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A recognized triumph in immunotherapy has been the development of neutralizing antibodies and Fc fusion proteins which inhibit the binding of tumour necrosis factor (TNF) to one or both of its receptors-TNF receptor 1 (TNFR1) and TNFR2. As a primary function of the TNF superfamily molecules is to regulate cell survival, inhibition of these interactions prevents the activation of signaling pathways downstream of the TNFRs, thereby minimizing the pro-inflammatory programme which initiates in immune cells and decreasing the pathology of autoimmune and inflammatory diseases.
Overview of TNF and TNFR superfamily
To date, a total of 19 ligands and 30 receptors have been identified as belonging to the TNF and TNFR superfamilies. In many cases, one ligand can bind several different receptors, thus leading to distinct signaling responses; conversely, different ligands often share the same receptor (Figure 1). The expression patterns of the TNFRs vary considerably, but in general, they are widely expressed on immune cells and are important in lymphocyte development. TNFRs are most often expressed at the cell surface, but another layer of signaling modulation can occur through protein cleavage at the membrane by metalloproteases or by alternative splicing to generate soluble forms of the extracellular domains (ECDs). The generation of soluble receptors plays an important role in buffering receptor activation. The TNF ligands are type II transmembrane proteins which can be active in this form, or also as soluble cytokines generated by proteolytic cleavage from the membrane or by intracellular cleavage via furin convertase. Lacking endogenous enzymatic activity, TNFRs rely on ligand-induced conformational changes to recruit adaptor molecules that initiate downstream signaling pathways.
Figure 1. TNFR superfamily members with disease-related amino acid substitutions highlighted on TNFR1, TACI, Fas and EDAR.
Some interactions that occur between TNF molecules and their receptors have gained prominence based on studies of animal models of immune function and disease. These studies indicate that interactions between TNF–TNFR molecules can positively regulate T-cell responses and mediate crosstalk between T cells and other cell types. The interactions between OX40 (also known as CD134 and TNFRSF4) and OX40 ligand (OX40L; also known as CD252 and TNFSF4) , 4-1BB (also known as CD137 and TNFRSF9) and 4-1BBL (also known as TNFSF9), CD27 (also known as TNFRSF7) and CD70 (also known as TNFSF7), and TL1A (also known as TNFSF15) and death receptor 3 (DR3; also known as TNFRSF25), have been the most extensively studied in terms of their direct effects on CD4+ and CD8+ T cells. In support of the importance of these interactions in controlling T-cell function, recent results have shown that promoting or blocking each one of these interactions in animal models of disease markedly affects the outcome of the disease.
Functional effects in immune cells
Each of the four TNF–TNFR interactions can stimulate conventional T cells and APCs, mediate communication between CD4+ and CD8+ T cells and promote immune responses. These ligand–receptor pairs also mediate interactions between NK cells and T cells, between NKT cells and APCs presenting lipid antigens, and between T cells and other types of immune or tissue cell.
Interactions between individual TNF–TNFR pairs control T-cell responses in two ways. First, they regulate the frequency of effector and/or memory CD4+ or CD8+ T cells that can be generated from naive T cells in response to antigen stimulation by providing proliferative and survival signals either directly to the T cells or to the APCs with which they interact. These molecules also regulate the frequency of effector memory T cells which are generated in recall responses. Second, they control T-cell function directly by promoting the production of cytokines such as IL-4 and IFNγ, or indirectly through stimulating the production of pro-inflammatory cytokines, such as IL-1 and IL-12, by professional or non-professional APCs. The interactions between these TNF superfamily members are primarily thought to deliver co-stimulatory signals, as their effects largely depend on antigen recognition and TCR signaling. Several models for how these interactions might contribute to T-cell responses are shown in Figure 2.
Figure 2. Control of T-cell proliferation by cooperative and sequential TNF–TNFR interactions.
The expression of OX40, CD27, 4-1BB and DR3 is either constitutive (in mice) or rapidly induced (in humans) on natural and inducible CD4+ or CD8+ TReg cells. Natural TReg cells express forkhead box P3 (FOXP3) and are selected in the thymus, whereas inducible TReg cells can differentiate from naive CD4+ or CD8+ T cells in the periphery in response to antigen and may or may not express FOXP3. Increasing the numbers or activity of TReg cells results in the suppression of immune responses, which is beneficial for the treatment of autoimmune and inflammatory conditions. By contrast, decreasing the numbers and function of TReg cells can enhance both innate and adaptive immune responses, which is beneficial for the treatment of cancer. Studies using mouse TReg cells have shown that ligation of the TNF superfamily members OX40 and 4-1BB affect these T-cell subsets (Figure 3).
Figure 3. TNF–TNFR family interactions regulate many cell types to amplify inflammation.
Targeting co-stimulatory and co-inhibitory receptors expressed by immune cells is a promising new approach for treating cancer. The FDA approval of the monoclonal antibody (mAb) against the inhibitory molecule CTLA-4 (ipilimumab) demonstrates the proof of principle that enhancing T cell function can have therapeutic effects in cancer patients. When TNF and TNFR superfamily protein are ligated either by their cognate receptor or by agonist antibodies, a broad range of cellular outcomes has been reported ranging from cell differentiation, proliferation, apoptosis and survival to increased production of cytokines and chemokines. In addition, the unique expression of some of the TNFR superfamily members on antigen-specific T cells has made these molecules ideal targets for novel immunotherapies.
There are many other costimulatory proteins that are required to generate optimal effector and memory T cells following antigen encounter. Several of these costimulatory proteins are members of the TNFR superfamily. Initially described to be expressed on activated T and B lymphocytes and APCs, ligation of some of these receptors is shown to promote cell division and survival, differentiation, maturation, and provide signals directly to T cells (Table 1). Because of the unique T cell activating features of these receptors, many groups have targeted TNFRs with agonist mAbs to enhance lymphocyte function, particularly in the context of tumor immunotherapy.
Accumulating evidence has shown that many TNF superfamily molecules have a central role in immune regulation, immune-mediated diseases and cancer. And both proapoptotic and costimulatory TNFL/TNFR ligand/receptor pairs hold considerable promise for immunotherapy of cancer, with various agonistic TNFR antibodies and recombinant soluble TNFLs poised for or undergoing clinical evaluation. Indeed, the host of ongoing preclinical studies and promising early clinical results suggests that targeting of the TNFL/TNFR axis will become part of clinical practice in the near future. However, the ubiquitous activation of proapoptotic or co-stimulatory TNFR signaling can have severe side-effects, as evidenced by the early clinical experience with systemic TNF infusion as well as the recent experience with systemic agonistic 4-1BB antibody treatment. Thus, tumor-restricted activation is being pursued in order to fully capitalize on the therapeutic potential of this regulatory axis.