DNA nanostructure‐programmed intermembrane spacing to modulate T‐cell immunity

1The Key Laboratory of Zhejiang Province for Aptamers and Theranostics, Zhejiang Cancer Hospital, Hangzhou Institute of Medicine (HIM), Chinese Academy of Sciences, Hangzhou, Zhejiang, China 2Molecular Science and Biomedicine Laboratory (MBL), State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Aptamer Engineering Center of Hunan Province, Hunan University, Changsha, Hunan, China 3Institute of Molecular Medicine (IMM), Renji Hospital, Shanghai Jiao Tong University School of Medicine, and College of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai, China


EFFECT OF INTERMEMBRANE SPACING ON TCR SIGNALLING
T-cell-mediated adaptive immunity plays a pivotal role in combating numerous diseases. This process is initiated when the T-cell receptor (TCR) binds to antigenic peptides presented by the major histocompatibility complex (pMHC) on the surface of antigen-presenting cells (APCs). 1 Upon TCR-pMHC-specific binding, a closecontact zone rapidly forms at the interface between the APC and T cell, with an axial distance of approximately 13 nm. 2 This close-contact zone facilitates the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic domains of the TCR complex by lymphocyte-specific protein tyrosine kinase (Lck), triggering a cascade of signalling pathways that lead to T-cell activation. Despite its crucial role in T-cell immunity, there is still controversy regarding the molecular mechanism of how TCR-pMHC binding initiates intracellular ITAM phosphorylation. 3 Previous studies have examined the spatial impact of the close-contact zone on TCR triggering process by genetically manipulating the length of pMHC ligand or other membrane proteins. 4,5 Whereas, these techniques involve modifications to protein structure and expression, and show difficulty to accurately assess the linear correlation between intermembrane distance and TCR signalling strength. While artificially engineered nanointerfaces can provide measurable systems, they have limitations in replicating real APCs and capturing the full complexity of this biological process, as they lack many intrinsic membraneassociated molecules and adaptive morphologies. 6 Additionally, the existing strategies have mainly focused on extending intermembrane spacing, with minimal exploration on the effects of compressing the intermembrane distance. This gap in research leaves room for further investigation into the underlying mechanisms.

PROGRAMMABLE DNA NANOJUNCTIONS FOR MODULATING INTERMEMBRANE SPACING WITH NANOMETER PRECISION
To overcome above limitations, we developed DNA nanojunctions (DNJs) of different sizes, by using cholesterollabelled DNA tetrahedrons (TDNs) as the cell membraneanchoring scaffolds 7 ( Figure 1A). TDNs were constructed by DNA self-assembly, displaying an overhang strand at the top vertex and cholesterol tags at the three bottom  vertices. Unlike conventional genetic engineering strategies, no protein modification was required in our system. The rapid, effective and stable membrane-anchoring capability of TDNs was also demonstrated by our previous work. 8 Meanwhile, the density of TDNs on the cell surface could be easily adjusted by modulating their incubation concentration. To control the intermembrane spacing, we designed three types of DNJs, namely, DNJ-7, DNJ-13 and DNJ-37, corresponding to the theoretical size of 9.6, 13.0 and 26.4 nm, respectively. These DNJs were constructed by hybridising the overhang strands between two TDNs of the same size. Based on total internal reflection fluorescence microscopy (TIRFM) and transmission electron microscopy (TEM) imaging, we showed that these DNJs can precisely tune the intermembrane spacing to the desired values ( Figure 1B,C). This capability allowed to extend, maintain and compress the spatial distance of TCR-pMHC-mediated contact zone in a real system of interaction between APCs and T cells.

POTENTIAL MOLECULAR MECHANISMS OF TCR SIGNALLING
By using DNJs to manipulate the axial dimension of the cell-cell interface, we observed a consistent trend in T-cell signalling amplitude: DNJ-7 > DNJ-13 > DNJ-37 ( Figure 2A). Additionally, we found that at a critical low surface density, DNJ-37, which is larger than the size of the TCR-pMHC complex, could weaken T-cell activation level by increasing the intermembrane distance and reducing CD45 segregation ( Figure 2B,C). On the other hand, DNJ-7, with a smaller size, could compress the intermembrane spacing at the APC-T-cell interface, resulting in a strict exclusion of CD45 and intensified conformational changes of TCR complex. This compression consequently led to a significant enhancement of T-cell activation. While DNJ-13 had a minimal effect on the intermembrane distance, it could effectively stabilise the TCR-pMHC-mediated cellular interface, leading to prolonged ITAM phosphorylation and stronger T-cell stimulation compared to DNJ-free control groups.

FUTURE OUTLOOK
These findings provide direct evidence for the pivotal role of the axial size of the contact zone in T-cell triggering. Notably, reducing the intermembrane distance at the APC-T-cell interface to less than 10 nm could significantly enhance T-cell activation, thus expanding the opportunities for the study of T-cell immunity and further applying for the development of novel therapies for immune-related disorders. Considering that cell-cell interactions through direct contact are prevalent in organisms and the interface dimension is important in modulating various signal transduction processes, this membrane-anchored DNA nanoplatform, with its high programmability, controllability, ease of operation, and good biocompatibility, 9,10 offers a promising way for studying these biochemical events. Moreover, it exhibits significant potential in promoting targeted T-cell responses in the field of immunotherapy.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare they have no conflicts of interest.