Characterization of GO and its affinity to cell membrane.
GO of different scales were synthesized as vehicles for the delivery of neuropeptides to DCs. Transmission electron microscopy (TEM) observations showed that GOs were characterized by irregular polygon of different lateral sizes (Figure 1A). The hydrodynamic radii of GOs were measured by dynamic light scattering (DLS) as 103.1 nm (marked as S-GO) and 1192.1 nm (marked as L-GO), respectively (Supplementary Figure 1). Atomic force microscopy (AFM) revealed the average thickness of 1-2 nm, which aligned with the structural characteristics of monolayer 2D materials (Figure 1B). In order to determine the maximum allowable concentration of GOs in delivery, flow cytometry was performed to analyze the survival rates of imDCs after incubation with GOs for 48 h. When the concentration of GOs was set as 15.6 µg/ml, GO showed little toxicity with the survival rate of imDCs higher than 90% compared to the control group (Supplementary Figure 2).
GO was proved to have strong affinities to cell membrane and mediate contact through membrane interactions. According to these characteristics, GO was chosen as a good nanoscaffold for variety biomedical applications. Confocal Raman results showed that after co-culture with S-GO or L-GO for 48h, the accumulation of GO was observed in cell membrane, suggesting that both S-GO and L-GO had nonspecific affinities to accumulate in cell-rich areas including RAW264.7, Hela and DC. L-GO were mostly adhered to the membrane while S-GO showed a preference of locating in cytoplasm (Figure 1C). Cytomembrane is rich in transmembrane receptors, which is a critical component in cell properties and subtype switching. However, whether GO’ affinities have any influence on membrane receptors or on the combination of ligands and receptors are still unknown. Here we chose RBCs as a lipid bilayer model because it has no organelles or nucleus, thus simplifying the interaction model. The surface of RBCs was first biotinylated to varying degrees to imitate receptors of different abundances on cytomembrane, and different concentrations of S-GO or L-GO were added to PE labeled avidin to allow physical adsorption. The binding process of avidin-biotin was used to simulate the combination that was dependent on the ligand and receptor (Figure 1D). The combining efficacy was reflected by the mean fluorescence intensity (MFI) of PE monitored by FACS. It is worth noting that even in the non-biotinylated group, the introduction of GO caused nonspecific affinity to RBCs (Supplementary Figure 3). Figure 1E gave direct evidence that GO-pretreatment could increase the binding affinity between PE-labeled avidin and biotinylated RBCs. Despite the presence of nonspecific binding, such improvement was much more significant in the case of L-GO than in its counterpart, especially in the low abundant biotin group (1 or 2 μg biotin added in the labelling system). The specific binding of biotin-avidin was measured after deduction of the nonspecific binding. When 8 μg GO was added to the pretreated avidin, FACS results showed that S-GO and L-GO both increased MFI at each concentration of biotin, especially at a lower concentration (especially 1 μg and 2 μg), and that L-GO showed stronger improvement in MFI than S-GO did (Figure 1E). The results after non-specific binding deduced are depicted as Figure 1F, in which case the GOs just acted as a promoter in the course of specific recognition of avidin and biotin. Take biotinylated RBCs (1 μg group) as an example. the addition of different concentrations (2, 4 and 8 μg) of S-GO showed 1.98-, 2.03- and 3.87-fold enhancements compared to the blank, respectively. Meanwhile, the corresponding results for the L-GO group were 2.58-, 3.87- and 4.71-fold, respectively. These results indicated that GOs’ high affinity to cytomembrane facilitated the specific combination between the ligand and receptor, and that the large-sized ones showed stronger enhancement (Figure 1F). As the current work was intended to explore TDCs-based immunotherapy in GVHD treatment, we then proceeded to the investigation of the interaction manner between GOs and DCs. Laser confocal was performed to validate the location of GOs on DC plasma membrane after 24 h of co-culture. S-GO and L-GO were labeled with FITC, and DCs were stained with rhodamine. Figure 1G shows that S-GO was mainly found in the cytoplasm of DCs, while L-GO was located at the edge of cells that were likely to correspond to the GOs associated with the plasma membrane, which was consistent with the phenomenon observed in the above RBCs-based model and literature. (Figure 1G).
