The immune regulatory effects of tetrahedral framework nucleic acid on human T cells via the mitogen‐activated protein kinase pathway

Abstract Objectives Autoimmune diseases are a heterogeneous group of diseases which lose the immunological tolerance to self‐antigens. It is well recognized that irregularly provoked T cells participate in the pathological immune responses. As a novel nanomaterial with promising applications, tetrahedral framework nucleic acid (TFNA) nanostructure was found to have immune regulatory effects on T cells in this study. Materials and Methods To verify the successful fabrication of TFNA, the morphology of TFNA was observed by atomic force microscopy (AFM) and dynamic light scattering. The regulatory effect of TFNA was evaluated by flow cytometry after cocultured with CD3+ T cells isolated from healthy donors. Moreover, the associated signaling pathways were investigated. Finally, we verified our results on the T cells from patients with neuromyelitis optica spectrum disorder (NMOSD), which is a typical autoimmune disease induced by T cells. Results We revealed the alternative regulatory functions of TFNA in human primary T cells with steady status via the JNK signaling pathway. Moreover, by inhibiting both JNK and ERK phosphorylation, TFNA exhibited significant suppressive effects on IFNγ secretion from provoking T cells without affecting TNF secretion. Similar immune regulatory effects of TFNA were also observed in autoreactive T cells from patients with NMOSD. Conclusions Overall, our results revealed a potential application of TFNA in regulating the adaptive immune system, as well as shed a light on the treatment of T cell–mediated autoimmune diseases.


| INTRODUC TI ON
Autoimmune diseases represent a family of complex diseases, which are characterized by the dysregulation of the host immune system leading to the damage of self-organs. 1 As the major components of the adaptive immune system, T cells recognize autoantigens and play important roles in promoting antigen-specific immune responses, thus triggering disease onset. It is well documented that those irregular provoked T cells also associated with cytokine and chemokine cascades, which might cause target organ damages, as well as disease progression and poor prognosis of patients. 2 Clinically, other than biological therapy, mounts of smallmolecular drugs are currently used in preventing and eliminating T cell-mediated autoimmune responses, including cell proliferation inhibitor cyclosporine A (CsA), IMPDH inhibitor mycophenolate mofetil, and lymphocyte homing inhibitor fingolimod. [3][4][5] Nevertheless, the above-mentioned immunosuppressive strategies exhibit a less selective manner and are often found to break the homeostasis of the host immune system. 6 Therefore, it is urgent to develop an efficient way not only decreases specific immune response induced by autoreactive T cells, but also protects regular host immune responses.
As a novel nanoscale material, tetrahedral framework nucleic acid (TFNA) is composed of four editable isometric single-strand DNA (ssDNA) molecules, which could self-assemble and form a stable three-dimensional DNA nanostructure with excellent endocytotic properties. 7,8 Additionally, the multi-biological activities of TFNA have already been revealed. [9][10][11][12] For instance, TFNA could polarize tissue-resident macrophages from M1 to M2 phenotypes that promotes the recovery of bisphosphonates-mediated jaw osteonecrosis in mice. 13 Similarly, TFNA significantly down-regulates IL-1β and IL-6 secretion by LPS-induced macrophages in attenuating bacterial inflammation. 14 Moreover, TFNA was also found to attenuate immune cell recruitment via blocking NF-κB activation by epithelial cells. 15 However, in spite of these remarkable functions in modulating innate immune responses at animal models, whether TFNA exhibits similar effects in human adaptive immunity remains unrevealed. Here, we aim to investigate the immune regulatory activities of TFNA in different types of primary human T cells under both physiological and pathological circumstances, and we also corroborate the negative regulatory effect of TFNA on T cells from neuromyelitis optica spectrum disorder (NMOSD) patients.

