Although the importance of CD40 was quickly recognized, it was less clear how it interacted with its ligand to convey a stimulus to responding cells. Crystallographic and molecular modeling studies produced a model in which the CD40L homotrimer fits into the three chains of the CD40 receptor (21
). However, the intracytoplasmic domain of CD40 does not interact with kinases or G proteins which raised questions about how ligand binding leads to downstream signaling. This problem was partially solved with the identification of adapter molecules called TNF Receptor Activation Factors or TRAFs (23
). The cytoplasmic tails of CD40 can form a supramolecular signaling complex composed of many TRAFs that in turn leads to the activation of NF-κB and other transcription factors. The next question was how CD40L/CD40 engagement leads to these downstream events.
The current model of CD40 activation is based on the idea that clustering of the receptor is needed to assemble the supramolecular intracytoplasmic signaling complex. Hard evidence for this model comes from studies of Fas, a related TNFRSF receptor. Using cultured cells engineered to express a fusion protein between Fas and yellow fluorescent protein (YFP), Siegel et al. studied the effects of engaging Fas with Fas ligand (FasL, CD95L). Schneider et al had previously shown that the effects of membrane FasL could be replicated by FLAG-tagged trimers of soluble FasL, but only if the trimers were crosslinked by anti-FLAG antibody (26
). Using Fas-YFP responder cells, Siegel et al showed that exposure to crosslinked FasL led to the rapid clustering of Fas-YFP into lipid rafts in the membrane. Under the fluorescent microscope, these receptor clusters were visualized as bright spots, reflecting the acronym given to them as Signaling Protein Oligomeric Transduction Structures or SPOTS (27
While a similar experiment has not yet been conducted for CD40, compatible data has been provided by Spencer et al. These investigators engineered cells in which the full transmembrane CD40 molecule was replaced with an engineered protein lacking the entire CD40L-binding extracellular domain but instead expressing a membrane-anchoring motif, the intracellular domain of CD40 needed for binding TRAFs, and two FKBP-related motifs capable of binding an FK-506-like small molecule. A expression cassette for this construct was then transduced into cultured DCs using an adenoviral vector. Following this, the investigators added AP1903, a small dumbbell-shaped chemical containing two FK-506-like moieties. This chemical resulted in the inducible clustering of the engineered CD40 intracytoplasmic domains and led to full DC activation, obtained entirely without the CD40 extracellular domain or CD40L engagement (28
). Combined with a tumor antigen, this system is now being tested in a clinical trial of prostate cancer immunotherapy (NCT00868595). In the context of this review, this work shows that clustering of the CD40 intracytoplasmic signaling domain is sufficient to activate DCs to initiates immune responses. Looking at the cell from the outside, clustering of CD40 receptor in the membrane by a ligand or antibody is the natural process whereby CD40 intracellular domains become clustered and capable of initiating downstream responses.
With this in mind, it is now possible to understand how agonistic anti-CD40 antibodies act to stimulate immune cells. An early clue came from studies of anti-CD40 antibodies as a stimulus for B cell proliferation. Although these antibodies were known to produce partial stimulation of B cells (29
), they did not lead to long-term B cell proliferation. Instead, Banchereau et al. found that it was necessary to add three components to the B cell cultures: anti-CD40 antibodies; a fibroblast line engineered to express the Fc receptor (FcR) for the antibody; and IL-4. Using this “B cell system,” cultured B cells could be massively expanded without the use of lectins or other artificial agents (30
). Yet it remained unclear why FcR-bearing fibroblasts were needed in this culture system. Evidently, simple immobilization of anti-CD40 antibody on culture plates or beads is unable to convey the same type of stimulus as antibody mounted on the surface of the FcR-bearing cells.
An important step in understanding agonistic anti-CD40 antibodies came in 2011 when the important role of FcRs was recognized. Similar to the Banchereau B cell system, four groups found that agonistic anti-CD40 antibodies only functioned in the presence of cells expressing IgG-binding FcγRs (31
). Ironically, the best FcγR is FcγRIIB, which is usually thought of as an inhibitory FcγR. Anti-CD40 monoclonal antibodies (MAbs) that bind to FcγRIIB exhibit strong CD40-stimulating activity but only if a cell bearing FcγRIIB is adjacent to the CD40-bearing cell to be stimulated (). This indicates an important spatial restriction on the effectiveness of anti-CD40 agonistic antibodies: a CD40-bearing cell that is not adjacent to an FcγR-bearing cell might not be effectively stimulated by these antibodies. Another scenario would be if the CD40-bearing cell itself also expressed FcγRIIB (a so-called cis
effect) and it has been proposed that this would operate on B cells that are known to express both CD40 and FcγRIIB (34
). However, the earlier results of Banchereau et al. in the B cell system (30
) indicate that the FcR must be on an adjacent cell and not on the CD40-bearing B cell that is itself being stimulated by the agonistic anti-CD40 antibody.
Agonistic anti-CD40 MAbs require a nearby FcγR-bearing cell to cluster CD40 and induce a signal