2022 Volume 45 Issue 12 Pages 1798-1804
OX40, a member of the tumor necrosis factor (TNF) receptor superfamily, is induced on activated T cells. Membrane-bound OX40 ligand (OX40L) expressed by activated antigen-presenting cells induces OX40 signaling, which promotes T cell immunity. OX40 agonism would be a potential target for immunotherapy, however, it remains unclear how the activity of OX40 can be successfully controlled by a designer OX40L protein. We prepared a soluble OX40L protein possessing a PA-peptide tag and a collagenous trimerization domain from mannose-binding lectin (MBL), and tested whether PA-MBL-OX40L fusion protein worked as an agonist for OX40. We found that the majority of recombinant PA-MBL-OX40L protein purified from culture supernatants displayed a trimer structure and bound to cell surface OX40 or OX40-Fc fusion protein in a dose-dependent manner. Upon stimulation of CD4+ T cells with TCR/CD3 without CD28, PA-MBL-OX40L displayed significantly increased proliferative and cytokine responses when compared with a benchmark agonistic monoclonal antibody for OX40. Both soluble and immobilized forms of PA-MBL-OX40L induced potent OX40 signaling in CD4+ T cells. Mice administered with PA-MBL-OX40L displayed significantly augmented T cell-mediated delayed-type hypersensitivity responses. Our results suggest that activity of OX40L could be engineered to elicit better T cell responses by rational design of its assembly and architecture.
Naïve or resting conventional CD4+ and CD8+ T cells do not express OX40 (CD134), a member of the tumor necrosis factor receptor (TNFR) superfamily. OX40 is induced on antigen-activated T cells, and it regulates long-lived effector and memory T cell immunity via interacting with OX40 ligand (OX40L, CD252) expressed by activated antigen-presenting cells (APCs), such as dendritic cells, B cells, and macrophages. A conserved C-terminal TNF homology domain (THD) of OX40L assembles into a homotrimer on the surface of APCs, and monomer OX40 on T cell is trimerized through binding to the membrane-bound OX40L trimer, resulting in organization of a quaternary hexamer complex at the T cell-APC interface. Upon T cell receptor (TCR) engagement, a second signal, termed cosignaling, provided by the interaction between OX40 and OX40L plays key roles in T cell-mediated immunity and diseases.1–5)
In a subgroup of TNF receptor family molecules including OX40, two or more their cognate TNF ligand trimers have been suggested to facilitate clustering of TNFRs on the cell membrane, which would be critical for inducing efficient signal transduction.6) The engagement of OX40 by the trimeric OX40L protein complex expressed by APCs or T cells promotes T cell activation, differentiation, survival, and the formation of effector and memory T cell pools.7,8) Agonistic immunoglobulin G (IgG) monoclonal antibodies with bivalent OX40 binding moieties have been frequently used in vitro and in vivo studies to activate OX40 cosignaling. However, it remains unclear how the architecture of OX40L molecule regulates assembly of OX40 signaling units on the surface of T cells. The question would be worth addressing and must be important for the rational design and development of agonists for OX40 and related TNFR family molecules.
Mannose-binding lectin (MBL) is a plasma protein produced by hepatocytes and contributes to the activation of the complement system. MBL displays an oligomeric structure composed of a collagen-like region followed by a carbohydrate-recognition domain (CRD). The collagen-like α-helical coiled-coil structure promotes trimerization of three polypeptides to build functional high-ordered structures.9,10) Given that MBL forms active oligomeric structures via the collagen-like domain, attachment of the collagenous structure of MBL to the THD of OX40L may support the formation of active trimer structure of OX40L that is needed for induction of OX40 signaling into a T cell. In this study, we replaced the CRD of MBL with the THD of OX40L and prepared a novel OX40L-fusion protein. This MBL-OX40L fusion protein was secreted as soluble oligomers, bound to cell surface OX40 and displayed superior agonistic activity toward OX40 expressed by activated CD4+ T cells. Our results demonstrate that the collagenous domain from MBL might be useful to make an active trimer structure of TNF ligand proteins.