The screening of neuropeptides aiming at inducing TDCs
Neuropeptides, mainly secreted by immune cells such as macrophages and monocytes, were considered capable of inducing immune tolerance with a broad anti-inflammatory effect. Neuropeptides UCN, α-MSH, VIP and AM were chosen as alternatives to induce the generation of TDCs firstly in a non-inflammatory microenvironment. Proinflammatory cytokines, such as IL-12p70, IL-6, IL-1β and TNF-α were measured by ELISA. It was found that neuropeptide incubation had little influence on cytokine secretion of DCs as shown in Supplementary Figure 4. Given the systemic inflammation in case of GVHD, we next investigated the performance of the four candidates in an inflammatory environment established by low-dose LPS co-incubation for 48 h. In the case of DCLPS group, stimulatory DCs (SDCs) were obtained with elevated proinflammatory cytokines secretion (Figure 2A). Meanwhile, in the presence of neuropeptide, the proinflammatory cytokines were found to be significantly decreased. Indeed, IL-12p70 secreted by DCs co-cultured with UCN declined by 61.8% percent compared with the LPS stimulated ones (85.2±10.8pg/ml). IL-6 showed 80.3% decrease, while IL-1β was 64.8% and TNF-α was 82.4%, indicating that UCN exhibited more effective suppression on proinflammatory cytokines secretion than other candidates (Figure 2A).
After UCN was selected as the most appropriate neuropeptides in the study, we stared to find out whether UCN was capable of inducing TDCs. Balb/c derived CD8+ T cells and C57BL/6J derived DCs were isolated separately. CD8+ T cells were stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) and co-cultured with DCs at ratios of 2:1, 4:1 and 8:1 for 48h. Results showed that DCs with UCN treatment exhibited significant inhibition on cytotoxic T cell proliferation. And the strongest suppression was observed at the ratio of 4:1with a decrease of 52.6% compared to DCLPS. Cytotoxic T-cell proliferation test demonstrated that TDCUCN decreased CD8+ T cell proliferation and induced T cell anergy (Figure 2B, 2C). CD40/80/86 were employed to characterize the maturity of DCs. It was found that after being co-cultured for 48h, the TDCUCN group showed lower expressions of costimulatory molecules than the mature ones. FACS was significantly different between TDCUCN and DCLPS with CD40 (1.7-fold) and CD80 (1.5-fold), which demonstrated that UCN induced TDCs could preserve the tolerant phenotype in an inflammatory environment (Figure 2D). Collectively, UCN was considered the most suitable one to induce TDCs.
Next, we attempted to construct a GO-based cell membrane targeted platform for the purpose of promoting the UCN delivery efficiency. UCN labeled by Cy5 incubated and physically adsorbed on to excessive S-GO or L-GO. Results of fluorescence imaging showed that the conjugation efficiency of UCN with S-GO was (89.57 ± 1.53) %, compared with (86.33 ± 2.01) % with L-GO (Figure 2E). The Fourier Transform Infrared (FT-IR) spectrometer was used to characterize the [email protected] complex. A notable absorption peak of the peptide bond was observed compared to S-GO or L-GO alone (Figure 2F), which demonstrated the neuropeptide UCN was successfully conjugated with GO nanosheets. Flow cytometry was performed to detect the survival rate of DCs after incubation with different concentrations of [email protected] complex for 48h. The survival rates of DCs varied significantly in a dose-dependent manner according to Figure 2G, which was why 15.6 μg/ml was chosen as the working concentration, for the corresponding survival rates in UCN-S and UCN-L group were both above 90% (90.02±2.95%, 90.10±1.98%).
GO as nanocarriers for UCN delivery and TDCs generation
After being established for UCN, the GO delivery system was further evaluated in terms of TDCs generation. After incubation with [email protected] for 48h, imDCs and the supernatant were collected separately. DCs were gated as CD11c+ population, CD40/80/86 showed no difference after incubation with free GO or [email protected] (Supplementary Figure 5). ELISA measurements indicated that proinflammatory cytokines did not change significantly compared with imDCs after incubation with free GO or [email protected] (Supplementary Figure 6). Further, the co-cultured DCs were added to CFSE labeled CD8+ T cells at a ratio of 1:4 for 48h. T cell proliferation rates of DCL-GO and DC[email protected] were 9.9% and 7.87%, separately, compared with 10.94% of imDCs (Supplementary Figure 7). Therefore, we confirmed that neither free GO nor [email protected] had any positive influence on DC maturity, which facilitated TDCs induction.