| Synthesis and characterization of tetrahedral framework nucleic acid
The TFNA nanostructure was synthesized based on a previous method. 16,17 Briefly, four specifically designed S1-S4 ssDNA molecules (Table 1) were synthesized by Takara Biotechnology. Equal concentrations of four ssDNA molecules were mixed in TM buffer (10 mM Tris-HCl, pH 8.0; 50 mM MgCl 2 ). The buffer was rapidly warmed to 95°C for 10 minutes and then rapidly cooled to 4°C for 20 minutes. The successful synthesis of TFNA was confirmed by 8% polyacrylamide gel and capillary electrophoreses. Dynamic light scattering using a Zetasizer Nano ZS90 (Malvern Instrument Ltd) was applied to examine the average size of TFNA. Atomic force microscopy measurements were performed in the tapping mode using a SPM-9700 instrument (Shimadzu).

| Isolation of T cells
Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood drawn from healthy volunteers and patients with NMOSD

| Cellular uptake of tetrahedral framework nucleic acid
To detect the uptake of TFNA in T cells, TFNA was fluorescently

| Flow cytometry
Cells were harvested and washed before staining for extracellular

| Western blotting
A whole-cell lysis kit (KeyGen) was used to harvest the total protein. Consecutively, 10% SDS-polyacrylamide gel electrophoresis was performed to separate target proteins, which were then trans- Quantification was performed by using the ImageJ software.

| Statistical analysis
Statistical analyses were performed using GraphPad Prism (Version 6.0). All data were presented as the mean ± SD, with n ≥ 3 replicates.
Means for all data were compared using a paired one-way ANOVA.

| Characterization of Synthesized TFNA
After gently mixing four selected ssDNA molecules in TM buffer, each ssDNA could automatically form a triangle by sharing the sides with other three paired ssDNA molecules and finally form the designated TFNA ( Figure 1A). The molecular weights of assembled TFNA were examined by polyacrylamide gel electrophoresis assay and capillary electrophoresis analysis. In consistent with its theoretical structure, the molecule weights of each ssDNA and TFNA are approximately 40 bp and 180 bp, respectively ( Figure 1B,C). Moreover, the size distribution and morphology of our generated TFNA were further identified by dynamic light scattering assay and atomic force microscopy ( Figure 1D,E). Accordingly, the TFNA was observed to have the particle size of approximately 10 nm with 2.2 nm in height ( Figure 1D.E). Altogether, above results confirmed that we successfully obtained the self-assembled TFNA with designated size and physical morphology.

| Identification of the endocytic efficiency
Previously studies revealed that TFNA could be rapidly taken up by cells through the caveolin-dependent pathway and subsequently transport to lysosomes. 19 To identify the endocytosis of TFNA in T cells, primary human T cells were obtained from peripheral blood of healthy donors and following treated by Cy5-labeled TFNA (TFNA-Cy5). After 6 hours co-cultivation, Cy5 signal could be detected by confocal laser scanning microscopy. As shown in

| IFNγ level was reduced in primary human T cells after TFNA treatment
To investigate whether TFNA could regulate the phenotype and functions of primary human T cells, we conducted an in vitro experiment to evaluate the activities of T cells after TFNA treatment.
T cells were sorted using magnetic nanobeads from peripheral blood However, the elevation of the Treg cell subgroup was only monitored in the CsA-treated group ( Figure 4B). Overall, TFNA down-regulated IFNγ secretion from T cells without changing their phenotypes.
Nevertheless, despite the surface marker expression, CsA showed a robust effect in eliminating both IFNγ and TNF secretion from CD8+ T cells ( Figure 4C), but TFNA only contributed to decrease the IFNγ level ( Figure 4D). Additionally, granzyme B expression in CD8+ T cells was affected by neither TFNA nor CsA ( Figure 4D). Taken together, TFNA displayed remarkable regulatory roles in restraining IFNγ secretion in both CD4+ and CD8+ T cells at steady status.