A vector containing cDNA encoding mouse Ox40l (Tnfsf4) was previously described.4) Based on cDNA sequences of mouse mannose-binding lectin 1 (Mbl1, NM_010775.2), mouse Cd70 (NM_011617.2), mouse 4-1bb (Tnfrsf9, NM_011612.2), and human OX40 (TNFRSF4, NM_003327.3), each cDNA of the entire coding region was amplified with PCR and ligated into the vector qCR™-Blunt II-TOPO™ (#450245, Thermo Fisher, Waltham, MA, U.S.A.). For construction of plasmids encoding PA-MBL-OX40L and PA-MBL-CD70, genes encoding a signal sequence of Igk (METDTLLLWVLLLWVPGSTGD), a PA-peptide tag (GVAMPGAEDDVV), the collagen-like domain of Mbl1 (18Ser-126Gly), and the extracellular domain of Ox40l (51Ser-198Leu) and Cd70 (45Ser-195Pro), respectively, were amplified with PCR and ligated into the vector qCR™-Blunt II-TOPO™. The TOPO vector was digested with EcoRI, and the insert containing PA-MBL-OX40L or PA-MBL-CD70 was cloned into the expression vector pCAGGS (LT727518.1). A plasmid encoding mouse Ox40 extracellular region (21Thr-211Pro) and human IgG1-Fc (mouse OX40-Fc) was previously described.11) For the construction of mouse 4-1bb and Fc chimeric plasmid (mouse 4-1BB-Fc), an extracellular region of cDNA of 4-1bb (24Val-187Leu) was used. For the construction of human OX40 and Fc chimeric plasmid (human OX40-Fc), an extracellular region of cDNA of OX40 (1Met-214Ala) was used. Preparation of recombinant proteins was previously described.11,12) Briefly, the vector was transfected into HEK293T cells by using polyethyleneimine (#408727, Merck Millipore, Burlington, MA, U.S.A.), and then cultured in Dulbecco modified Eagle medium (043-30085, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) containing 2% fetal calf serum (FCS), 100 U/mL penicillin, and 100 µg/mL streptomycin for 5 to 7 d. Alternatively, a DG44-CHO cell (ATCC, CRL-9096) clone stably expressing PA-MBL-OX40L was established to produce PA-MBL-OX40L protein. Recombinant Fc fusion protein was purified from the culture supernatants by HiTrap™ rProtein A FF column (#17507901, Cytiva, Tokyo, Japan). Recombinant PA-MBL-OX40L was purified from the culture supernatants by Anti-PA tag antibody beads (012-25841, FUJIFILM). Recombinant protein was concentrated with Amicon Ultra-4 (UFC801024, Merck Millipore). Concentration of recombinant protein was determined by bicinchoninic acid (BCA) assay (297-73101, FUJIFILM Wako Pure Chemical Corporation). PA-MBL-OX40L protein was mixed with 4× Laemmli sample buffer (240 mM Tris–HCl, pH 6.8, 40% Glycerol, 8% sodium dodecyl sulfate (SDS), and 0.1% bromophenol blue) and separated by SDS-polyacrylamide gel electrophoresis (PAGE).
Flow CytometryT cell hybridoma cells derived from CD4+ T cells from Ox40−/− mice expressing mouse Ox40 were previously described.4,13,14) For detection of OX40-OX40L interactions, T cells were firstly incubated with PA-MBL-OX40L, secondary with anti-PA antibody (NZ-1, 016-25863, FUJIFILM Wako Pure Chemical Corporation), and then thirdly with goat anti-rat IgG-fluorescein isothiocyanate (FITC) (#55745, MP biomedicals, Irvin, CA, U.S.A.). Data were acquired on a FACSCanto II (BD Bioscience, Franklin Lakes, NJ, U.S.A.) and analyzed with FlowJo (Tree Star, Ashland, OR, U.S.A.) or Flowing (http://flowingsoftware.btk.fi/) software.
Enzyme-Linked Immunosorbent Assay (ELISA)ELISA plate (#439454, Thermo Fisher) was coated with 0.5 µg/mL of OX40-Fc. The binding between OX40-Fc and PA-MBL-OX40L was visualized with anti-PA antibody and anti-rat IgG-HRP (112-035-167, Jackson ImmunoResearch, West Grove, PA, U.S.A.).
Cytokines in culture supernatants were assessed by a sandwich ELISA assay with anti-interleukin-2 (IL-2) (JES6-1A12, 503701) capture antibody, biotin-anti-IL-2 (JES6-5H4, 503803) detection antibody, anti-interferon-γ (IFN-γ) (R4-6A2, 505702) capture antibody, biotin-anti-IFN-γ (XMG1.2, 505804) detection antibody, anti-IL-4 (11B11, 504101) capture antibody, biotin-anti-IL-4 (BVD6-24G2, 504201) detection antibody, anti-IL-17A (TC11-18H10.1, 506901) capture antibody, biotin-anti-IL-17A (TC11-8H4, 507001) detection antibody, HRP streptavidin (405210), and 3,3′, 5,5′-tetramethyl benzidine (421101) from BioLegend, San Diego, CA, U.S.A. The absorbance was measured at 450 nm using FilterMax F5.
