TNF receptor agonists induce distinct receptor clusters to mediate differential agonistic activity

Monoclonal antibodies (mAb) and natural ligands targeting costimulatory tumor necrosis factor receptors (TNFR) exhibit a wide range of agonistic activities and antitumor responses. The mechanisms underlying these differential agonistic activities remain poorly understood. Here, we employ a panel of experimental and clinically-relevant molecules targeting human CD40, 4-1BB and OX40 to examine this issue. Confocal and STORM microscopy reveal that strongly agonistic reagents induce clusters characterized by small area and high receptor density. Using antibody pairs differing only in isotype we show that hIgG2 confers significantly more receptor clustering than hIgG1 across all three receptors, explaining its greater agonistic activity, with receptor clustering shielding the receptor-agonist complex from further molecular access. Nevertheless, discrete receptor clustering patterns are observed with different hIgG2 mAb, with a unique rod-shaped assembly observed with the most agonistic mAb. These findings dispel the notion that larger receptor clusters elicit greater agonism, and instead point to receptor density and subsequent super-structure as key determinants.

T-Cell Receptor Clustering Methods Used for Single Cell Analysis: Potential Application in Oncology

We know that T-cell activation and effector function is integral for cancer cell destruction in immunotherapeutic treatment in oncology. The fundamental behaviour of T-cells at the time of activation is poorly understood but is likely to be central to this action. Cellular clustering occurs on at least two levels: gathering of multiple mobile cells of similar type, and aggregation between different cell types. Receptors are implicated in both of these processes. Analysis of receptor clustering is a different process whereby receptors form clusters on the cell membrane surface and can be studied to determine their relationship to immune activation. Receptor clustering has been shown to occur in some (perhaps all) cell types, but little is known about this phenomenon, particularly in T-lymphocytes. T-Cell Receptors (TCRs) which are important for the activation of T-lymphocytes. T-cell receptors, also known as cluster of differentiation 3 (CD3) molecules, bind specific antigen to create intracellular signaling in the process of T-cell activation as part of the immune response. The detail of how TCRs physically behave on the T-lymphocyte surface and specifically how they cluster remains unclear, including during the early phases of initiation of immune activation in the T-cell response. The aim of this review is to investigate how receptor clustering that has been studied, can be more effectively studied in the future and what the current evidence suggests about TCR clustering/T-cell activity relationships.