A method for in situ visualization and biological function research of cell pd-l1 glycosylation based on hybridization chain reaction

Abstract

The application discloses a cell PD-L1 glycosylation in-situ visualization and biological function research method based on hybridization chain reaction. The application adopts a hybridization chain reaction strategy (HCR) of a logic "AND" gate activated by nucleic acid aptamer recognition and metabolic glycan labeling, and constructs a new method for a cell PD-L1 glycosylation in-situ visualization and biological function research method. Through HCR signal amplification induced by adjacent double recognition, the detection sensitivity of PD-L1 glycosylation on different cells is significantly improved, and subtle fluctuations in the PD-L1 glycosylation level can be accurately captured, so as to provide an analysis tool for revealing the dynamic changes under different physiological or pathological conditions. Meanwhile, the application first clarifies the immune regulation function of the PD-L1 glycosylation of dendritic cells, and further expands the research scope of tumor immune checkpoints.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical medical research technology, specifically relating to an in situ visualization and biological function study method for cellular PD-L1 glycosylation based on hybridization chain reaction. Background Technology

[0002] Programmed death ligand 1 (PD-L1), a transmembrane protein, inhibits T cell activation by binding to programmed death receptor-1 (PD-1) on the surface of T cells. The PD-L1 / PD-1 pathway is known to be a key mechanism of immune regulation and has become an important target for cancer immunotherapy. Currently, several drugs targeting this pathway have been approved for clinical trials or are in clinical trials. However, clinical data show that only 20%-40% of patients respond to this type of treatment, and the proportion of patients achieving long-term remission is even lower. Furthermore, the expression level of PD-L1 in tumor tissue cannot consistently predict the efficacy of anti-PD-L1 / PD-1 therapy. Clinical observations show that approximately half of PD-L1-positive tumor patients do not respond to PD-L1 / PD-1 blockade therapy, while a small number of PD-L1-negative tumor patients can still benefit. This indicates that the understanding of the regulatory mechanisms of PD-L1 expression and function is still insufficient, and further research is necessary.

[0003] Post-translational modifications are a crucial mechanism regulating PD-L1 immune function. Glycosylation, as one of the most common post-translational modifications, involves the covalent linkage of glycans to target molecules. Studies have shown that PD-L1 primarily undergoes N-glycosylation. In tumor cells, N-glycosylation can enhance PD-L1 stability by inhibiting its degradation and promote its binding with CD8. + PD-1 binding on T cells. The transmembrane ubiquitin-like protein TMUB1 inhibits PD-L1 ubiquitination and induces its glycosylation, thereby promoting tumor immune escape. Furthermore, intervention on N-glycosylation of PD-L1 enhances the blocking effect of anti-PD-L1 antibodies, suggesting that this modification is associated with the efficacy of immune checkpoint blockade and could serve as a novel biomarker for predicting treatment response. These studies indicate that the working mechanism of PD-1 / PD-L1 signaling may be more complex.

[0004] Furthermore, studies have found that PD-L1 is not only expressed on tumor cells but also widely expressed on various immune cells in the tumor microenvironment. Dendritic cells (DCs), as important antigen-presenting cells, are one of the key sources of PD-L1 in the tumor microenvironment. However, whether PD-L1 on DCs undergoes glycosylation modification, and what role this modification plays in DC-mediated immune regulation, remains unclear. Common methods for studying protein-specific glycosylation include Western blotting, mass spectrometry, and lectin microarray technology. However, because these strategies require the isolation of target proteins from cells before detection, they cannot observe the glycosylation status on the surface of living cells in situ, and are difficult to directly use to study the biological functions of this modification at the living cell level.

[0005] Therefore, it is necessary to establish a method for in situ imaging and functional analysis of PD-L1 glycosylation on the surface of various cells, especially dendritic cells, under physiological conditions, in order to clarify its expression level and its immunomodulatory role in the tumor microenvironment. Summary of the Invention

[0006] The purpose of this invention is to provide a method for in situ visualization and biological function study of cellular PD-L1 glycosylation based on hybridization chain reaction.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for in situ visualization detection of cell surface PD-L1 glycosylation based on hybridization chain reaction includes the following steps:

[0009] (1) Acetylated azide-modified mannosamine derivatives were used to label metabolic glycans in cells;

[0010] (2) The DBCO-T1 probe and Aptamer-T2 probe were bound to the glycan and PD-L1 protein on the cell surface, respectively, and unbound probes were washed away.

[0011] (3) Add Connector probe, H1 hairpin probe and H2 hairpin probe to make DBCO-T1 and Aptamer-T2 adjacent to each other, thereby triggering the cascade hybridization chain reaction of H1 and H2 hairpin probes to achieve signal amplification;

[0012] (4) PD-L1 glycosylation was visualized in situ by flow cytometry or laser confocal fluorescence microscopy.

[0013] Furthermore, the sequence of Aptamer-T2 is shown in SEQ ID NO: 1; the sequence of DBCO-T1 probe is shown in SEQ ID NO: 2; the sequence of Connector probe is shown in SEQ ID NO: 3; the sequence of H1 hairpin probe is shown in SEQ ID NO: 4; and the sequence of H2 hairpin probe is shown in SEQ ID NO: 5.