CD4+ T CellsC57BL/6 mice (Japan SLC) were bread under specific pathogen-free condition. Animal experimental protocols were approved by the Animal Care and Use Committee of the University of Toyama (Approved No. A2021PHA-11) and conducted in accordance with the Institutional Animal Experiment Handling Rules of the University of Toyama. Splenic CD4+ T cells separated with CD4 (L3T4) microbeads (130-117-043, Miltenyi Biotec, Bergisch Gladbach, Germany) or Naive CD4+ T Cell Isolation Kit (130-104-453, Miltenyi Biotec) were cultured in RPMI1640 medium (189-02025, FUJIFILM Wako Pure Chemical Corporation) with 10% FCS, penicillin, streptomycin, 2 mM L-alanyl-L-glutamine, and 50 µM 2-ME. T cells were plated in 96-well culture plates and stimulated with plate-bound anti-CD3ε (low-endotoxin, azide free; 145-2C11; 100340, BioLegend) in the presence or absence of soluble PA-MBL-OX40L or anti-OX40 agonistic antibody (OX86, #562181, BD Bioscience, or 119431, BioLegend). Proliferation was assessed with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) (M2128, MilliporeSigma, Burlington, MA, U.S.A.) assay. The water-insoluble MTT formazan was solubilized with dimethyl sulfoxide (DMSO), and the absorbance was measured with a plate reader at 540 nm using FilterMax F5 (Molecular Devices, San Jose, CA, U.S.A.).
For stimulation with microbeads, anti-PA antibody (3 µg) or OX86 (3 µg) was firstly immobilized on the surface of Dynabeads protein G (4 × 107 beads, 10004D, ThermoFisher,). After washing with sterile phosphate buffered saline (PBS), anti-PA coated beads were incubated with PA-MBL-OX40L or PA-MBL-CD70. CD4+ T cells were mixed with the beads at 1 : 60 ratios and cultured in anti-CD3 coated 96-well U-bottomed plates for 3 d.
Delayed-Type Hypersensitivity (DTH) ResponseDTH response was evaluated as previously described.12) C57BL/6 mice were immunized subcutaneously at the tail with 200 µL of 1.25 mg/mL methyl bovine serum albumin (mBSA) (A1009, Merck) emulsified with complete Freund’s adjuvant (CFA) (F5881, Merck) on day 0. Seven days after the immunization, mice were challenged subcutaneously in a footpad with 30 µL of 7 mg/mL mBSA in PBS plus 10 µL of PBS or 500 µg/mL PA-MBL-OX40L or 500 µg/mL of OX86. An equal volume of PBS was injected another footpad as a control. One day after the challenge, footpad thickness was measured with a digital caliper (Shinwa Rules Co., Ltd., Japan). The magnitude of the DTH response was determined as follows: [footpad swelling (%)] = ([footpad thickness of mBSA-injected footpad (mm)]−[footpad thickness of PBS-injected footpad (mm)]) ÷ [footpad thickness of PBS-injected footpad (mm)] × 100.
Statistical AnalysisStatistical significance was assessed with Student t test with two-sided distributions (* p < 0.05, ** p < 0.01, *** p < 0.001).
For control of T cell responses, it would be important to clarify what type of protein structure of OX40L is effective for the induction of potent OX40 signaling that supports the activation of T cells. In this study, we hypothesized that the collagen-like domain derived from MBL could be utilized for making an active trimer structure of OX40L. We attached a PA-peptide tag and the collagen-like domain to the N-terminal of the THD of mouse OX40L (Fig. 1A). The PA-MBL-OX40L protein purified from culture supernatants displayed dimer and trimer structures in SDS-PAGE (Fig. 1B, (−), without boil and reduction).
(A) The domain structures of mannose-binding lectin (MBL), OX40 ligand (OX40L), and MBL-OX40L fusion protein with PA peptide tag, PA-MBL-OX40L. (B) SDS-PAGE (10%) of PA-MBL-OX40L under non-boiling/non-reducing (−) and boiling/reducing (+) conditions.