To simulate the environment where GVHD occurs, [email protected] pretreated TDCs were further challenged in an inflammatory microenvironment induced by low-dose LPS. As a member of the CRF family, UCN mainly binds to CRHR2 expressed on cell membrane and affects inflammation response. CRHR2, a G protein-coupled receptors, is mainly expressed on cell membrane. BioGPS, a gene annotation portal, can provide complete gene information, including patterns of gene expressions in different cells or tissues. Data from BioGPS showed that CRHR2 expressions in the immune system, like BDCA4+ DCs, CD19+ B cells, CD8+ T cells, CD56+NK cells and CD14+ monocytes, is under median / were below the median value (Supplementary Figure 8). It was speculated that the low abundance of CRHR2 expressions on DC membrane might be the key to restricting the efficiency of TDCs induction. Therefore, improving the enrichment of UCN on the cell surface and promoting the binding efficacy of UCN and CRHR2 seemed to be an alternative solution to the limited production of TDCs.
We went on to explore whether GO loading would benefit the recognition of UCN by CRHR2 as expected. UCN was labeled with 5-TAMER and coupled with GOs, respectively. After DC incubation, FACS results indicated that [email protected] tended to be adhered to the cytomembrane compared with [email protected] or free UCN (29.3% verse 20.0% and 14.8%) (Figure 3A). 5-TAMER labeled UCN was further pre-conjugated with FITC-GOs, while DCs were stained with Cell TrackerTM Blue. Confocal imaging observation showed that most of the UCNs could be internalized in DCs cytoplasm without any GO carrier, which might fail to bind to the membrane receptor CRHR2. [email protected] appeared to be in the cell cytoplasm, while [email protected] were mostly located on the surface of the cell membrane (Figure 3B), which conformed with the manner in which the carrier GO alone interacted. These results suggested that [email protected] experienced a normal endocytosis process during incubation with DCs, while the larger lateral diameter of L-GO might probably benefit the concentration of UCN molecules on plasma membrane and the binding event with its receptor. The deduction was further verified by the co-location results of UCN and CRHR2 observed by the laser confocal (Supplementary Figure 9).
The downstream signaling molecular expressions were further examined by Western blot to explore the mechanism of TDC generation. Results showed that the presence of LPS had little influence on PKA C or CREB phosphorylation, so did single S-GO or L-GO treatment. UCN and [email protected] complex exhibited a similar increase of PKA C phosphorylation to DCLPS. UCN incubation elicited more phosphorylation of the downstream CREB (1.81-fold), which indicated that UCN could effectively mediate TDCs production. And higher expressions of p-CREB (3.07-fold or 3.49-fold) were detected in the [email protected] or [email protected] group, suggesting that the enrichment of UCN on DC membrane, which resulted from GO coupling, could trigger cAMP/PKA pathway activation more effectively (Figure 3C and 3D). The above results served as evidence that GO had positive effects on the targeted delivery of UCN and promoted the combination of the ligand and receptor on the plasma membrane of DCs.
Once the cAMP/PKA/CREB signal pathway was activated, the CBP (CREB binding protein, CBP) molecules were translocated into the nucleus and further activated NF-κB, resulting in a decrease of pro-inflammatory cytokine expressions and co-stimulatory molecular levels of DCs. Thus, the levels of cytokine secretion and CD40/80/86 phenotype of TDCs were examined after LPS challenge. It was observed that the concentration of IL-12p70 in the supernatant showed no significant difference in S-GO or L-GO treated groups compared with mature DCs. The concentration of IL-12p70 decreased to 54.85±4.99 pg/mL after [email protected] pretreatment, and to 50.49±0.52 pg/mL after [email protected] incubation, which was significantly lower than in the free UCN group (60.06±3.91 pg/mL) and mature DC group (85.97±2.19 pg/mL). The amounts of IL-1β, IL-6 and TNF-α secreted by DCs co-cultured with [email protected] and [email protected] also showed notable decreases compared to the free UCN group. These inflammatory cytokine levels demonstrated that the UCN-GO complex was more efficient in inducing TDCs than free UCN treatment (Figure 3E). Although S-GO or L-GO had little influence on expressions of costimulatory molecules, S-GO enhanced the efficiency of UCN in inhibiting the expression of surface costimulatory molecules CD40 (1.2-fold) and CD80 (1.3-fold) according to FACS results, compared with 1.5-fold, 1.7-fold, and 1.1-fold after [email protected] pretreatment, which exhibited a notable decrease in DCs maturity (Figure 3F). Given the positive role that GO played in promoting ligand and receptor combination and delivery of UCN, it could be speculated that the [email protected] process enhanced UCN’s ability to induce TDCs. Furthermore, it was obvious that [email protected] outperformed [email protected] in TDC generation. Thus, [email protected] was chosen as an inducer of TDCs for follow-up experiments.
