Primary Leukocyte Screens for Innate Immune Agonists

Abstract

The innate immune system of mammals is a key defense mechanism against invading foreign pathogens. Innate immune stimulants may have applications as vaccine adjuvants as well as in the treatment of cancer and some viral diseases, and clinical studies have been performed using agonists of Toll-like receptors (TLRs) 7, 8, and 9. The high-throughput screens for such agonists have typically relied on the overexpression of a single TLR gene in an immortalized cell line and are inherently artificial systems that are restricted to the identification of agonists for a single receptor. The authors describe 2 assays for the identification of immunostimulants that employ primary human leukocytes cocultured with hepatitis C virus (HCV) replicon-expressing cells. In these assays, stimulation of innate immune pathways in leukocytes induces interferon (IFN) expression, which acts to inhibit HCV replication, providing a high-throughput and low-cost readout for leukocyte activation. These assays are highly sensitive and provide a physiologically relevant system for the identification of a broad range of immunostimulant agents.

Toll-like receptors in immunity and inflammatory diseases: Past, present, and future

“…Similarly, Immunomodulatory oligonucleotides (IMOs), called IMO-2055 and IMO-2125, targeting TLR9 have also been developed and are used as single therapeutic agents or as in combination with other cancer chemotherapeutic drugs in certain cancers including non-small cell lung cancer (NSCLC) and colorectal cancer along with viral infections (i.e. Hepatitis C virus-infected patients) [452][453][454]. Thus, so far only three TLR agonists have been approved in different countries for different diseases [455].…”

Sequence determinants of innate immune activation by short interfering RNAs

“…These primary cells have the additional advantage that, unlike TLR7 transfected immortalised cell lines, they appear to fully recapitulate in vivo TLR signalling [ 30 ]. We and others [ 32 , 33 ], had shown that the human hepatoma cell line Huh7, harbouring an HCV replicon that expresses a luciferase reporter, is a sensitive and easily assayed system for detection of IFN. Therefore, we chose to employ an assay system in which innate immunostimulation of PBMCs by siRNAs is detected using inhibition of HCV replication as a surrogate marker for IFN production.…”

“…Therefore, we chose to employ an assay system in which innate immunostimulation of PBMCs by siRNAs is detected using inhibition of HCV replication as a surrogate marker for IFN production. Such assay systems have been shown to be dependent on immune stimulation of the leukocytes (since treatment of Huh7 cells with a broad range of TLR agonists in the absence of leukocytes does not inhibit replication of the HCV replicon) and to be dependent upon secretion of IFNα from the activated leukocytes (as shown by neutralizing mAb experiments) [ 32 , 33 ].…”

“…PBMCs or pDCs (84.5% CD303+, data not shown) were stimulated with either siRNA or known TLR agonists for 24 h prior to supernatant transfer to Huh7-Luc cells, following previously determined experimental protocols [ 32 , 33 ]. Unless otherwise indicated 50,000 PBMCs plated in 96-well plates in 80 μL antibiotic-free 10%FCS-RPMI were stimulated with TLR agonist (final volume of 100 μL/well; maximum 1% DMSO per well) or transfected with siRNA complexed with DOTAP (Roche) (final volume of 100 μL/well; maximum 100 nM siRNA per well).…”

Roles of toll-like receptors in Cancer: A double-edged sword for defense and offense

“…IMO-2055 became Idera Pharmaceuticals' first IMO drug candidate to enter clinical development. This drug had anticancer activity in a mouse model when used as a monotherapy, and its activity was amplified when used in combination with chemotherapeutic agents (Goodchild et al, 2009). In a Phase I trial that evaluated the safety and immunological activity of IMO-2055 alone and in combination with chemotherapy agents, IMO was found to be much more efficacious when used in combination.…”