We previously prepared a T cell hybridoma cell clone, derived from effector T cells from Ox40−/− mice, and this cell was transfected to express Ox40 to establish OX40-positive cells4,13,14) (Fig. 2A, left). PA-MBL-OX40L bound to OX40-positive cells but not to OX40-negative cells (Fig. 2A, right), in a dose-dependent manner (Fig. 2B). PA-MBL-OX40L also bound to mouse OX40-Fc, but not to mouse 4-1BB-Fc, immobilized on the plastic surface (Figs. 2C, D). TNF ligand CD70 with PA tag and MBL, PA-MBL-CD70, did not bind to mouse OX40-Fc (Fig. 2C). Mouse OX40L has been shown to cross-react with human OX40.15) Indeed, we could detect the interaction between PA-MBL-OX40L and human OX40-Fc (Fig. 2D). These results show that the collagen-like domain of MBL facilitates the formation of active structure of OX40L that is permissive for binding to OX40.
(A) Binding activity of PA-MBL-OX40L to cell surface OX40 evaluated by flow cytometry. OX40-positive and -negative T cell hybridoma cells were stained with anti-OX40 antibody, OX86 (left), or PA-MBL-OX40L (right). Number adjacent to outline areas indicate percent OX40+ cells. (B) Dose–response curve of PA-MBL-OX40L to OX40-positive T cell hybridoma cells evaluated by flow cytometry. (C) Binding specificity of PA-MBL-OX40L to plate absorbed mouse OX40-Fc evaluated by ELISA. (D) Binding activity of PA-MBL-OX40L to both mouse OX40-Fc and human OX40-Fc evaluated by ELISA.
In addition to the primary TCR/CD3 signaling, the secondary cosignaling is needed to promote proliferation and cytokine production of T cells. To evaluate functional significance of PA-MBL-OX40L in terms of inducing OX40 cosignaling, naïve and total CD4+ T cells were stimulated with immobilized anti-CD3 agonistic antibody, without anti-CD28 agonistic antibody, in the presence or absence of soluble PA-MBL-OX40L. Soluble PA-MBL-OX40L significantly augmented cell proliferation and IL-2 production induced by anti-CD3 (Figs. 3A, B). Importantly, those enhanced responses mediated by PA-MBL-OX40L were significantly higher than those of OX86, a benchmark agonistic IgG for OX40 (Figs. 3A, B). Soluble PA-MBL-OX40L dose dependently promoted the production of IL-2 from activated total CD4+ T cells (Fig. 3B). These results indicate that the collagenous structure of MBL assembles an active structure of OX40L that supports the induction of potent OX40 cosignaling into T cells.
(A) Naïve CD4+ T cells (1 × 105 cells/well) were cultured in 96-well flat-bottomed culture plates precoated with 10 µg/mL of anti-CD3 antibody in the absence (None) or presence of 3 µg/mL of soluble PA-MBL-OX40L or 3 µg/mL of soluble agonistic antibody to OX40 (OX86) for 3 d. (B) Total CD4+ T cells (0.5 × 105 cells/well) were cultured with 10 µg/mL of plate-bound anti-CD3 antibody in the presence of indicated concentrations of soluble PA-MBL-OX40L or soluble OX86 for 3 d. Cell proliferative response was measured by MTT assay. Concentrations of IL-2 in the culture supernatants were determined by ELISA. Data are mean ± standard deviation (n = 3) and from one experiment representative of at least two independent experiments with similar results. * p < 0.05, ** p < 0.01, *** p < 0.001 (Student t test).
OX40L is expressed on the surface of activated APCs. We thought that PA-MBL-OX40L presented on microparticles might mimic cell-associated OX40L. Thus, we next attempted to evaluate T cell responses mediated by PA-MBL-OX40L immobilized on microbeads. Firstly, we confirmed that CD4+ T cells proliferated vigorously and produced greater amounts of IL-2, IFN-γ and IL-4, in response to a fixed amount of soluble PA-MBL-OX40L and increasing doses of anti-CD3 (Fig. 4A). To prepare microbeads bearing PA-MBL-OX40L, anti-PA tag IgG was initially immobilized on protein G-microbeads, and then the beads were incubated with PA-MBL-OX40L. The microbeads with PA-MBL-OX40L also significantly increased proliferative and cytokine responses induced by anti-CD3, as compared with control beads (anti-PA only) (Fig. 4B). The beads bearing OX86 did not induce sufficient proliferative and cytokine responses (Fig. 4B). Thus, both soluble and bead-associated PA-MBL-OX40L could promote productive OX40 cosignaling in activated T cells.
