrosine phosphorylation in untreated and HCQ-treated cell populations gave similar results (data not shown).
Frederick D. Goldman et al. Blood 2000;95:
©2000 by American Society of Hematology

4. HCQ does not alter TCR-induced PLCγ1 tyrosine phosphorylation
HCQ does not alter TCR-induced PLCγ1 tyrosine phosphorylation.Jurkat T cells were treated with varying concentrations of HCQ for 24 hours, stimulated with C305 for 15 minutes, and then lysed in NP-40 detergent.
HCQ does not alter TCR-induced PLCγ1 tyrosine phosphorylation.Jurkat T cells were treated with varying concentrations of HCQ for 24 hours, stimulated with C305 for 15 minutes, and then lysed in NP-40 detergent. PLCγ1 was immunoprecipitated from 5 × 107 cell equivalents, and immunoprecipitates were blotted with either antiphosphotyrosine (upper panel) or anti-PLCγ1 (lower panel), and immunoreactive bands were visualized by enhanced chemiluminescence. Representative data from more than 3 similar experiments are shown.
Frederick D. Goldman et al. Blood 2000;95:
©2000 by American Society of Hematology

5. HCQ does not inhibit TCR-induced inositol phosphate production
HCQ does not inhibit TCR-induced inositol phosphate production.Jurkat T cells were treated with varying concentrations of HCQ for 24 hours and then loaded with [3H] myoinositol for 3 hours.
HCQ does not inhibit TCR-induced inositol phosphate production.Jurkat T cells were treated with varying concentrations of HCQ for 24 hours and then loaded with [3H] myoinositol for 3 hours. Cells were left untreated (none) or were stimulated with C305 for 15 minutes, and were then lysed in chloroform. Soluble inositol phosphates were extracted by anion exchange and quantitated by liquid scintillation counting. Mean data from 3 separate experiments are expressed as stimulation indices: (cpm stimulated/cpm unstimulated) ± SD.
Frederick D. Goldman et al. Blood 2000;95:
©2000 by American Society of Hematology

6. HCQ does not alter MAP kinase activation
HCQ does not alter MAP kinase activation.Jurkat T cells were treated with varying concentrations of HCQ for 24 hours, stimulated with C305 for 0 to 5 minutes, and then lysed in NP-40 detergent.
HCQ does not alter MAP kinase activation.Jurkat T cells were treated with varying concentrations of HCQ for 24 hours, stimulated with C305 for 0 to 5 minutes, and then lysed in NP-40 detergent. Proteins from whole-cell lysates were subjected to 12% SDS-PAGE and were transferred to nitrocellulose, and membranes were immunoblotted with anti-ERK-2. Immunoreactive bands were visualized by enhanced chemiluminescence. Representative data from more than 3 similar experiments are shown.
Frederick D. Goldman et al. Blood 2000;95:
©2000 by American Society of Hematology

7. HCQ inhibits antigen-receptor–dependent intracellular calcium mobilization and alters the size of intracellular calcium stores.Jurkat T cells or Ramos B cells were pretreated with varying concentrations of HCQ for 24 hours and then loaded with the calcium-s...
HCQ inhibits antigen-receptor–dependent intracellular calcium mobilization and alters the size of intracellular calcium stores.Jurkat T cells or Ramos B cells were pretreated with varying concentrations of HCQ for 24 hours and then loaded with the calcium-sensitive dye calcium green-1. Changes in intracellular Ca++ concentration (fluorescence) versus time (seconds) were monitored by bulk spectrofluorometry. (A) Jurkat T cells were activated with the clonotypic anti-TCR mAb C305 (as indicated by the arrow) followed by addition of 1 μmol/L ionomycin (arrowhead). (B) Ramos B cells were activated by IgM crosslinking (arrow) followed by the addition of 1 μmol/L ionomycin (arrowhead). (C) Jurkat cells were stimulated with 1 μmol/L thapsigargin, as indicated by arrow, in the presence of 1.8 mmol/L EGTA. Residual calcium response was evaluated by addition of 1 μmol/L ionomycin (arrowhead). Results are representative of more than 5 separate experiments.
Frederick D. Goldman et al. Blood 2000;95:
©2000 by American Society of Hematology

8. HCQ inhibits intracellular calcium mobilization in primary T cells
HCQ inhibits intracellular calcium mobilization in primary T cells.Peripheral blood T cells from healthy volunteers were pretreated with varying concentrations of HCQ for 18 hours and were then loaded with calcium-sensitive dyes calcium green-1 and fura-red.
HCQ inhibits intracellular calcium mobilization in primary T cells.Peripheral blood T cells from healthy volunteers were pretreated with varying concentrations of HCQ for 18 hours and were then loaded with calcium-sensitive dyes calcium green-1 and fura-red. Cells were stimulated, as indicated by arrow, with (A) biotinylated OKT3 (40 μg/mL) plus avidin (10 μg/mL) or (B) 1 μmol/L ionomycin. Changes in intracellular Ca++ concentration (fluorescence) versus time (seconds) were monitored by fluorescence-activated cell sorter, and results are representative of 3 separate experiments.
Frederick D. Goldman et al. Blood 2000;95:
©2000 by American Society of Hematology