[0014] Furthermore, the cascade hybridization chain reaction adopts a two-step reaction method, first incubating with DBCO-T1 and Aptamer-T2, then washing, and then adding Connector, H1 and H2 to carry out the reaction.

[0015] Furthermore, the reaction conditions for the second step are: incubation time of 20-60 min, incubation temperature of 4-37 ℃; final concentration of Connector of 500 nM-2.5 μM, and final concentrations of H1 and H2 of 100 nM-1 μM.

[0016] Furthermore, the cells include human melanoma cells A375, human lung adenocarcinoma cell line A549, mouse dendritic cell line DC2.4, mouse bone marrow-derived dendritic cells BMDCs, and mouse macrophage line RAW264.7.

[0017] The above detection method is applied in the preparation of cellular PD-L1 glycosylation detection reagents or tumor immune checkpoint function research kits.

[0018] An application of the above detection method in studying the biological function of PD-L1 glycosylation on the surface of dendritic cells includes the following applications:

[0019] (1) Using the cascade hybridization chain reaction detection system, compare the binding ability of PD-L1 and PD-1 protein on the surface of dendritic cells in different glycosylation states;

[0020] (2) Using the aforementioned cascade hybridization chain reaction detection system, assess the effect of PD-L1 glycosylation level on the surface of dendritic cells on CD8 cells co-cultured with them. + Regulatory role of T cell proliferation.

[0021] Furthermore, the assembly of the cascade hybridization chain reaction detection system does not affect the binding ability of PD-L1 protein on the surface of dendritic cells; after treatment with PNGase F enzyme to downregulate PD-L1 glycosylation, the binding ability of PD-L1 to PD-1 was significantly reduced.

[0022] Furthermore, mouse bone marrow-derived dendritic cells with CD82 that have been treated with PNGase F enzyme to downregulate PD-L1 glycosylation were compared with those of CD82. +T cells were co-cultured, and the detection method described above was used to verify that downregulation of PD-L1 glycosylation levels could enhance dendritic cells' resistance to CD8+. + The proliferative effect of T cells.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] (1) For the first time, in-situ detection and imaging of surface PD-L1 glycosylation in living cells was achieved.

[0025] Existing methods for detecting protein glycosylation (such as gel electrophoresis and mass spectrometry) require pre-isolation and purification of glycoproteins, which disrupts cell integrity and makes it impossible to study glycosylation modifications under physiological conditions. While lectin microarray methods can partially preserve cell structure, the weak affinity of lectins makes it difficult to accurately detect the glycosylation level of specific proteins (such as PD-L1). In contrast, this invention employs a strategy combining glycan metabolic labeling with specific nucleic acid aptamer recognition to label glycan chains and PD-L1 protein separately. Hybridization chain reaction (HCR) assembly is induced in a reaction buffer that maintains cell viability, enabling signal amplification and in-situ imaging of PD-L1 glycosylation on different cells. This method has advantages such as small molecular weight of chemical groups, high biocompatibility, and ease of modification, and it is simple to operate without the need for pre-isolation of glycoproteins.

[0026] (2) Significantly improves the detection sensitivity of PD-L1 glycosylation, and can accurately quantify its dynamic changes.

[0027] Based on the HCR signal amplification strategy induced by proximity dual recognition, this invention significantly improves the detection sensitivity of PD-L1 glycosylation on different cells, and can accurately capture subtle fluctuations in PD-L1 glycosylation levels, providing a highly sensitive analytical tool for studying its dynamic changes under different physiological or pathological conditions.

[0028] (3) The immune regulatory function of PD-L1 glycosylation on DCs was revealed for the first time.

[0029] This invention is the first to demonstrate at the functional level that PD-L1 glycosylation on DCs plays a key role in regulating PD-L1 / PD-1 interactions, and that this glycosylation modification may inhibit CD8. + T cell proliferation participates in immunosuppression in the tumor microenvironment. This discovery breaks through the limitation of existing technologies that can only detect the presence or absence of glycosylation, and for the first time directly links PD-L1 glycosylation on DCs with functional T cell immune responses. Attached Figure Description

[0030] Figure 1 This is a schematic diagram illustrating the experimental principle of the present invention.

[0031] Figure 2To verify the feasibility of HCR assembly in a buffer system, Figure (a) shows the observation of HCR products by agarose gel electrophoresis, and Figures (bc) show the comparison of fluorescence intensity of each group by fluorescence spectrophotometer.

[0032] Figure 3 To validate PD-L1 expression and glucose metabolism labeling, (a) flow cytometry was used to detect the PD-L1 expression levels in A375 and MCF-7 cells; (b) Western blotting was used to detect the expression levels of glycosylated and non-glycosylated PD-L1 proteins in A375 cells before and after PNGase F enzyme treatment; and (c) flow cytometry was used to detect the glucose metabolism labeling efficiency in A375 and MCF-7 cells before and after Ac4ManNAz labeling.

