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Complement Activation by CpG in a Human Whole Blood Loop System: Mechanisms and Immunomodulatory Effects
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology.
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia.
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2009 (English)In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 183, no 10, 6724-6732 p.Article in journal (Refereed) Published
Abstract [en]

Phosphorothioate oligodeoxynucleotides can activate complement, and experimental murine studies have revealed differential effects upon simultaneous TLR stimulation and complement activation compared with either event alone. We set out to investigate the immune stimulatory effects of CpG 2006 in fresh non-anticoagulated human blood with or without presence of active complement. We also sought to elucidate the mechanism behind complement activation upon stimulation with phosphorothioate CpG 2006. In a human blood loop system, both backbone and sequence-specific effects by CpG were counteracted by selective inhibition of C3. Furthermore, DNA backbone-mediated CD40 and CD83 expression on monocytes and sequence-specific IL-6 and TNF production were reduced by complement inhibition. CpG-induced complement activation occurred via either the classical or the alternative pathway and deposits of both IgM and properdin, two activators of complement, were detected on CpG after incubation with EDTA plasma. Quartz crystal microbalance with dissipation monitoring demonstrated alternative pathway convertase build-up onto CpG as a likely pathway to initiate and sustain complement activation. Specific inhibition of C3 suppressed CpG 2006 uptake into monocytes indicating that C3 fragments are involved in CpG internalization. The interplay between complement and TLR9 signaling demonstrated herein warrants further investigation.

Place, publisher, year, edition, pages
The American Association of Immunologists, Inc. , 2009. Vol. 183, no 10, 6724-6732 p.
Keyword [en]
CpG, complement, compstatin, properdin, whole blood loop system, QCM, alternative pathway
National Category
Medical and Health Sciences
Research subject
Immunology
Identifiers
URN: urn:nbn:se:uu:diva-110143DOI: 10.4049/jimmunol.0902374ISI: 000271765700077PubMedID: 19864604OAI: oai:DiVA.org:uu-110143DiVA: diva2:275716
Available from: 2009-11-06 Created: 2009-11-05 Last updated: 2015-03-01
In thesis
1. Immunological Checkpoint Blockade and TLR Stimulation for Improved Cancer Therapy
Open this publication in new window or tab >>Immunological Checkpoint Blockade and TLR Stimulation for Improved Cancer Therapy
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
TLR-stimulering och CTLA-4 samt PD-1 blockad för förbättrad cancerterapi
Abstract [en]

This thesis concerns the investigation of novel immunotherapies for cancer eradication. CpG therapy was used in order to target antigen-presenting cells (APCs), facilitating antigen presentation and activation of T cells. Blockade of the two major immune checkpoint regulators (CTLA-4 and PD-1) was also studied to ensure proper and sustained T cell activation. The therapies were investigated alone and compared to BCG, the standard immunotherapy in the clinic today for bladder cancer. In addition, CpG as well as BCG was combined with CTLA-4 or PD-1 blockade to examine if the combination could improve therapy.

Single and combination strategies were assessed in an experimental bladder cancer model. In addition, one of the therapies (local aCTLA-4 administration) was evaluated in an experimental pancreatic cancer model. To be able to study the effects of CpG in humans, a human whole blood loop system has been used. This allowed us to dissect the potential interplay between CpG and complement.

CpG was found to be superior to the conventional therapy, BCG, in our experimental model and T cells were required in order for effective therapy to occur. Used as a monotherapy, CTLA-4 blockade but not PD-1 blockade, prolonged survival of mice. When CTLA-4 or PD-1 blockade was combined with CpG, survival was enhanced and elevated levels of activated T cells were found in treated mice. In addition, Treg levels were decreased in the tumor area compared to tumors in control treated mice. CTLA-4 blockade was also effective when administrated locally, in proximity to the tumor. Compared to systemic CTLA-4 blockade, local administration gave less adverse events and sustained therapeutic success.

When CpG was investigated in a human whole blood loop system it was found to tightly interact with complement proteins. This is an interesting finding which warrants further investigation into the role of TLRs in complement biology. Tumor therapy could be affected either negatively or positively by this interaction.

The results presented herein are a foundation for incorporating these combination therapies into the clinic, specifically for bladder cancer but in a broader perspective, also for other solid tumors such as pancreatic cancer.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 95 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 506
Keyword
CpG ODNs, TLR-9, CTLA-4, PD-1, PD-L1, B7. H1, immunotherapy, checkpoint blockade, bladder cancer, cancer, complement, TLRs, Compstatin, properdin, C3, experimental animal model, whole blood loop system, combination therapies
National Category
Immunology in the medical area Immunology in the medical area
Research subject
Immunology
Identifiers
urn:nbn:se:uu:diva-110147 (URN)978-91-554-7675-5 (ISBN)
Public defence
2010-01-29, Rudbeck laboratory, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
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Supervisors
Available from: 2009-12-23 Created: 2009-11-05 Last updated: 2009-12-23Bibliographically approved

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Mangsbo, Sara M

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