How the engagement of T-cell receptor (TCR) to antigenic peptide-loaded major histocompatibility complex (pMHC) on antigen-presenting cells (APCs) initiates intracellular signaling cascades in T cells is not well understood. The dimension of the cellular contact zone is regarded as one of the key determinants. Upon TCR-pMHC ligation, multiple accessory molecules are recruited to form regions of close contact at the interface between APCs and T cells. The small size of the TCR-pMHC complex holds the cellular contact at a short intermembrane distance of ~13 nm. Although the spatial impact of the contact zone on T cell signaling has been reported, these strategies mainly relied on reductionist models or involved changes in protein structure. It is difficult to achieve a relatively comprehensive map of this complex biological process. To address these issues, we used cholesterol-conjugated TDNs as the membrane-anchored scaffold and constructed DNA nanojunctions (DNJs) of distinct sizes to manipulate the intermembrane spacing around the TCR-pMHC dimension in a real APC-T cell interaction system, with no need to involve protein modification (Figure 1).
Figure 1. Schematic illustration of DNJ-based precise manipulation of the close contact zone for studying TCR signaling
These DNJs were formed through DNA hybridization between two membrane-anchored tetrahedral DNA nanostructures (TDNs), which were constructed by DNA self-assembly with a 17-nt overhang at the top vertex and cholesterol tags at the three bottom vertices. To precisely tune the intermembrane distance, three types of DNJs, termed as DNJ-7, DNJ-13, and DNJ-37, were constructed by combining two complementary TDNs of the same size, and their theoretical heights were calculated to be 9.8, 13.0, and 26.4 nm, respectively. We first verified the successful construction of these three TDNs and their good performance for effective cell-surface engineering. Based on variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) and transmission electron microscopy (TEM) imaging, the intermembrane spacing generated by DNJ-7, DNJ-13 and DNJ-37 was matched well with the expected value (Figure 2).
Figure 2. VA-TIRFM imaging and TEM imaging of the intermembrane spacing mediated with DNJ-7, DNJ-13, and DNJ-37.
We next tested the impact of DNJs on T cell activation using the cellular interaction system of OVA peptide/LPS-pretreated APCs and OT-I T cells. At a critical low surface density of TDNs (~180 constructs per μm2), DNJ-37 induced obvious inhibition on T cell signaling, compared with the normal activation group. In contrast, enhancement on T cell activation were caused by DNJ-7 and DNJ-13, while DNJ-7 performed the best. To further test the impact of intermembrane spacing on T cell signaling under the premise of stable and equal antigenic ligation, we constructed an artificial T cell activation system with precise control over the number of ligated TCR complex in each group. A similar T cell stimulation tendency of DNJ-7 > DNJ-13 > DNJ-37 was obtained, revealing that the DNJ effect on T cell activation was imposed through modulating the intermembrane spacing (Figure 3).
Figure 3. Modulating T cell activation with DNA nanojunction-mediated intermembrane distance
We also studied the possible molecular mechanism of T-cell receptor signaling. Our experiment results showed that ZAP70 could be rapidly accumulated at the cytoplasmic microdomain of the ligated TCR complex, and these small clusters showed poor colocalization of CD45, as consistent with the kinetic segregation model. Images of TIRFM confirmed that CD45 was excluded in the periphery of the ligated TCR complex and displayed a ring-like pattern. The diameter of the CD45 ring was shrunk by nearly three times after incorporation of DNJ-37, revealing that DNJ-37 could facilitate CD45 diffusion into the contact zone, agreeing well with its inhibitory effect on T cell stimulation. For the contact zone mediated with DNJ-13, negligible change on CD45 distribution was observed. DNJ-7, on the other hand, allowed the strict exclusion of CD45 with the ring diameter increased by ~1.5 fold, providing an explanation for the superior performance of DNJ-7 in promoting T cell activation. Interestingly, the occupancy of DNJs within the contact zone displayed a size-inverse tendency of DNJ-7 > DNJ-13 > DNJ-37.
We next used TEM to directly visualize the interface between APCs and OT-I T cells. The contact zone mediated by DNJ-7 showed a spindle-shaped pattern, in which the axial distance in the middle was ~10.02 nm and approached to its theoretical size of ~7.8 nm at both ends. For DNJ-13, uniform intermembrane gaps with a dimension similar to that of the TCR-pMHC complex (~12.8 nm) were observed. With incorporation of DNJ-37, however, the intermembrane spacing of the middle was held at ~13.60 nm under the TCR-pMHC ligation, while the two ends were enlarged to more than 20 nm, showing a dumbbell-like shape. Based on these results, we speculated that DNJ-7 with its capacity to axially compress the contact zone might generate additional mechanical forces to aggravate conformational change of the TCR complex. By combining this force effect with strict CD45 exclusion, DNJ-7 was able to effectively promote T cell signaling. While DNJ-13 induced little effect on the intermembrane distance, it could stabilize the pMHC-engaged interface between APCs and T cells, thus enabling prolonged ITAM phosphorylation for stronger T cell stimulation. These findings proved that T cell signaling was also regulated by the stability of the close contact zone, consistent with the model of serial triggering. With the capability to stretch the contact zone in the premise of not affecting antigenic ligation, DNJ-37 could induce a pulling force to strengthen TCR signaling though, it might also facilitate the access of CD45 to the ligated TCR complex, where the latter effect overwhelmed the former one, as inferred from its inhibitory effect on T cell activation.
In summary, using cholesterol-conjugated TDNs as the membrane-anchored scaffold, we constructed DNJs of distinct sizes for precise control over the interface between APCs and T cells. Based on this programmable membrane-anchored DNA nanoplatform, we have, for the first time, manipulated the intermembrane spacing of the close-contact zone around the TCR-pMHC dimension in a real APC-T cell interaction system. Our study supported direct evidence that the axial dimension of the close contact zone plays an important role in T cell triggering. With the ability to shorten the APC-T cell interface, we showed that a wider research window could be applied to the study of T cell immunity.
The link: https://www.nature.com/articles/s41565-023-01333-2
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