[0033] Figure 4 To verify the cell binding of DBCO-T1 to Aptamer-T2 and detect the FRET effect, (a) flow cytometry was used to detect the binding efficiency of DBCO-T1 to A375 and MCF-7 cells labeled with Ac4ManNAz (+) / unlabeled (-), with a DBCO-free T1 probe as the control group; (b) flow cytometry was used to detect the binding efficiency of Aptamer-T2 to A375 and MCF-7 cells labeled with Ac4ManNAz (+) / unlabeled (-), with a PD-L-free T1 probe as the control group. 1. The T2 probe of nucleic acid aptamer MJ5C was used as a control group; (c) Laser confocal fluorescence microscopy was used to verify the binding of DBCO-T1 and Aptamer-T2 aptamers to Ac4ManNAz-labeled A375 cells and to detect the fluorescence resonance energy transfer (FRET) effect between the two aptamers. Multiple control groups with missing connector, missing Aptamer-T2, and missing DBCO-T1 were set up. The specificity of FRET signal was verified by Cy3, Cy5 and FRET channel signals.

[0034] Figure 5 To detect the HCR assembly efficiency of cells under different conditions by flow cytometry, (a) different incubation temperatures, (b) different connector concentrations, H1 and H2 probe concentrations, and (c) different reaction times.

[0035] Figure 6 To verify the amplification of HCR on the surface of A375 cells; (a) shows the amplification of HCR on the surface of A375 cells by laser confocal fluorescence microscopy imaging with multiple missing control groups; (b) shows the normalized relative fluorescence intensity quantitative statistics of Figure (a); (c) shows the amplification of HCR on the surface of A375 cells by flow cytometry with multiple missing control groups.

[0036] Figure 7The amplification of HCR on the surface of MCF-7 cells was detected by laser confocal fluorescence microscopy; (a) shows the confocal imaging results; (b) shows the normalized relative fluorescence intensity quantitative statistics of Figure (a).

[0037] Figure 8 The study aimed to verify PD-L1 expression, glycosylation modification, and HCR amplification in DC2.4 cells. Specifically, (ab) flow cytometry and Western blot were used to verify high PD-L1 expression and glycosylation modification in DC2.4 cells; and (ce) laser confocal imaging, quantitative fluorescence, and flow cytometry were used to verify the amplification of HCR on the surface of DC2.4 cells.

[0038] Figure 9 Experimental results were used to verify the performance of the cell surface glycoprotein detection method based on aptamer recognition and hybridization chain reaction; (a) confocal detection of HCR signal on DC2.4 surface pretreated with PNGase F for different times; (b-d) specificity results; (e) method stability.

[0039] Figure 10 The effects of HCR amplification and PD-L1 glycosylation on the PD-1 binding ability of DC2.4 cells were investigated. (a) Flow cytometry was used to detect the association between HCR amplification and PD-1 binding. (b) Schematic diagram of glycosylation / deglycosylation assessment. (cd) Flow cytometry was used to detect changes in HCR assembly and PD-1 binding after treatment with PNGase F and tunicamycin. (e) Pearson correlation analysis was used to verify the correlation between HCR signal and PD-1 binding signal.

[0040] Figure 11 Induced maturation of bone marrow-derived dendritic cells (BMDCs) and PD-L1 glycosylation of CD8 + The effects of PD-L1 glycosylation on T cell proliferation were analyzed. (a) Schematic diagram of the experimental procedure for obtaining immature and mature bone marrow mononuclear cells (BMDCs) from C57BL / 6 mouse bone marrow mononuclear cells; (b) Microscopic observation of morphological changes in BMDCs during maturation; (c) Flow cytometry analysis of PD-L1 expression levels on the surface of immature and mature BMDCs; (d) Flow cytometry detection of HCR assembly on the surface of mature BMDCs; and (e) Evaluation of the effect of PD-L1 glycosylation on CD8+ in the in vitro co-culture system based on HCR. + (f) Flow cytometry analysis of HCR assembly on the surface of BMDCs treated with or untreated with PNGase F; (g) Flow cytometry analysis of CD8-mediated proliferation by allogeneic BMDCs pretreated with or untreated with PNGase F. + T cell proliferation.

[0041] Figure 12To verify the universality and applicability of the HCR method in other PD-L1 positive tumor and immune cell types; (a) flow cytometry verified that the method could be successfully triggered on the surface of A549 cells and RAW264.7 cells; (b) flow cytometry verified that the method could be used for dynamic analysis of PD-L1 glycosylation in RAW264.7 cells. Detailed Implementation

[0042] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0043] Example 1: Establishment of an in situ detection method for PD-L1 glycosylation on the surface of dendritic cells based on hybridization chain reaction (HCR)

[0044] 1. Experimental Methods

[0045] 1.1 Assembly of HCR in buffer system

[0046] (1) DNA sequence synthesis: The DNA required for HCR assembly was synthesized by Shanghai Sangon Biotech Co., Ltd. The DNA sequence is shown in Table 1:

[0047] Table 1 DNA Sequence

[0048]

[0049] (2) DNA stock solution preparation and denaturation

[0050] The DNA lyophilized powder stored at -20 °C was centrifuged at 5000 rpm for 1 min at 4 °C, and an appropriate amount of DEPC water was added to dissolve the DNAs involved in Table 1 into a 100 μM storage solution. The storage solutions of DBCO-T1, Aptamer-T2, and Connector were mixed with an equal volume of DPBS (pH=7.2) containing 50 mM Mg(Ac)2, heated at 95 °C for 5 min, and cooled to room temperature on ice to obtain denatured DNA solutions. The storage solutions of H1-5'FAM-3'BHQ and H2 were mixed with an equal volume of DPBS containing 50 mM Mg(Ac)2, heated at 60 °C for 10 min, and cooled to room temperature on ice to obtain denatured DNA solutions.