| Tetrahedral framework nucleic acid downregulates IFNγ secretion by inhibiting JNK phosphorylation
We further investigated the phosphorylation levels of -Jun Nterminal kinase (JNK), p38 and extracellular signal-regulated kinase (ERK; Figure 4E,F). It is well-recognized that JNK, p38, and ERK be-

| Tetrahedral framework nucleic acid exhibits regulatory effects in eliminating activated T cells from patients with Neuromyelitis optica spectrum disorder
Neuromyelitis optica spectrum disorder represents a relapsing autoimmune disease that preferentially affects the central nervous system including the brain, optic nerve and spinal cord where patients suffer from severe visual impairment and motor disability. 21 Recent studies indicated that autoreactive T cells played important roles in NMOSD relapsing and progression. 22 Here, in order to further dissect the regulatory roles of TFNA, T cells were sorted from peripheral blood of admitted NMOSD patients with the acute phase from April 2019 to January 2020 at the Department of Neurology, West China Hospital (Table 2).
In consistent with our previously studies, after 12 hours ex vivo cultivation, TFNA illustrated a robust effect in blocking IFNγ secretion from Th1 cells, whereas Th2 cell population remained virtually unchanged among three groups ( Figure 6B, E).
Meanwhile, although TFNA failed to affect the phenotypes of CD8+ T cells from patients ( Figure 6A, D), the IFNγ and TNF secretion levels of CD8+ T cells were dramatically reduced after 12 hours co-cultivation ( Figure 6C, F). It is worth mentioning that a less intensive impact of TFNA in TNF secretion from CD8+ T cells was observed by comparing with the CsA group, which decreased approximately 95% of TNF secretion ( Figure 6F). Indeed, this finding emerged a distinct manner of TFNA from currently immunosuppressive-therapeutic strategies, including both CsA and steroids, which decrease all cytokines and/or chemokines from T cells. In summary, TFNA could serve as a more manageable immune modulator that not only controls disease progression, but also protects host immune responses. Meanwhile, a similar immune suppressive effect of TFNA was observed in primed T cells as well ( Figure 5). It is also noteworthy that TFNA had no effect on T cell differentiation at neither primed nor resting T cells ( Figure 3). Additionally, our ex vivo studies revealed a significantly blockade of IFNγ and TNF secretion in T cells from NMOSD patients after TFNA treatment ( Figure 6). By using T-cell activation inhibitor CsA as a positive control, we further dissected that TFNA down-regulated JNK/ERK phosphorylation to block MAPK signaling pathway-dependent cytokine secretion (Figures 4-5).

| D ISCUSS I ON
Altogether, our current findings suggested that TFNA could be used as a novel immune regulator to prevent excessive pro-inflammatory cytokine secretion from provoked T cells, which is supposed to be the major signs of autoimmune responses. Neuromyelitis optica spectrum disorder is a typical autoimmune disease that affects the central nerve system and T cells play pivotal roles in disease relapsing. 32 In particular, the Th1/Th2 ratio in the relapsing phase of NMOSD patients was higher than that in NMOSD patients in the remitting phase. 33 Our latest results demonstrated that circulating CD8+ T cells produce IL-2 and IFNγ, which are important cytokines for NMOSD relapsing and play crucial roles in disease progression as well. 20 Unfortunately, although current immunosuppressive therapies concomitant with glucocorticoids are considered as the frontline treatments for controlling the progression of NMOSD, there were still 30%-53% of patients with advanced disease or relapsing. 34 Simultaneously, many NMOSD patients have to suffer severe infection due to extremely low host immunity during the treatment. 35 Hence, it is urgent to develop novel strategies in targeting activated T cells of patients with NMOSD. Here, by involving NMOSD patients, ex vivo studies were designed to further confirm the selective regulatory manners of TFNA in inhibiting both IFNγ and TNF secretion by T cells. Indeed, because of the small sample size, more NMOSD patients are needed to be involved in our further cohort. In addition, accumulating evidence has already mentioned that effect/memory and short-lived effector T cells lead to autoimmune responses in situ. 36 Thus, we plan to look insight into the regulatory effects of TFNA in autoantigen-specific T cells with different subsets. Overall, we illustrated the immune regulatory function of TFNA by downregulating the expression of IFNγ in human T cells without affecting the phenotype and TNF secretion levels. In addition, it showed a more selective manner in modulating the immunity than the immunosuppressors, which made us believe that TFNA could be used as an immunoregulator in the treatment of T cell-mediated autoimmune diseases, including NMOSD and other diseases.

| CON CLUS ION
Self-assembled TFNA was found to be capable of entering and accumulating in the cytoplasm of primary human T cells. On the basis of this finding, we revealed that TFNA regulates T cell-mediated

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.