bioRxiv preprint doi: https://doi.org/10.1101/2020.12.30.424816; this version posted January 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Simultaneous inhibition of human CD4 and 4-1BB 2 biogenesis suppresses cytotoxic T lymphocyte proliferation 3 4 Elisa Claeys1, Eva Pauwels1, Stephanie Humblet-Baron2, Dominique 5 Schols1, Mark Waer3, Ben Sprangers4, Kurt Vermeire1* 6 7 1KU Leuven Department of Microbiology, Immunology and Transplantation, Laboratory of Virology and 8 Chemotherapy, Rega Institute, Leuven, B-3000, Belgium 9 2KU Leuven Department of Microbiology, Immunology and Transplantation, Laboratory of Adaptive 10 Immunology, Leuven, B-3000, Belgium 11 3KU Leuven Department of Microbiology, Immunology and Transplantation, Laboratory of Tracheal 12 Transplantation, Leuven, B-3000, Belgium 13 4KU Leuven Department of Microbiology, Immunology and Transplantation, Laboratory of Molecular 14 Immunology, Leuven, B-3000, Belgium 15 16 *For correspondence: [email protected] 17 ORCID iD: Kurt Vermeire: 0000-0003-1123-1907 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.30.424816; this version posted January 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 18 ABSTRACT 19 The small molecule cyclotriazadisulfonamide (CADA) down-modulates the human CD4 20 receptor, an important factor in T cell activation. Here, we addressed the immunosuppressive 21 potential of CADA using in vitro activation models. CADA inhibited lymphocyte proliferation in 22 a mixed lymphocyte reaction, and when human PBMCs were stimulated with CD3/CD28 beads 23 or phytohemagglutinin. The immunosuppressive effect of CADA involved both CD4+ and CD8+ 24 T cells but was, surprisingly, most prominent in the CD8+ T cell subpopulation where it inhibited 25 cell-mediated lympholysis. We discovered a direct down-modulatory effect of CADA on 4-1BB 26 (CD137) expression, a survival factor for activated CD8+ T cells. More specifically, CADA 27 blocked 4-1BB protein biosynthesis by inhibition of its co-translational translocation across the 28 ER membrane in a signal peptide-dependent way. This study demonstrates that CADA, as 29 potent down-modulator of human CD4 and 4-1BB, has promising in vitro immunomodulatory 30 characteristics for future in vivo exploration as immunosuppressive drug. 31 32 33 KEYWORDS 34 Cyclotriazadisulfonamide, CADA, CD4 receptor, T cell activation, immunosuppression, 4-1BB, 35 CD137, signal peptide, ER, co-translational translocation 36 37 ABBREVIATIONS 38 CADA, cyclotriazadisulfonamide; CD, cluster of differentiation; CTPS1, cytidine triphosphate 39 synthase 1; ER, endoplasmic reticulum; hCD4, human CD4; IL, interleukin; Lck, lymphocyte 40 C-terminal Src kinase; mCD4, murine CD4; MLR, mixed lymphocyte reaction; MMF, 41 mycophenolate mofetil; PBMC, peripheral blood mononuclear cell; PHA, phytohemagglutinin; 42 pSTAT5, phosphorylated signal transducer and activator of transcription 5; sCD25, soluble 43 CD25; SP, signal peptide 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.30.424816; this version posted January 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 44 INTRODUCTION 45 The cluster of differentiation 4 (CD4) receptor is a type I integral membrane protein consisting 46 of four extracellular immunoglobulin-like domains, a spanning transmembrane region and a 47 short cytoplasmic tail (Maddon et al., 1985). The lymphocyte C-terminal Src kinase (Lck) 48 non-covalently interacts with the cytoplasmic tail of CD4 (Shaw et al., 1989). Next to its function 49 in CD4 signaling, Lck inhibits endocytosis of the CD4 receptor by preventing the entry of CD4 50 into clathrin-coated pits (Pelchen-Matthews et al., 1992). Several immune cell types express 51 the CD4 receptor with T helper cells expressing the highest levels, followed by monocytes that 52 express already 10- to 20-fold less CD4 compared to T cells (Collman et al., 1990). Studies in 53 CD4 null mice underline the role of the CD4 receptor in positive thymic selection and 54 development of helper T cells (Rahemtulla et al., 1991). 