[0051] (3) Group setup and incubation reaction

[0052] To investigate whether DNA probes could successfully trigger HCR in DPBS buffer (containing 25 mM Mg(Ac)2), six experimental groups (L1-L6) were set up. 2 μL of denatured DNA solution was added sequentially according to the group, as shown in Table 2 below. Reaction conditions: In the first stage, after adding DBCO-T1, Aptamer-T2, Connector, and reaction buffer, the mixture was incubated at 4 ℃ for 1 h; in the second stage, after adding H1-5'FAM-3'BHQ and H2, the mixture was incubated at 4 ℃ in the dark for 1.5 h.

[0053] Table 2 Grouping of L1-L6 samples

[0054]

[0055] Note: "+" indicates that a sample is added, and "-" indicates that no sample is added.

[0056] 1.2 Detection of HCR Assembly in Buffer System by Agarose Gel Electrophoresis

[0057] Place the 3% agarose gel flat in the electrophoresis tank. Add 5 μL of the amplified L1-L6 samples and 1 μL of 6×DNA Loading Buffer premix to the wells from left to right. At the same time, add 5 μL of DNA marker on both sides as molecular weight reference. Finally, run the gel electrophoresis apparatus, controlling the voltage at 60-80 V and the current at 40 mA or higher.

[0058] 1.3 Assembly of HCR in the detection buffer system of fluorescence spectrophotometer

[0059] The samples used for fluorescence spectrophotometry detection were the same as those used in the agarose gel electrophoresis method described above. After HCR amplification, 50 μL of sample was taken for fluorescence spectrophotometry detection. The fluorescence excitation wavelength was 488 nm. The fluorescence spectra of L1-L6 products in the range of 500-800 nm were detected, and the L4 blank group was used as background subtraction, while the L3 control group was used for normalization analysis.

[0060] 1.4 In vitro cell culture and glucose metabolism labeling

[0061] Human melanoma cell line A375, human lung adenocarcinoma cell line A549, and mouse macrophage cell line RAW264.7 were selected as PD-L1 positive cells (highly expressing glycosylated PD-L1 protein). PD-L1 negative human breast cancer cell line MCF-7 was selected as PD-L1 negative cells. All cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution at 37°C in a 5% CO2 incubator. DC2.4, an immortalized dendritic cell line derived from the bone marrow of C57BL / 6 mice, was cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin solution under the same environmental conditions. Glycochemical labeling was performed by removing the medium and replacing it with 50 μM Ac4ManNAz complete medium for 48-60 h after cell adhesion and a confluence density of 60-70%.

[0062] 1.5 Flow cytometry detection of PD-L1 expression, glucose metabolism markers, and binding to DNA probes in cells

[0063] (1) Detection of PD-L1 expression in cells

[0064] Take 1×10 6 Single-cell suspensions were washed three times with pre-chilled phosphate-buffered saline (PBS, pH 7.2) and centrifuged at 2000 rpm for 5 min at 4 °C. The supernatant was discarded, and the cell pellet was resuspended in PBS with 5 μL of APC-labeled anti-PD-L1 antibody added. The mixture was incubated at 4 °C in the dark for 30 min. After incubation, the cells were washed three times with PBS to remove unbound antibodies. The cells were then analyzed by flow cytometry, with isotype-matched antibodies used as negative controls. Ten samples were recorded for each group. 4 Each particle.

[0065] (2) Characterizing the labeling efficiency of cellular metabolic glycans

[0066] Ac4ManNAz-labeled cells were collected, with untreated cells serving as a control group. Cells were washed three times with pre-chilled PBS and centrifuged at 2000 rpm for 5 min at 4°C. The cell pellet was then collected, labeled with DBCO-Alexa Fluor 488 (AF488) (final concentration 10 μM), and brought to a final volume of 100 μL using DPBS reaction buffer containing 5 mM MgCl2, 4.5 g / L glucose, 1 mg / mL BSA, and 0.1 mg / mL yeast tRNA. The mixture was gently vortexed and incubated at room temperature in the dark for 1 h. After incubation, the cells were centrifuged at 2000 rpm for 5 min at 4°C. The supernatant was discarded, and the cells were washed twice with DPBS wash buffer containing 5 mM MgCl2 and 4.5 g / L glucose. The cell pellet was resuspended in wash buffer, and the fluorescence intensity of the AF488 channel was immediately detected using flow cytometry. Ten cells were recorded for each sample. 4 Each particle.

