RESUMO
The CD3/T cell receptor (TCR) complex is responsible for antigen-specific pathogen recognition by T cells, and initiates the signaling cascade necessary for activation of effector functions. CD3 agonistic antibodies are commonly used to expand T lymphocytes in a wide range of clinical applications, including in adoptive T cell therapy for cancer patients. A major drawback of expanding T cell populations ex vivo using CD3 agonistic antibodies is that they expand and activate T cells independent of their TCR antigen specificity. Therapeutic agents that facilitate expansion of T cells in an antigen-specific manner and reduce their threshold of T cell activation are therefore of great interest for adoptive T cell therapy protocols. To identify CD3-specific T cell agonists, several RNA aptamers were selected against CD3 using Systematic Evolution of Ligands by EXponential enrichment combined with high-throughput sequencing. The extent and specificity of aptamer binding to target CD3 were assessed through surface plasma resonance, P32 double-filter assays, and flow cytometry. Aptamer-mediated modulation of the threshold of T cell activation was observed in vitro and in preclinical transgenic TCR mouse models. The aptamers improved efficacy and persistence of adoptive T cell therapy by low-affinity TCR-reactive T lymphocytes in melanoma-bearing mice. Thus, CD3-specific aptamers can be applied as therapeutic agents which facilitate the expansion of tumor-reactive T lymphocytes while conserving their tumor specificity. Furthermore, selected CD3 aptamers also exhibit cross-reactivity to human CD3, expanding their potential for clinical translation and application in the future.
RESUMO
State-of-the-art super-resolution microscopy allows researchers to spatially resolve single proteins in dense clusters. However, accurate quantification of protein organization and stoichiometries requires a general method to evaluate absolute binder labeling efficiency, which is currently unavailable. Here we introduce a universally applicable approach that uses a reference tag fused to a target protein of interest. By attaching high-affinity binders, such as antibodies or nanobodies, to both the reference tag and the target protein, and then employing DNA-barcoded sequential super-resolution imaging, we can correlate the location of the reference tag with the target molecule binder. This approach facilitates the precise quantification of labeling efficiency at the single-protein level.
RESUMO
Dendritic cells (DCs) regulate immune priming by expressing programmed death ligand 1 (PD-L1) and PD-L2, which interact with the inhibitory receptor PD-1 on activated T cells. PD-1 signaling regulates T cell effector functions and limits autoimmunity. Tumor cells can hijack this pathway by overexpressing PD-L1 to suppress antitumor T cell responses. Blocking this inhibitory pathway has been beneficial for the treatment of various cancer types, although only a subset of patients responds. A deepened understanding of the spatial organization and molecular interplay between PD-1 and its ligands may inform the design of more efficacious nanotherapeutics. We visualized the natural molecular PD-L1 organization on DCs by DNA-PAINT microscopy and created a template to engineer DNA-based nanoclusters presenting PD-1 at defined valencies, distances, and patterns. These multivalent nanomaterials were examined for their cellular binding and blocking ability. Our data show that PD-1 nano-organization has profound effects on ligand interaction and that the valency of PD-1 molecules modulates the effectiveness in restoring T cell function. This work highlights the power of spatially controlled functional materials to unravel the importance of multivalent patterns in the PD-1 pathway and presents alternative design strategies for immune-engineering.