TDC[email protected] effectively deleted cytotoxic T cells and generated Tregs.
Then, the T cell subtype and function were detected after co-culture with TDCUCN or TDC[email protected]. CD8+ T cells isolated from Balb/c mice were labeled with CFSE and incubated with different TDCs at the ratio of 4:1. After 48h of incubation, FACS results showed that both TDCUCN and TDC[email protected] exhibited robust inhibition of cytotoxic T cell proliferation caused by LPS with a decrease of 39.4% and 25.1%, respectively (Figure 4A). Next, the cytotoxic function of CD8+ T cells was evaluated by CD44, CD69 and CD107a. FACS demonstrated that CD8+ T cells lost their cytotoxic functions after incubation with TDCUCN or TDC[email protected], for TDC[email protected] suppressed the CD44/69/107a expression with a decrease of 79%, 75% and 64% compared with mature DC, respectively (Figure 4B).
According to primary research, despite the inhibition of CD8+ T cells through PD-L1 expression6, TDCs also mediated CD80/86 combination with CTLA4, leading to Treg generation. The proportion of Treg cells was further investigated in the population of CD4+ T cells. Notably, more CD4+ T cells obtained Treg phenotype after TDCUCN or TDC[email protected] pretreatment, which was characterized by a remarkably increase in CD25 (1.38-fold and 1.63-fold) and CTLA4 (3.42-fold and 4.18-fold) compared with mature DCs (Figure 4C). Collectively, these results indicated that [email protected] complex was a promising candidate in TDC generation that led to anergy of cytotoxic T cells and Treg expansion.
[email protected] complex triggered TDCs cytoskeleton rearrangement to promote the homing ability in vivo
Another important feature of TDCs is the ability to migrate, for they have to home to lymph nodes to produce a notable influence. Despite the lateral size, another important discrepancy in physicochemical property between L-GO and S-GO is the content of carbon free radicals. According to electron paramagnetic resonance (EPR) results, more carbon free radicals would be measured on the surface of L-GO compared to S-GO, which can be remarkably blocked by FBS (Supplementary Figure 10). Thus, it stands to reason to expect more ROS production in the DCL-GO or TDC[email protected] co-incubation group, which has been further verified by FACS analysis as shown in Figure 5A. This indicated that ROS was accumulated during interactions between GOs and plasma membrane. Moreover, the expression of CCR7 on DC membrane showed the same tendency as ROS after co-incubation (Figure 5B). Collectively, we deduced that the adherence of L-GO to DC membrane was crucial to the activation of Rho A/ROCK pathway which existed in the downstream of ROS and participated in cell migration. Thus, the phosphorylation of Rho A/ROCK signals was further evaluated. L-GO amplified the phosphorylated levels of ROCK and MLC of DCs by 2.04–fold and 2.40-fold compared to the control DC. Although free UCN significantly suppressed p-ROCK and p-MLC, L-GO binding exhibited robust reversal (Figure 5C&D). Because of the remarkable changes in p-ROCK and p-MLC, L-GO was considered an effective initiator of cytoskeleton rearrangement. Simultaneously, we observed a significant decrease in p-IKBα and p-P65 in TDCUCN and TDC[email protected] groups, suggesting that TDCs remained the tolerant phenotype even in the presence of LPS.
Since cytoskeletal is the executor of cell migration, the rearrangement of DC cytoskeleton was observed by immunofluorescence (IF) in advance.