(A) Total CD4+ T cells (0.5 × 105 cells/well) were cultured in 96-well U-bottomed culture plates precoated with indicated concentrations of anti-CD3 antibody in the presence or absence of 10 µg/mL of soluble PA-MBL-OX40L for 3 d. (B) Total CD4+ T cells (0.5 × 105 cells/well) were cultured in 96-well U-bottomed culture plates precoated with indicated concentrations of anti-CD3 antibody in the presence of 2.8 µm Dynabeads Protein G (3 × 106 particles/well) preabsorbed with anti-PA or anti-PA/PA-MBL-OX40L or OX86 for 3 d. Cell proliferative and cytokine responses were evaluated by MTT assay and ELISA, respectively. Data are mean ± standard deviation (n = 3) and from one experiment representative of at least two independent experiments with similar results. * p < 0.05, ** p < 0.01, *** p < 0.001 (Student t test).
We next prepared another PA-MBL-TNF ligand fusion protein to evaluate whether this molecule can also display a similar function for CD4+ T cells. The TNF family molecule CD70 is also expressed by activated APCs and promotes the T cell cosignaling via the TNF receptor family molecule CD27.1) Both PA-MBL-CD70 and PA-MBL-OX40L on the beads equivalently increased the proliferative and cytokine responses in CD4+ T cells (Fig. 5), suggesting that the methodology used in this study is applicable to other TNF family proteins.
Total CD4+ T cells (0.5 × 105 cells/well) were cultured in 96-well U-bottomed culture plates precoated with indicated concentrations of anti-CD3 antibody in the presence of 2.8 µm Dynabeads Protein G (3 × 106 particles/well) preabsorbed with anti-PA or anti-PA/PA-MBL-OX40L or anti-PA/PA-MBL-CD70 for 3 d. Cell proliferative and cytokine responses were evaluated by MTT assay and ELISA, respectively. Data are mean ± standard deviation (n = 3) and from one experiment representative of at least two independent experiments with similar results. ** p < 0.01, *** p < 0.001 (Student t test).
Lastly, we investigated whether PA-MBL-OX40L increased the activation of T cells in vivo. DTH response, classified as type IV hypersensitiveness, is mediated by cellular immunity, and T-helper 1 (Th1) and T-helper 17 (Th17) cells play dominant roles for inducing inflammatory responses. OX40 cosignaling induced by PA-MBL-OX40L in effector Th1 and Th17 cells may promote DTH responses. To evaluate this possibility, mice were immunized with mBSA on day 0 and challenged with mBSA on day 7 to induce DTH responses. PA-MBL-OX40L or agonistic mAb for OX40 (OX86) in combination with mBSA was injected into a footpad on day 7 to evaluate the activity of PA-MBL-OX40L in the effector stage after Th1/17 differentiation (Fig. 6A). We found that mice administered with PA-MBL-OX40L, but not OX86, exhibited significantly increased DTH responses as determined by footpad swelling (Fig. 6B). Splenocytes from mice injected with mBSA/PA-MBL-OX40L displayed significantly higher proliferative and IFN-γ/IL-17 responses to mBSA, as compared to those from mBSA/PBS or mBSA/OX86 injected group (Fig. 6C). This result shows that PA-MBL-OX40L effectively activates effector T cell responses in vivo.
(A) Schematic diagram of the experimental schedule for inducing DTH response, challenged with mBSA only or mBSA plus PA-MBL-OX40L or mBSA plus OX86. (B) Footpad swelling on day 8. Data are mean ± standard error of the mean of three mice per group. (C) mBSA-specific T cell proliferative and cytokine responses. Pooled spleen cells from three C57BL/6 mice immunized and challenged with mBSA were cultured with indicated concentrations of mBSA for 3 d. Cell proliferation was assessed by MTT assay. IFN-γ and IL-17A levels were determined by ELISA. The average and standard deviation of three wells are shown. * p < 0.05, ** p < 0.01, *** p < 0.001 (Student t test).
Collectively, PA-MBL-OX40L fusion protein potently enhances OX40-driven T cell activation. These results suggest that oligomerized TNF family molecules via the collagen-like domain of MBL coordinate clustering of cognate TNFRs on the T cell membrane and effectively augment TNFR family cosignaling in T cells.
