Figure 1. 4-1BB/4-1BBL signaling pathway
TCR ligation of T cells transiently induces expression of 4-1BB (also known as CD137 or TNFRSF9, encoded by TNFRSF9), a fellow of the TNF receptor superfamily. 4-1BB was originally identified in mice by a modified differential screening procedure. The human homologue of 4-1BB (CD137) was cloned from activated human T-cell leukemia virus type 1-transformed human T-lymphocytes library. Human 4-1BB resides on chromosome 1p3613, in a cluster of related genes including TNFR2, CD30, OX40 and TAMP/Apo3, which have been shown to be mutated in several malignancies. From previous studies, we can know that the human 4-1BB contains 255 a.a. with two potential N-linked glycosylation sites. Hydrophobicity analysis predicted that 4-1BB has a putative signal peptide, an extracellular domain, a transmembrane region and a short intracellular domain. The molecular weight of the protein was calculated to be 27 kDa and was shown to be 60% identical to murine 4-1BB. In the cytoplasmic domain, five regions of a.a. sequences were conserved between mice and human, indicating that these residues might be important for 4-1BB function.
The ligand of 4-1BB was first confirmed in the EL4 cell line through its binding with a fusion 4-1BB/Fc protein. The 4-1BBL gene was then cloned through the screening of an EC1 cDNA expression library. Human 4-1BBL was first isolated in 1994 by Alderson and his colleagues. They used a fusion protein consisting of the extracellular portion of human 4-1BB coupled to the Fc region of human immunoglobulin (Ig) G1 to identify and clone the gene for human 4-1BBL from an activated CD4+ T-cell clone using a direct expression cloning strategy. Sequence analysis showed that human 4-1BBL consisted of 254 a.a. and shared a 36% identity with murine 4-1BBL. Fluorescence in situ hybridization analyses or FISH analysis showed that the human 4-1BBL gene mapped to chromosome 19 in the region 19p13.3. Hydrophobicity analysis predicted that human 4-1BBL has a signal hydrophobic domain, while the absence of a signal sequence suggested that 4-1BBL was a type II membrane glycoprotein with a single predicted transmembrane segment.
The function of pathway
4-1BB is a co-stimulatory molecule with roles in expansion, acquisition of effector function, survival, and development of T cell memory. In addition to being expressed on activated T cells, 4-1BB is also expressed on several cell types in the hematopoietic lineage, as well as endothelial and epithelial cells. 4-1BBL (TNFSF9), the primary ligand of 4-1BB, is expressed predominantly on professional APC populations, B cells, macrophages and other cell types and binds with 4-1BB to induce signaling through TRAF1 and TRAF2 to activate the NF-κB, AKT, p38 MAPK, and ERK pathways. These signaling pathways induce the expression of survival genes encoding survivin, Bcl-2, Bcl-XL, and Bfl-1 and decrease the expression of pro-apoptotic Bim. Therefore, 4-1BB/4-1BBL signaling pathway can promote different type cells to live. Interestingly, the phenotype in response to virus challenge differs between 4-1BB and its ligand. For example, 4-1BBL-deficient mice show reduced accumulation of memory CD8+ T cells, whereas 4-1BB deficient mice have decreased memory but enhanced accumulation of acute CD8+ T cells. This type of result implicates bidirectional signaling or a distinct receptor signaling mechanism. Other models of infectious disease have shown that the effect of 4-1BB blockade depends upon the context and disease features. For instance, infections that are quickly cleared or produce minimal inflammation are often unaffected by 4-1BB deficiency, whereas chronic or highly inflammatory disease requires 4-1BB for memory development and/or viral clearance.
4-1BB has also been involved in autoimmune disorders and graft versus host disease (GVHD). Studies in multiple models of inflammatory and autoimmune disease have all demonstrated a reduced disease severity following therapy that targets the 4-1BB-4-1BBL signaling interaction. Antibody blockade or genetic knockout of 4-1BB has improved disease pathology and decreased production of cytokines by CD4+ T cells. Clinical studies have demonstrated an increase in serum levels of soluble 4-1BB in patients with multiple sclerosis and rheumatoid arthritis, suggesting that 4-1BB might be a useful biomarker for disease severity, as well as a target for clinical trial. Interestingly, stimulation of 4-1BB signaling with an agonist monoclonal antibody (mAb) also ameliorates allograft response and autoimmune disorders in preclinical models. Agonist 4-1BB antibodies appear to work both by inducing apoptosis of over-stimulated, pathogenic CD8+ T cells and by expanding indoleamine 2,3-dioxygenase-expressing DCs that induce differentiation of suppressive Treg cell populations. Paradoxically, stimulation of 4-1BB with agonist antibodies also inhibits inflammation in many murine models of autoimmunity, which may be due to augmenting regulatory CD8 T cell activity and/or driving death of pathogenic CD4 T cells. These results indicate that the context of 4-1BB blockade or stimulation will be essential for identifying the best therapeutic approach for each disorder. 4-1BB may also control sepsis and biliary cirrhosis. 4-1BB-deficient mice exhibit lower mortality in a model of sepsis driven by cecal ligation and puncture, and similar results are produced by blocking 4-1BBL in wild-type mice. In patients with primary biliary cirrhosis, there is additionally a positive correlation between expression of soluble and membrane 4-1BBL and mRNA for 4-1BBL, with serum levels of disease markers such as bilirubin, γ-glutamyltransferase, and interleukin (IL) -18.
Besides, stimulating 4-1BB has also been an attractive candidate for application to cancer immunotherapy, particularly because 4-1BB agonists can enhance the rescue of T cells tolerized by chronic antigen exposure and reduce the burden of established tumors in preclinical models. Recent studies in murine models have suggested that using a lower dose of agonist anti-4-1BB mAb in combination with another immune modulator, such as anti-PD-1 or anti-CTLA-4 mAb, might be active against tumors without inducing toxicity. The signaling domain of 4-1BB is also a common component of the chimeric antigen receptor (CAR) construct for T cell immunotherapy in clinical trials and development. This approach uses genetic engineering to introduce a tumor-specific antibody fragment linked to intracellular TCR CD3ζand 4-1BB signaling domains to activate cytotoxic activity of CD8+ T cells after antibody-antigen recognition. Use of the 4-1BB signaling domain leads to prolonged survival of CAR T cells in vitro and the development of a central-memory phenotype associated with increased oxidative metabolism. Importantly, incorporation of 41BB in the CAR construct avoids the toxic effects associated with agonist mAb therapy.