[0067] (3) Characterizing the binding of cellular DNA probes

[0068] Before the experiment, the stock solutions of DBCO-T1-FAM, S-T1-FAM, Aptamer-T2-FAM, and S-T2-FAM were mixed with DPBS solution containing 5 mM MgCl2, denatured at 95 ℃ in the dark for 5 min, and then cooled to room temperature on ice. Cells with or without Ac4ManNAz labeling were collected, and DNA solutions of DBCO-T1-FAM (final concentration 500 nM), S-T1-FAM (final concentration 500 nM), Aptamer-T2-FAM (final concentration 200 nM), and S-T2-FAM (final concentration 200 nM) were added respectively. The volume was adjusted to 100 μL using DPBS reaction buffer containing 5 mM MgCl2, 4.5 g / L glucose, 1 mg / mL BSA, and 0.1 mg / mL yeast tRNA. The mixture was gently vortexed and incubated at 37 ℃ in the dark for 30 min. After incubation, the cells were then... Centrifuge at 2000 rpm for 5 min at ℃, discard the supernatant, and wash the cells twice with DPBS washing buffer containing 5 mM MgCl2 and 4.5 g / L glucose. After resuspending the cell pellet with an appropriate amount of washing buffer, immediately detect the fluorescence intensity of the FAM channel of the sample using flow cytometry. Record 10 samples per group. 4 Each particle.

[0069] 1.6 Western blot analysis to characterize PD-L1 glycosylation levels in cells

[0070] (1) Collect A375 and DC2.4 cells in the logarithmic growth phase, centrifuge, collect the precipitate, add twice the volume of RIPA protein lysis buffer containing protease inhibitors to the precipitate, and vortex thoroughly to mix; sonicate on ice and lyse the cells at 4 °C for 30 min, vortex once every 5 min, repeat 6 times, centrifuge at 12000 g at 4 °C for 20 min; aspirate the supernatant into a pre-cooled new EP tube, and detect the protein concentration by BCA method.

[0071] (2) Take 30 µg of protein sample, add 2 μL of glycoprotein denaturation buffer, add distilled water to the system to 20 μL, denature at 100℃ for 10 min, then add buffer, NP-40 and PNGase F, and incubate at 37 ℃ for 1 h; use the same treated sample without PNGase F as a control. Add loading buffer to all samples and boil to denature, then store at -80 ℃ for later use.

[0072] (3) Take 30 µg of denatured protein for SDS-PAGE electrophoresis. After separation by constant voltage at 80 V and 120 V, transfer the protein to a PVDF membrane. After transfer at constant current of 210 mA, block the membrane with 5% blocking solution at room temperature for 2 h.

[0073] (4) After blocking, wash the membrane with TBST, add diluted primary antibody and incubate overnight at 4 ℃; after washing thoroughly with TBST, add diluted secondary antibody and incubate at room temperature for 1 h, wash the membrane again, and use chemiluminescence imaging to detect the PD-L1 glycosylation expression level.

[0074] 1.7 Laser confocal fluorescence microscopy was used to verify the proximity linkage of DNA probes on PD-L1 on the cell surface.

[0075] DBCO-T1-Cy5 and Aptamer-T2-Cy3 were synthesized, and the binding and adjacency connectivity of the two probes on the same glycogen metabolism-labeled cell surface PD-L1 were observed by fluorescence resonance energy transfer (FRET). Cells were seeded in complete medium containing 50 μM Ac4ManNAz and cultured for 48 h. DBCO-T1-Cy5 (final concentration 500 nM), Aptamer-T2-Cy3 (final concentration 200 nM), and Connector (final concentration 500 nM) were added according to the groupings, and the volume was adjusted to 200 μL using DPBS reaction buffer. Cells were incubated at 37 ℃ in the dark for 30 min. After incubation, the supernatant was discarded, and the cells were gently washed twice with washing buffer. After fixation, washing, DAPI staining, DPBS washing, and mounting, images of the Cy3 (Ex: 543 nm, Em: 560-610 nm), Cy5 (Ex: 638 nm, Em: 650-700 nm), and FRET (Ex: 543 nm, Em: 650-700 nm) channels were captured using a laser scanning confocal fluorescence microscope to observe the binding of DNA probes and the formation of FRET.

[0076] 1.8 Parameter optimization for HCR assembly efficiency on the surface of live cells

[0077] Before the experiment, the stock solutions of DBCO-T1, Aptamer-T2, and Connector were mixed with DPBS solution containing 5 mM MgCl2, and denatured at 95 ℃ in the dark for 5 min before cooling to room temperature. The stock solutions of H1-FAM and H2 were mixed with DPBS solution containing 5 mg Cl2 and denatured at 60 °C in the dark for 10 min, then cooled to room temperature. Ac4ManNAz-labeled cells were collected and incubated with denatured DBCO-T1 and Aptamer-T2 cells in DPBS reaction buffer at 37 °C for 30 min. After incubation, the supernatant was discarded, and the precipitate was washed twice with DPBS washing buffer. The cell pellet was resuspended in an appropriate amount of reaction buffer. DNA solutions of Connector (final concentration 500 nM or 2.5 μM), H1 and H2 (final concentration 1 μM or 100 nM), and an appropriate amount of reaction buffer were added according to the cell group. After mixing, the mixture was gently shaken and incubated in the dark at 4 °C or 37 °C for different incubation times: 0 min, 20 min, 40 min, and 60 min. min; after incubation, the supernatant was discarded and the cells were washed. The fluorescence intensity of the FAM channel of the samples was detected by flow cytometry to compare the differences in HCR assembly efficiency between the H2-free group and the HCR group under different incubation times, temperatures and DNA probe concentrations.

