Abstract
The transmembrane protein (TM) from retroviral envelope proteins contains a highly conserved immunosuppressive (ISU) motif, which has been reported to play a key role in escaping the immune system of the host. Such ISU motifs delay or prevent a reaction of the adaptive immune system not only against the virus but also against cells that display the viral envelope protein. In TM of gammaretroviruses such as murine leukemia virus (MLV) this immunosuppressive ability can be abrogated by two amino acid substitutions E14R and A20F in the ISU motif. This immunosuppressive property makes ISU a potential biotherapeutic agent for autoimmune disease therapy.
TM adopts a homotrimeric structure through coiled-coil interaction. It should be
noted that monomer ISU-peptides have no function except when coupled to a carrier or synthesized as a dimer. The synthetic ISU peptide (CKS-17) was reported to downregulate proinflammatory cytokines (like TNF-α) and upregulate antiinflammatory cytokines (like IL-10) when coupled to a carrier. CKS-25, which is longer than CKS-17 with a more stable structure, has a therapeutic effect in an eczema animal model when formed as dimer.
In this project, we take use of trimerization (TIE) domain derived from human collagen XIII to construct a trimerbody TIE-ISU-WT/Mut. Trimerbody expressing cancer cells would be used to establish immune escaping animal model. Trimerbodies can be expressed well in 293T and NIH-3T3 cells after transfection or transduction. However, we failed to stably modify target tumor cells in the same way. Since soluble CKS-25 trimerbody was found to induce TNF-α in the human monocytic cell line THP-1 in parallel work, we decided to use 293T and NIH-3T3 cells to set up co-culture experiments with THP-1 cells. We found a significant increase of TNF-α after co-cultivating transfected 293T cells with lipopolysaccharide (LPS) pretreated THP-1 cells. A transwell assay indicated that TNF-α was induced by direct interaction between transfected 293T cells and THP-1 cells. In addition, the expressed TM or TIE-ISU have a synergistic effect with LPS on TNF-α production. By co-culture with MyD88 knock out THP-1 cells, we find that this induction was MyD88 dependent. Interestingly, we found that stimulation of TNF-α was abrogated when STING was knocked out. This suggests that STING is playing a key role in the TNF-α induction pathway. Moreover,TM or TIE-ISU transduced NIH-3T3 cells failed to induce IL-10 production in murine splenocytes.
Collectively, the establishment of an animal model was abandoned because of
transduction problems. Contrary to the expectations transfected 293T cell induced instead of inhibited TNF-α production when co-cultivated with THP-1 cells. This suggests another role of the ISU domain in modulating immune responses. In THP-1 cells TNF-α production is not only MyD88 dependent but also STING dependent.
TM adopts a homotrimeric structure through coiled-coil interaction. It should be
noted that monomer ISU-peptides have no function except when coupled to a carrier or synthesized as a dimer. The synthetic ISU peptide (CKS-17) was reported to downregulate proinflammatory cytokines (like TNF-α) and upregulate antiinflammatory cytokines (like IL-10) when coupled to a carrier. CKS-25, which is longer than CKS-17 with a more stable structure, has a therapeutic effect in an eczema animal model when formed as dimer.
In this project, we take use of trimerization (TIE) domain derived from human collagen XIII to construct a trimerbody TIE-ISU-WT/Mut. Trimerbody expressing cancer cells would be used to establish immune escaping animal model. Trimerbodies can be expressed well in 293T and NIH-3T3 cells after transfection or transduction. However, we failed to stably modify target tumor cells in the same way. Since soluble CKS-25 trimerbody was found to induce TNF-α in the human monocytic cell line THP-1 in parallel work, we decided to use 293T and NIH-3T3 cells to set up co-culture experiments with THP-1 cells. We found a significant increase of TNF-α after co-cultivating transfected 293T cells with lipopolysaccharide (LPS) pretreated THP-1 cells. A transwell assay indicated that TNF-α was induced by direct interaction between transfected 293T cells and THP-1 cells. In addition, the expressed TM or TIE-ISU have a synergistic effect with LPS on TNF-α production. By co-culture with MyD88 knock out THP-1 cells, we find that this induction was MyD88 dependent. Interestingly, we found that stimulation of TNF-α was abrogated when STING was knocked out. This suggests that STING is playing a key role in the TNF-α induction pathway. Moreover,TM or TIE-ISU transduced NIH-3T3 cells failed to induce IL-10 production in murine splenocytes.
Collectively, the establishment of an animal model was abandoned because of
transduction problems. Contrary to the expectations transfected 293T cell induced instead of inhibited TNF-α production when co-cultivated with THP-1 cells. This suggests another role of the ISU domain in modulating immune responses. In THP-1 cells TNF-α production is not only MyD88 dependent but also STING dependent.
| Original language | English |
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| Publisher | Århus Universitet |
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| Number of pages | 104 |
| Publication status | Published - Apr 2019 |