T Cell Costimulation with Anti-CD137 Monoclonal Antibodies Is Mediated by K63–Polyubiquitin-Dependent Signals from Endosomes

Abstract

Agonist anti-CD137 (4-1BB) mAbs enhance CD8-mediated antitumor immunity. Agonist anti-human CD137 mAbs binding to four distinct epitopes on the CD137 glycoprotein costimulated T cell activation irrespective of the engaged epitope or its interference with CD137L binding. CD137 perturbation with all these agonist mAbs resulted in Ag and Ab internalization toward an endosomal vesicular compartment. Internalization was observed in activated T lymphocytes from humans and mice, not only in culture but also in Ab-injected living animals. These in vivo experiments were carried out upon systemic i.v. injections with anti-CD137 mAbs and showed CD137 internalization in tumor-infiltrating lymphocytes and in activated human T cells transferred to immunodeficient mice. Efficient CD137 internalization required K63 polyubiquitination and endocytosed CD137-containing vesicles recruited TNFR-associated factor (TRAF) 2 and were decorated with K63 polyubiquitins. CD137 stimulation activates NF-κB through a K63-linked polyubiquitination-dependent route, and CD137-associated TRAF2 becomes K63 polyubiquitinated. Consistent with a role for TRAF2 in CD137 signaling, transgenic mice functionally deficient in TRAF2 showed delayed immunotherapeutic activity of anti-CD137 mAbs. As a whole, these findings advance our knowledge of the mechanisms of action of anti-CD137 immunostimulatory mAbs such as those currently undergoing clinical trials in cancer patients.

The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer

“…Such a trade off in FcγR affinity could also apply to other immunomodulatory antibodies that target TNFRs (CD27, OX40, GITR, CD137), which are also expressed on critical immune cells. In addition it has been shown that for CD137 antibody mediated internalisation of receptor can play a role in driving signalling [29], and it is unclear how changes in Fc isotype might affect this property.…”

Lys63-polyubiquitination by the E3 ligase casitas B-lineage lymphoma-b (Cbl-b) modulates peripheral regulatory T cell tolerance in patients with systemic lupus erythematosus

“…As prior studies have illustrated numerous roles of Lys63-linked (K63) polyubiquitylation in immune responses [39][40][41][42][43][44][45], as well as its relationship with Cbl-b [27], we determined if the deficiency in this E3 ubiquitin ligase could be associated with an abnormal polyubiquitin profile of T regs from SLE patients in a K63-dependent manner. According to our hypothesis, we observed a differential polyubiquitination profile distinguished by a decreased expression of K63 substrates in lupus T regs , but not in effector T cells.…”

“…Lysine-63-specific protein ubiquitination has been implicated in the regulation of signal transduction in immune receptors [39][40][41][42][43][44][45], which is mediated by the E2 ubiquitinconjugating enzyme Ubc13, as shown by multiple studies, and directs conjugated proteins to lysosomes [44]. As Ubc13 inducible knock-out mice specific for T regs display a reminiscent pathological phenotype of the T reg -deficient mice [46], we hypothesized that Cbl-b deficiency would modify the ubiquitin profile of T regs from SLE patients in a K63-dependent manner, which could contribute to resistance to suppression.…”

Nivolumab and Urelumab Enhance Antitumor Activity of Human T Lymphocytes Engrafted in Rag2−/−IL2Rγnull Immunodeficient Mice

“…Fluorochrome-conjugated mAbs to the following human antigens were used: CD3 (UCHT1), CD4 (OKT4), CD8 (RPAT8), CD45 (HI30), FoxP3 (150D), Ki-67 (B56), CD279/PD-1 (EH12.2H7), TIM-3 (F38-2E2), and PD-L1/B7-H1 (29E.2A3) from BioLegend; CD25 (BC96), Eomes (WD1928), and LAG-3 (3DS223H) from eBioscience, CD137 (5D1; ref. 35) and Perforin (deltaG9) from BD Pharmigen. FACSCanto II and FACSCalibur (BD Biosciences) were used for cell acquisition and data analysis was carried out using the FlowJo software (TreeStar Inc.).…”