[0078] 1.9 Verification of cell surface HCR assembly

[0079] Ac4ManNAz-labeled A375, MCF-7, and DC2.4 cells were collected. According to the grouping (Table 3), DNA solutions of DBCO-T1 (final concentration 500 nM) and Aptamer-T2 (final concentration 200 nM) and DPBS reaction buffer were added, respectively. After gentle mixing, the cells were incubated at 37 °C for 30 min. After incubation, the supernatant was discarded, and the precipitate was washed twice with DPBS washing buffer. The cell pellet was resuspended in an appropriate amount of reaction buffer, and DNA solutions of Connector (final concentration 500 nM), H1 (final concentration 1 μM; H1-FAM was used for flow cytometry, and H1-AF488 for confocal microscopy), and H2 (final concentration 1 μM) and an appropriate amount of reaction buffer were added. After mixing, the cells were incubated at 4 °C with gentle shaking for 1 h. After incubation, the supernatant was discarded, and the cells were washed twice. The fluorescence intensity of the FAM channel in the samples was detected using flow cytometry. Finally, images of the AF488 (Ex: 488 nm, Em: 505-525 nm) channel were captured using a confocal microscope to observe the assembly of the HCR.

[0080] Table 3 HCR Experimental Groups

[0081]

[0082] 2. Experimental Results

[0083] 2.1 HCR Principle

[0084] First, cells were cultured using a small-molecule metabolic marker, acetylated azide, to modify a mannosamine derivative (Ac4ManNAz) for metabolic glycan labeling (MGL). Subsequently, a copper-free bioorthogonal reaction was used to effectively label the dibenzocyclooctyne (DBCO)-conjugated anchoring trigger probe (DBCO-T1) onto the cell surface glycan. Simultaneously, another trigger probe (Aptmer-T2) conjugated to the PD-L1 aptamer (MJ5C) specifically recognized a non-functional epitope of the PD-L1 protein. Next, a connector probe was added, which hybridized to both DBCO-T1 and Aptamer-T2, bringing the two trigger probes closer together. This proximity effect promoted complementary binding, forming a functional DNA double-stranded structure that could open the fluorescently labeled H1 hairpin probe. Finally, the activated H1 and H2 probes triggered HCR, achieving signal amplification. This cascade reaction enables in-situ signal amplification, thereby allowing for sensitive in-situ detection of PD-L1 glycosylation on the cell surface. Figure 1).

[0085] 2.2 Successful induction of HCR assembly in buffer system

[0086] After the reaction in the buffer system, firstly, agarose gel electrophoresis was used to observe that the extended probes of DBCO-T1 and Aptamer-T2 triggered the generation of HCR products under the action of the connector. Figure 2 a, L1). Conversely, in the absence of DBCO-T1 or Aptamer-T2 or other HCR probes, no HCR products were observed in the other lanes. Figure 2 a, L2-L6); Next, the fluorescence intensity of each group was compared using a fluorescence spectrophotometer. The results showed that when DBCO-T1 and Aptamer-T2 hybridized, the HCR assembly of H1 and H2 was triggered, thereby opening the hairpin structure of H1, relieving the quenching effect of the BHQ probe on FAM fluorescence, and increasing the fluorescence intensity (a, L2-L6); Figure 2 (bc). The above results indicate that the designed DNA probe combination can successfully induce HCR assembly in a buffer system.

[0087] 2.3 Validation of PD-L1 and its glycosylation expression, and cellular glucose metabolism markers in A375 cells.

[0088] Flow cytometry confirmed that A375 cells were PD-L1 positive, while MCF-7 cells were PD-L1 negative. Figure 3 a); Next, the total protein of A375 cells was deglycosylated by PNGase F enzyme. Western blotting showed a decrease in the molecular weight of PD-L1 protein, indicating that N-glycosylation modification exists on the PD-L1 protein of A375 cells. Figure 3 b); Then, A375 and MCF-7 cells were labeled with metabolic glycans using Ac4ManNAz. Cells were tagged with AF488 fluorescently via a copper-free bioorthogonal reaction between DBCO-AF488 and Ac4ManNAz on the labeled cells. Fluorescence intensity was detected by flow cytometry. The results showed that glucose metabolism labeling was successfully achieved on A375 and MCF-7 cells. Figure 3 c).