The OX40-OX40L system plays important roles in immune regulation mediated by T cells.1–3,16) The aim of this study is to test whether OX40L activity can be engineered to elicit effective T cell responses by rational molecular design of OX40L. We found that the collagenous domain of MBL organized active OX40L oligomers and that soluble PA-MBL-OX40L effectively promoted OX40 cosignaling. PA-MBL-OX40L immobilized on the surface of microbeads also displayed pronounced OX40-driven T cell responses. The information obtained here would be valuable for designing functional TNF family molecules that favor immunotherapy.
Membrane-bound OX40L plays a dominant role for controlling inflammation and immunity mediated by OX40+ T cells. Inflammatory signals mediated by TLRs, CD40, TSLP, type I IFN, IL-18, and PGE2 enhance the expression of OX40L on APCs and other cell types. OX40L is highly expressed by cells at inflammatory sites, which promotes local inflammation. Elevated membrane-bound OX40L in the inflammatory milieu can be cleaved to generate a soluble OX40L, which is detected in patients with rheumatoid arthritis at ng/mL levels.17) OX40 engagement with membrane-bound OX40L is effective for stimulating conventional T cells and inhibiting Treg cells.4,8,18) Overexpression of membrane-bound OX40L in tumors and dendritic cells augments anti-tumor immunity.19,20) These studies demonstrate that cell surface OX40L is a potent stimulator for OX40.
It has been proposed that membrane-bound TNF family ligands are more effective in clustering their cognate receptors on the target cell, as compared to soluble TNF ligands.6,21) An intriguing issue would be how to establish a potent structure of oligomerized TNF trimers that can facilitate the clustering of TNF receptors on the T cell membrane. In this study, we selected a collagenous structure from mouse MBL1 to trimerize OX40L. The structure of MBL is similar to that of C1q, the primary component of the classical pathway of complement. It has revealed a structural and evolutionary link between TNF and C1q proteins.22,23) Mouse MBL1 consists of an N-terminal cross-linking region, a collagen-like domain, a neck region and a C-terminal CRD. To construct a trimer structure of OX40L, we replaced the globular CRD of MBL1 with the THD of OX40L, and additionally attached a PA-peptide tag to the N-terminal end. We detected 1–10 µg/mL of PA-MBL-OX40L protein in the culture supernatants of HEK293T and CHO cells transfected with an expression vector coding for the PA-MBL-OX40L gene. The majority of purified PA-MBL-OX40L was trimer, but it contained a minor amount of dimer. It has been demonstrated that native full-length MBL in the blood or recombinant MBL displays oligomeric forms with different molecular sizes, which contain dimer structures,24–26) suggesting that the collagenous domain of mouse MBL1 forms not only a trimer structure but also other oligomeric structures including a dimer.
PA-MBL-OX40L bound to cell surface OX40 and significantly promoted in vitro and in vivo T cell responses. PA-MBL-OX40L showed greater activity for stimulation of T cells than OX86, a benchmark agonist IgG for OX40. We also prepared PA-MBL-CD70 protein, and PA-MBL-OX40L and PA-MBL-CD70 had comparable activity for T cells. These results demonstrate that the collagen-like domain of MBL would be a suitable framework to create TNF trimers and that the type of T cell regulation engineered in this study might apply for augmenting TNFR cosignaling in general. However, there is still the issue of whether hyperactivating stimuli mediated by the TNF-TNFR clusters are effective for inducing T cell longevity required for establishing long-term immunological memory. Combined signals mediated by TNFRs, CD28, γ-chain cytokine receptors, and co-inhibitory receptors may be the key for inducing effective T cell immunity that would require for therapeutic intervention for infections and cancers. This needs to address in the future studies.
In summary, we provide the first description of MBL-OX40L fusion protein that strongly promotes T cell activation. Our results demonstrate that intrinsic activity of TNF family ligands for their cognate receptors is controllable by engineering oligomeric molecular status and that the information presented in this study will aid the design of biomolecules that favor TNFR-targeted immunotherapies.
We thank the Life Science Research Center, University of Toyama for technical support. We thank members of Laboratory of Molecular Cell Biology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama and Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, Tohoku University for their assistance and help.
This work was supported by Japan Society for the Promotion of Science KAKENHI Grants JP15H04640 (T.S.) and JP18H02572 (T.S.), the Tamura Science and Technology Foundation.
The authors declare no conflict of interest.