To simulate the inflammatory microenvironment in the process of GVHD, low-dose LPS was added to the co-culture system as a stimulus to TDC challenge. Obviously, L-GO materials had positive effect on the rearrangement of DC cytoskeleton characterized by increased expressions of F-Actin and β-tubulin. In contrast, free UCN showed a notable decrease, which might have been brought about by inflammatory inhibition. However, after L-GO binding, TDC[email protected] displayed remarkable restoration of F-Actin and β-tubulin expressions. To be more specific, the fluorescence intensity of F-Actin and β-tubulin in the TDC[email protected] group was 2.31-fold and 1.42-fold higher than that of TDCUCN, respectively (Figure 5E&F). In the absence of LPS, a similar tendency was also observed as shown in Supplementary Figure 11. Thus, the strong migration ability of TDCs induced by [email protected] complex was expected. Combined with the Western blotting results in Figure 5C, it could be concluded that the GO-based cytomembrane-philic delivery platform participated in cytoskeletal rearrangement via ROS generation and ROCK phosphorylation.
Living cell imaging was carried out to track and analyze a time-laps cell migration pathway and length. After 6h of observation, it was found that the L-GO had a stronger ability to induce DCs migration and longer displacement in contrast to UCN (Figure 5G). And the complex of [email protected] could significantly restore the migration ability of TDCs impaired by UCN. L-GOs treatment also accelerated cell migration velocity. Unlike the disordered movements of the control DCs, DCs incubated with [email protected] showed faster track velocity with a dominant motile mode of classical amoebic movement, which displayed a more efficacy migration mode (Figure 5H).
Furthermore, we employed the “footpad injection model” to confirm the ability of DCs to home to lymph nodes (LNs) after being co-cultured with UCN and [email protected] Mice were observed at time points of 24 h, 48 h and 72 h after footpad inoculation of Fluc+ DCs using the fluorescence imaging system. The migration rate of DCs was measured by the ratio of fluorescence signals in PLNs (popliteal lymph nodes, PLNs) to the signals remaining on the foot pad. LPS was employed as a positive control. After L-GO treatment, DCs acquired a much stronger ability to home to PLNs than immature DCs, while free UCN treatment contributed to promotion. TDC[email protected] exhibited a more significant migration rate than TDCUCN. Comparisons between the groups demonstrated that in the TDC[email protected] group, the ratio of DCs migrating to PLNs at 24 h, 48 h and 72 h were respectively 17.75 ± 4.11, 19.75 ± 5.09 and 25.24 ± 5.07, which was the highest among these groups. (Figure 6A&B).
We also investigated the distribution of Fluc+ DCs after intravenous administration in vivo. Fluorescence signals were detected using the fluorescence imaging system at 4 h, 24 h and 48 h post injection. Results showed that the entire fluorescence intensities were hardly different between these groups at different time points (Figure 6C&D). Organs were collected at 72 h post injection and imaged by the fluorescence system. The LPS group exhibited much stronger migration to lymphoid tissue than the control did. Since L-GOs treatment also facilitated the migration of DCs, TDC[email protected] showed more enhanced migration to lymphoid tissues than free UCN treatment, as was illustrated by analysis of fluorescence intensities (Figure 6E). Statistical analysis also demonstrated that [email protected] treatment accelerated cell migration to lymphoid tissues more significantly than free UCN co-cultured ones (Figure 6F).
Moreover, the isolated organs were separated into two groups, solid organs and lymphoid ones. Results confirmed that TDC[email protected] exhibited stronger migration capacity to spleen and lymph nodes than TDCUCN, which probably benefitted the interaction and regulation with T cells (Figure 6G).
[email protected] treated TDCs regulated the phenotype of donor T cells and protected recipients from lethal GVHD
An acute GVHD mouse model was established to help find out whether the TDC[email protected] could notably inhibit cytotoxic T cell proliferation or generate adequate Treg cells in GVHD relief.