[0089] 2.4 Characterizing the binding and adjacency linkage of DNA probes on the cell surface

[0090] First, the binding of cells to the FAM-labeled DBCO-T1 was investigated. Flow cytometry revealed that, compared to the FAM-labeled control group without DBCO(T1), A375 and MCF-7 cells labeled with Ac4ManNAz were able to bind to DBCO-T1 more effectively. Figure 4a); Then, the binding of cells to the FAM fluorescently labeled Aptamer-T2 was analyzed. The results showed that, compared to the FAM fluorescently labeled control group without PD-L1 aptamer (T2), A375 cells were recognized by Aptamer-T2 with or without Ac4ManNAz labeling, while PD-L1-negative MCF-7 cells were not effectively recognized. Figure 4 b); Finally, laser confocal fluorescence microscopy revealed that the DBCO-T1-Cy5 and Aptamer-T2-Cy3 fluorescent probes successfully bound to the cell surface and elicited fluorescence resonance energy transfer (FRET) signals, indicating that both fluorescent probes bound to the same PD-L1 protein and its glycosylation structure. Furthermore, compared to the group without a connector, the FRET signal was enhanced in the connector group, suggesting that the connector probes can enhance the FRET between the DBCO-T1-Cy5 and Aptamer-T2-Cy3 fluorescent probes. Figure 4 c). In summary, cells can effectively bind to Aptamer-T2 and DBCO-T1, providing a basis for in situ detection of PD-L1 glycosylation on the cell surface.

[0091] 2.5 Parameter optimization for HCR assembly efficiency on the surface of live cells

[0092] Next, we further optimized the assembly efficiency of HCR on the surface of live cells. To ensure detection specificity, a two-step reaction procedure was adopted: metabolically labeled cells were first incubated with DBCO-T1 and Aptamer-T2, unbound probes were washed away, and then Connector and H1 and H2 were added to induce neighbor recognition and trigger HCR; subsequently, key parameters were optimized. Temperature optimization revealed that, compared with the group without H2, the second-step incubation temperature of 4 ℃ was more conducive to obtaining higher HCR efficiency than 37 ℃. Figure 5 a); Concentration optimization revealed that when the final reaction concentrations of Connector, H1, and H2 were 500 nM and 1 μM, respectively, the HCR group obtained more significant fluorescence signals, indicating that the HCR assembly efficiency was effectively improved. Figure 5 b); Furthermore, flow cytometry revealed that the HCR assembly efficiency was highest when the second incubation time was 60 minutes. Figure 5 c).

[0093] 2.6 In situ visualization of PD-L1 glycosylation on PD-L1 positive model cells based on HCR strategy

[0094] After optimizing the reaction conditions, the feasibility of achieving in situ visualization and signal amplification of PD-L1 glycosylation in PD-L1 positive model cells based on the HCR method was further explored. First, the HCR signal on A375 cells was detected using laser confocal fluorescence microscopy. The results showed that, compared with the H2-free group, the HCR assembly synergistically induced by nucleic acid aptamers and glycogen metabolism markers enhanced the signal of PD-L1 glycosylation on the cell surface by approximately 16.87 times, while the background signal in each control group remained at a low level. Figure 6 ab). Flow cytometry also confirmed that A375 cells in the HCR group showed significantly enhanced fluorescence signals ( Figure 6 c). In contrast, no significant HCR signal was observed in PD-L1-negative MCF-7 cells, either in the H2-free group or the HCR group. Figure 7 This suggests that the HCR strategy can achieve specific assembly on the surface of PD-L1 positive cells. In summary, the adjacent dual-label induced HCR strategy can achieve in situ detection and signal amplification of PD-L1 glycosylation.

[0095] 2.7 In situ visualization of PD-L1 glycosylation on dendritic cells (DCs) based on HCR strategy

[0096] The results showed that DC2.4 cells were PD-L1 positive ( Figure 8 a). The peptide-N-glycosidase F treatment experiment further confirmed that PD-L1 in DC2.4 cells exhibits N-glycosylation modification (a). Figure 8 b). Based on this, the feasibility of the HCR strategy in DC2.4 cells was evaluated. The results showed that, compared with the H2-free control group, HCR enhanced the in situ fluorescence signal of PD-L1 glycosylation on DC2.4 cells by approximately 13.25 times (b). Figure 8 cd). Flow cytometry results also confirmed the enhanced fluorescence signal produced by HCR on the surface of DC2.4 cells (cd). Figure 8 e). In summary, PD-L1 in DC2.4 cells exhibits N-glycosylation modification, and the proximity-dual recognition-induced HCR strategy can also effectively achieve in situ detection and signal amplification of PD-L1 glycosylation in DC2.4 cells.

[0097] 2.8 Performance Validation of HCR-Based PD-L1 Glycosylation Detection Method on DC Surface

[0098] Further verification of the performance of the HCR detection method established in the aforementioned study yielded the following results: Figure 9 As shown.

[0099] Figure 9The results showed that as the PNGase F treatment time increased from 0 h to 24 h, the intensity of the green fluorescence signal of HCR on the cell surface decreased significantly in a time-dependent manner, confirming that the HCR signal can sensitively respond to the downregulation of glycosylation level of cell surface glycoproteins.

[0100] Subsequently, the target specificity of the method was verified through aptamer competition experiments, and the results were as follows: Figure 9 As shown in b~d, with the increase of MJ5C dose, the aptamer recognition signal ( Figure 9 b) and subsequent HCR amplification signal ( Figure 9 c) All showed a dose-dependent decrease, indicating that the signal output of this detection system depends on the specific recognition of the aptamer by the PD-L1 target. Furthermore, the HCR fluorescence intensity of the Ac4ManNAz-treated group was significantly higher than that of the untreated group ( Figure 9 d) This directly confirms that the generation of the HCR signal depends on the successful labeling of Ac4ManNAz.

