a0c97badf80bba711afff4638283cb37 pmc_34943995.pdf aefcc35bdb7fdc2b327f54ce1961b9f59019c4b3 pmc_34943995.pdf 00ae07164b495ba82043f4c31a779923df6b92a91af21d93081983ee86244194 pmc_34943995.pdf Title: Immunomodulatory Effects Associated with Cladribine Treatment Subject: Cladribine is a synthetic deoxyadenosine analogue with demonstrated efficacy in patients with relapsing-remitting multiple sclerosis (MS). The main mechanism of action described for cladribine is the induction of a cytotoxic effect on lymphocytes, leading to a long-term depletion of peripheral T and B cells. Besides lymphocyte toxicity, the mode of action may include immunomodulatory mechanisms affecting other cells of the immune system. In order to induce its beneficial effects, cladribine is phosphorylated inside the cell by deoxycytidine kinase (DCK) to its active form. However, the mechanism of action of cladribine may also include immunomodulatory pathways independent of DCK activation. This in vitro study was designed to explore the impact of cladribine on peripheral blood mononuclear cells (PBMC) subsets, and to assess whether the immunomodulatory mechanisms induced by cladribine depend on the activation of the molecule. To this end, we obtained PBMCs from healthy donors and MS patients and performed proliferation, apoptosis and activation assays with clinically relevant concentrations of cladribine in DCK-dependent and -independent conditions. We also evaluated the effect of cladribine on myeloid lineage-derived cells, monocytes and dendritic cells (DCs). Cladribine decreased proliferation and increased apoptosis of lymphocyte subsets after prodrug activation via DCK. In contrast, cladribine induced a decrease in immune cell activation through both DCK-dependent and -independent pathways (not requiring prodrug activation). Regarding monocytes and DCs, cladribine induced cytotoxicity and impaired the activation of classical monocytes, but had no effect on DC maturation. Taken together, these data indicate that cladribine, in addition to its cytotoxic function, can mediate immunomodulation in different immune cell populations, by regulating their proliferation, maturation and activation. Keywords: multiple sclerosis; cladribine; immune-regulation; flow-cytometry Author: Nicolás Fissolo, Laura Calvo-Barreiro, Herena Eixarch, Ursula Boschert, Carmen Espejo, Xavier Montalban and Manuel Comabella Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Dec 10 10:13:26 2021 CET ModDate: Fri Dec 10 10:20:30 2021 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 13 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1598941 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- AIQCCL+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 BMWNIW+VnURWPalladioL Type 1 Custom yes yes no 15 0 QQUKJG+URWPalladioL-Ital Type 1 Custom yes yes no 19 0 YGVXCH+URWPalladioL-Bold Type 1 Custom yes yes no 24 0 LCGRCS+URWPalladioL-Roma Type 1 Custom yes yes no 28 0 YFFKLH+CMSY10 Type 1 Builtin yes yes no 32 0 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yes yes yes 333 0 OMCENO+Arial CID TrueType Identity-H yes yes yes 399 0 OMCGJE+Calibri,Bold CID TrueType Identity-H yes yes yes 405 0 OMCCPB+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 411 0 OMCCPC+PalatinoLinotype CID TrueType Identity-H yes yes yes 414 0 OMCCPD+PalatinoLinotype TrueType WinAnsi yes yes no 420 0 OMCDAF+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 423 0 OMCEBA+TimesNewRoman CID TrueType Identity-H yes yes yes 426 0 OMCECA+Arial,Bold CID TrueType Identity-H yes yes yes 432 0 OMCENO+Arial CID TrueType Identity-H yes yes yes 506 0 OMCGJE+Calibri,Bold CID TrueType Identity-H yes yes yes 512 0 OMCCPB+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 518 0 OMCCPC+PalatinoLinotype CID TrueType Identity-H yes yes yes 521 0 OMCCPD+PalatinoLinotype TrueType WinAnsi yes yes no 527 0 OMCDAF+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 530 0 OMCECA+Arial,Bold CID TrueType Identity-H yes yes yes 533 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-10-21 02:40:18 CEST RepresentationInformation: pmc_34943995.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-03-31 12:06:32 CEST Size: 1598941 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 732 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Children: Item: Title: Subjects Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Cell Isolation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Media and Reagents Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: In Vitro Stimulation of PBMCs and Functional Assays Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Children: Item: Title: Proliferation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Apoptosis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Activation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: In Vitro Stimulation of Monocytes and Activation Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Flow Cytometry Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Children: Item: Title: Assessment of the Functional In Vitro Dose of Cladribine Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Inhibition of Cell Proliferation Caused by Cladribine Depends on Prodrug Activation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: Cladribine Induces Apoptosis in a DCK-Dependent Manner Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: Cladribine Impairs Cell Activation Both Dependently and Independently of Deoxycytidine Kinase Activity Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Cladribine Shows a Marginal Effect on Monocyte Activation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Cladribine Does Not Affect the Differentiation Process of DCs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Info: Title: Immunomodulatory Effects Associated with Cladribine Treatment Author: Nicolás Fissolo, Laura Calvo-Barreiro, Herena Eixarch, Ursula Boschert, Carmen Espejo, Xavier Montalban and Manuel Comabella Subject: Cladribine is a synthetic deoxyadenosine analogue with demonstrated efficacy in patients with relapsing-remitting multiple sclerosis (MS). The main mechanism of action described for cladribine is the induction of a cytotoxic effect on lymphocytes, leading to a long-term depletion of peripheral T and B cells. Besides lymphocyte toxicity, the mode of action may include immunomodulatory mechanisms affecting other cells of the immune system. In order to induce its beneficial effects, cladribine is phosphorylated inside the cell by deoxycytidine kinase (DCK) to its active form. However, the mechanism of action of cladribine may also include immunomodulatory pathways independent of DCK activation. This in vitro study was designed to explore the impact of cladribine on peripheral blood mononuclear cells (PBMC) subsets, and to assess whether the immunomodulatory mechanisms induced by cladribine depend on the activation of the molecule. To this end, we obtained PBMCs from healthy donors and MS patients and performed proliferation, apoptosis and activation assays with clinically relevant concentrations of cladribine in DCK-dependent and -independent conditions. We also evaluated the effect of cladribine on myeloid lineage-derived cells, monocytes and dendritic cells (DCs). Cladribine decreased proliferation and increased apoptosis of lymphocyte subsets after prodrug activation via DCK. In contrast, cladribine induced a decrease in immune cell activation through both DCK-dependent and -independent pathways (not requiring prodrug activation). Regarding monocytes and DCs, cladribine induced cytotoxicity and impaired the activation of classical monocytes, but had no effect on DC maturation. Taken together, these data indicate that cladribine, in addition to its cytotoxic function, can mediate immunomodulation in different immune cell populations, by regulating their proliferation, maturation and activation. 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