To determine the amount of L-GOs that model mice would be exposed to or caused by DCs immunotherapy, FITC labelled L-GO or [email protected] were incubated with DCs for 48 h. The supernatant and cells were collected separately for fluorescence detection. Results in Supplementary Figure 12 illustrated that the residual L-GOs that adhered onto the cytomembrane of DCs were less than 30% of the incubated amount with the excessive L-GO or [email protected] mainly in the cell culture medium. A widely accepted visualized GVHD murine model was established by infusion of a cell mixture composed by T cell-deleted allogenic bone marrow cells and Fluc+ T cells. And the entire fluorescence intensity could represent the proliferation level of T cells in vivo. The group labeled as “BM+T” meant no successive immunotherapy treatment. After transplantation, 4×106 DCs, DCL-GO, TDCUCN and TDC[email protected] were infused intravenously respectively (Supplementary Figure 13). On the 14th day post transplantation, imaging analysis showed the “BM+T” group had the highest fluorescence intensity, which indicated massive activation and acute proliferation of T cells. Injection of DCL-GO resulted in little difference compared with the DCs group, while TDCUCN displayed a 2.37-fold decrease in fluorescence intensity. Furthermore, the TDC[email protected] group showed 2.8-fold reduction compared with the TDCUCN group, suggesting the positive effect of L-GOs binding in inhibiting T cell proliferation (Figure 7A&B). The LNs, lung, thymus, spleen, pancreas, liver, intestine and kidney were isolated and imaged. Results showed that the BM+T group had extensive T cell proliferation in multiple tissues, especially in the spleen, mesenteric lymph nodes (MLNs), thymus and small intestine (SI), which indicated rapid progress of GVHD. The infusion of control DCs or DCL-GO displayed relief to a certain extent. As expected, TDCUCN and TDC[email protected] groups both exhibited a significant decrease in organ fluorescence intensities, especially the latter, which induced apoptotic deletion of donor CD8+ T cells (Figure 7C&D). ELISA was performed to analyze the cytokine levels in serum. It was found that after transplantation, such proinflammatory cytokines as IL-1β, IL-6 and MCP-1 were robustly increased, while the anti-inflammatory cytokines of IL-10 and TGF-β both significantly decreased compared to the irradiation control, which was a sign of the explosive inflammation in vivo. Infusion of control DCs and DCL-GO had slight effect on cytokine secretion, while TDCUCN and TDC[email protected] induced an obvious decrease in these proinflammatory cytokines and a notable increase in the anti-inflammatory ones. (Figure 7E).
TDCs also play non-redundant roles in promoting expansion and function of Tregs. As primary studies have showed, TDCs can induce T cell tolerance broadly via two categories: intrinsic and extrinsic. TDCs produce PD-L1 and CD80/86, which bind to PD-1 and CTLA-4 respectively, leading to the deletion of cytotoxic T cells and expansion of Tregs that can obstruct the proliferation and survival of those activated T cells. Moreover, TDCs can suppress the antigen-reactive T cells and promote Treg generation through IDO production6. We further isolated the MLNs and spleens to evaluate the proportion of Tregs. FACS showed that the proportion of Treg increased dramatically both in the spleen and MLNs after TDC treatment. According to the FACS results, TDC[email protected] injection mediated 1.67-fold and 9.23-fold increases of CD4+CD154+ and 66.7% and 73.7% decreases of CD4+CTLA4+ in the spleen and MLNs separately, compared to BM+T group, which both corresponded to the immune suppressive T cells (Figure 7F).
Cytotoxic T cell apoptotic deletion and Tregs generated by TDCs both played a vital role in GVHD relief by mitigating tissue damage. Hematoxylin and eosin (HE) staining of the involved organs confirmed the positive effect. The BM+T group was attacked by the donor T cells in multiple organs and had massive lymphocyte infiltration, which were symptoms of severe GVHD. DCs and DCL-GO infusion both showed limited remission, while TDCUCN and TDC[email protected] displayed notable improvement of multiple organs involved such as the spleen, liver, MLN, thymus and SI (Figure 7G, Supplementary Figure 14), especially the latter. Also, mice which received TDCs treatment, especially those in TDC[email protected] group, showed remarkable abrogation in symptoms caused by severe GVHD, including hunched posture, skin and ear inerythema, alopecia and weight loss. Altogether, these results indicated that [email protected] treatment was extremely efficient for TDC generation, which was probably the key to altering T cell subtype and the resultant GVHD relief in vivo.