Absence of IL-10 production by human PBMCs co-cultivated with human cells expressing or secreting retroviral immunosuppressive domains

Abstract

Immunosuppression by retroviruses including the human immunodeficiency virus—1 (HIV-1) is well known, however the mechanisms how retroviruses induce this immunosuppression is not fully investigated. It was shown that non-infectious retroviral particles as well as retroviral or recombinant retroviral transmembrane envelope (TM) proteins demonstrated immunosuppressive properties. The same was shown for peptides corresponding to a highly conserved domain in the TM protein. This domain is called immunosuppressive (ISU) domain and it induces modulation of the cytokine release of peripheral blood mononuclear cells (PBMCs) from healthy donors. In addition, it changes the gene expression of these cells. Common indications for the immunosuppressive activity were tumour growth in vivo and interleukin—10 (IL-10) release from human PBMCs in vitro. Single mutations in the ISU domain abrogated the immunosuppressive activity. In order to develop a new model system for the expression of the ISU domain and presentation to PBMCs which is not prone to possible endotoxin contaminations, two expression systems were developed. In the first system, designated pOUT, retroviral proteins containing the ISU domain were expressed and released into the cell culture medium, and in the second system, tANCHOR, the ISU domain was presented by a tetraspanin-anchored sequence on the cell surface of human cells. Both systems were exploited to express the wild-type (wt) ISU domains of HIV-1, of the porcine endogenous retrovirus (PERV) and of the murine leukaemia virus (MuLV) as well as to express mutants (mut) of these ISU domains. PERV is of special interest in the context of virus safety of xenotransplantation using pig organs. Expression of the TM proteins was demonstrated by confocal laser scanning microscopy, ELISA and Western blot analyses using specific antibodies. However, when cells expressing and releasing the ISU were co-incubated with human PBMCs, no increased production of IL-10 was observed when compared with the mutants. Similar results were obtained when the released TM proteins were concentrated by immunoprecipitation and added to PBMCs. We suggest that the absence of IL-10 induction can be explained by a low amount of protein, by the lack of a biologically active conformation or the absence of additional factors.

tANCHOR cell-based ELISA approach as a surrogate for antigen-coated plates to monitor specific IgG directed to the SARS-CoV-2 receptor-binding domain

“…This consideration is relevant because an emerging variant could possibly cause an altered expression when fused to the tANCHOR system. Since similar effects have not yet been observed for other proteins displayed using the tANCHOR display technology, we can only presume that further SARS-CoV-2 RBD variants will likewise be displayed on the cell surface, as this study has shown [ 24 , 27 , 48 , 58 ]. Antibody-binding capability is another significant drawback of this kind of developed assay.…”

“…In this study, we focused on detecting antibodies derived from clinical samples directed against the RBD of SARS-CoV-2. Although we previously demonstrated in principle that it is possible to detect purified antibodies directed against epitopes 2F5/4E10 and the immunosuppressive domain (ISU) of HIV-1 by using the tANCHOR system, it was still unclear whether clinical samples could also be screened [ 23 , 24 ]. Therefore, we optimized this cell-based ELISA approach by employing the tANCHOR system to monitor IgG from clinical samples by using serum samples containing antibodies against the S protein of SARS-CoV-2.…”

“…We developed a cell-based IgG RBD-specific ELISA system to avoid the need to express, purify, and coat ELISA plates with the RBD antigen from future strains. This ELISA system is based on the tANCHOR display system, which is suitable to present proteins on the surface of human cells [ 23 , 24 ]. The tANCHOR system is based on the use of tetraspanin-derived protein sequences without the large extracellular loop (LEL).…”

tANCHOR-cell-based assay for monitoring of SARS-CoV-2 neutralizing antibodies rapidly adaptive to various receptor-binding domains

“…20 , 21 In particular, the transmembrane anchors derived from the Tspan CD82 showed best performance for displaying proteins on the cell surface of human embryonic kidney 293T (HEK293T) or HeLa cells, where the protein of interest is fused and expressed as a chimeric-membrane-bound unit connected by optimized linker sequences between the third and fourth TM. 21 , 22 For testing the neutralization activity of an SARS-CoV-2 variant, only the coding DNA sequence in the tANCHOR vectors has to be adjusted. This enables rapid adaptation to upcoming SARS-CoV-2 variants.…”

tANCHOR fast and cost-effective cell-based immunization approach with focus on the receptor-binding domain of SARS-CoV-2

“…Glycosylation of proteins is very critical as shown for HIV when neutralizing antibodies should be raised against native viral protein structures [ 14 ]. To meet the requirements for a fast and efficient immunization approach including posttranslational modification of the antigen, we employed the tetraspanin (Tspan) anchor (tANCHOR) system [ 15 , 16 ]. This system is based on the use of transmembrane domains derived from the Tspan superfamily where a protein of interest is fused and expressed as a chimeric membrane-bound protein displayed on the surface of human cells.…”