55 The CD4 receptor is also crucial for proper immune function, especially during T cell activation 56 in which it can fulfil several roles (Claeys et al., 2019). The CD4 receptor can exert an 57 intercellular adhesion function by stabilizing the interaction between the T cell receptor on 58 CD4+ T cells and the major histocompatibility complex class II on antigen-presenting cells 59 (Janeway, 1989). More important are the signaling function of the CD4 receptor in T cell 60 activation through Lck and the enhancement of T cell sensitivity to antigens mediated by CD4 61 (Konig et al., 2004; Krogsgaard et al., 2005). Besides its role in T cell activation, the CD4 62 receptor is suggested to be involved in peripheral T cell differentiation towards the T helper 2 63 subset and in the chemotactic response of CD4+ T cells towards interleukin (IL)-16 (Cruikshank 64 et al., 1991; Fowell et al., 1997). Additionally, different functions are attributed to the CD4 65 receptor in other types of immune cells including natural killer and dendritic cells (Bernstein et 66 al., 2006; Bialecki et al., 2011). The important role of the CD4 receptor in the immune system 67 has been further demonstrated by the in vitro and in vivo immunosuppressive potential of 68 non-depleting anti-CD4 monoclonal antibodies (Mayer et al., 2013; Schulze-Koops et al., 2000; 69 Winsor-Hines et al., 2004). 70 In the field of virology, attachment of viral gp120 of human immunodeficiency virus (HIV) to the 71 cellular CD4 receptor initiates HIV infection of target cells (Dalgleish et al., 1984; Klatzmann et 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.30.424816; this version posted January 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 72 al., 1984). From an antiviral screen, the small molecule cyclotriazadisulfonamide (CADA) was 73 identified as a potent inhibitor of HIV infection (Vermeire et al., 2002). The antiviral effect of 74 this synthetic macrocycle is due to down-modulation of the CD4 protein, the primary entry 75 receptor for HIV (Vermeire et al., 2003). This down-modulating activity of CADA is reversible 76 in vitro: when treatment is ceased, cellular CD4 expression is rapidly restored to normal levels 77 (Vermeire et al., 2007). Additionally, CADA does not compromise cellular viability as was 78 demonstrated by long-term (about 1 year) exposure of a T cell line to CADA, with full recovery 79 of CD4 expression when treatment was terminated (Vermeire et al., 2014). The sensitivity of 80 the CD4 receptor to CADA is species-specific, as expression of murine CD4 (mCD4) was not 81 affected by CADA, while primary T cells of macaques responded in a similar way as human T 82 cells. Mechanistically, CADA was shown to inhibit endoplasmic reticulum (ER) co-translational 83 translocation of the human CD4 (hCD4) pre-protein in a signal peptide (SP)-dependent way 84 (Vermeire et al., 2014). CADA selectively binds to the SP of hCD4, thereby locking it in an 85 intermediate conformation inside the Sec61 translocon channel during co-translational 86 translocation through the ER membrane, finally resulting in proteasomal degradation in the 87 cytosol of the mistranslocated hCD4 precursor molecules. The CADA-sensitive region of hCD4 88 consists primarily of the hydrophobic core of the hCD4 SP, although the presence of charged 89 residues in the N-terminal portion of the mature protein enhances sensitivity (Van Puyenbroeck 90 et al., 2020). Almost no binding of CADA to the mCD4 SP was detected, explaining the 91 observed resistance of mCD4 to CADA (Vermeire et al., 2014). 92 Thus, CADA down-modulates the CD4 receptor, a key component in T cell activation. 93 Therefore, we explored in this study whether CADA has a potential immunomodulatory 94 capacity. Here, CADA was evaluated in several in vitro models of T cell activation and was 95 found to exert a clear immunosuppressive effect. Furthermore, in addition to the earlier 96 reported CD4 receptor, we identified 4-1BB – a crucial co-stimulatory factor in T cell activation 97 of mainly cytotoxic lymphocytes – as a new target of CADA. 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.30.424816; this version posted January 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 98 RESULTS 99 100 CADA down-modulates the human CD4 receptor and has an immunosuppressive effect 101 in the mixed lymphocyte reaction 102 In line with our previous report (Vermeire et al., 2014), the small molecule CADA (Figure 1A) 103 dose-dependently down-modulated the hCD4 receptor on Jurkat T cells as well as on human 104 peripheral blood mononuclear cells (PBMCs) (Figure 1B). At a concentration of 10 μM CADA, 105 the cell surface hCD4 expression level was greatly reduced in both cell types: 86% reduction 106 in hCD4 expression for Jurkat cells and 74% for PBMCs, as compared to untreated control 107 cells (IC50 values of 0.41 µM and 0.94 µM, respectively).