[0101] Finally, the stability of the HCR detection method was verified by flow cytometry. Figure 9 (e) The results showed that this method is resistant to minor fluctuations in temperature and incubation time, ensuring the reproducibility and comparability of experimental results. Furthermore, the HCR method does not affect DC cell survival or phenotype.

[0102] Example 2: Study on the biological function of PD-L1 glycosylation on the surface of DCs based on HCR

[0103] 1. Experimental Methods

[0104] 1.1 Investigating the effect of glycosylation on the PD-1 binding ability of PD-L1 on the surface of DCs based on HCR

[0105] First, Ac4ManNAz-labeled DC2.4 cells were incubated for 24 h at 37°C in a 5% CO2 incubator in complete culture medium containing different concentrations of PNGase F (0 U / mL, 100 U / mL, 200 U / mL) or tunicamycin (TM) (0 nM, 10 nM, 100 nM) to inhibit N-glycosylation modification. Then, the treated cells were incubated with DBCO-T1 and Aptamer-T2 in reaction buffer at 37°C for 30 min. After washing, Connector, H1-FAM, and H2 were added, and the cells were gently shaken at 4°C for 1 h to achieve HCR assembly. To detect the PD-1 binding capacity of PD-L1 cells, the incubated cells were collected, washed, and incubated with 2 μg / mL mouse biotinylated PD-1 protein at room temperature for 1 h. After washing, streptavidin-labeled PE (SA-PE) was added, and the cells were incubated at room temperature in the dark for 30 h. The cells were washed twice with washing buffer after a minimum of 1 minute. The fluorescence intensity of the FAM and PE channels was detected by flow cytometry. The fluorescence intensity of the FAM channel reflects the glycosylation level of PD-L1 on the cell surface, while the fluorescence intensity of the PE channel reflects the binding of PD-L1 to PD-1 protein on the cell surface. A DNA probe-deficient control was included in the experiment. Pearson correlation analysis was used to analyze the correlation between the fluorescence intensity of HCR signal and PD-1 binding signal on the DC2.4 cell surface, and the Pearson correlation coefficient was calculated.

[0106] 1.2 Bone marrow-derived dendritic cells (BMDCs) and CD8 + Preparation of T cells

[0107] Mononuclear cells (BMDCs) were isolated from the bone marrow of 6-8 week old C57BL / 6 mice and seeded in RPMI 1640 medium containing 20 ng / mL GM-CSF, 20 ng / mL IL-4, 10% FBS, and 1% penicillin-streptomycin. The medium was replaced with fresh medium containing cytokines every 3 days (half the volume was replaced). Immature BMDCs were harvested on day 7, stained with APC-labeled anti-CD11c antibody, and cell purity was assessed by flow cytometry. To induce maturation, immature BMDCs were further stimulated with 100 ng / mL LPS and 20 ng / mL IFN-γ for 48 hours. Cell morphology was observed under a light microscope, and the maturation status of dendritic cells was analyzed by flow cytometry using APC-anti-CD11c, PE-anti-CD80, and FITC-anti-CD86 antibody staining. Furthermore, EasySep was used to analyze the cell maturation status. TM mouse CD8 + CD8 cells were isolated and purified from the spleen of C57BL / 6 mice using a T-cell sorting kit, following the manufacturer's instructions. +T cells; after staining with APC-labeled anti-CD8 antibody, CD8 was detected by flow cytometry. + T cell purity; Dynabeads were used to maintain their proliferative capacity. TM Mouse T activator CD3 / CD28 stimulates CD8 + T cells were cultured in RPMI 1640 medium containing 30 U / mL IL-2, 0.05 mM β-mercaptoethanol, 1 mM sodium pyruvate, 10 mM HEPES, 10% FBS, and 1% penicillin-streptomycin. CD8 cells were collected at specified time points. + T cells were used in subsequent experiments.

[0108] 1.3 Analysis of the effect of PD-L1 glycosylation on BMDCs-mediated CD8 + Regulation of T cell proliferation

[0109] To prepare tumor antigen-specific tumor cell lysates (BMDCs), the mouse leukemia cell line C1498 was treated with a 5-cycle freeze-thaw cycle to prepare tumor cell lysates (TCL). Mature BMDCs were then co-incubated with TCL (200 µg protein / 1 × 10⁻⁶). 6 Cells / mL), along with PNGase F, were incubated in a 37 °C, 5% CO2 incubator for 24 hours; subsequently, the collected BMDCs were incubated with CD8... + T cells were seeded at a 1:5 ratio in 24-well plates and co-cultured. After 3 days of co-culture, cells were collected and subjected to APC-anti-CD8 surface staining and PE-anti-Ki67 intracellular staining. After washing with PBS, CD8+ was detected by flow cytometry. + T cell proliferation was analyzed, and the effect of PD-L1 glycosylation on CD8 on BMDCs was examined. + Regulation of T cell proliferation.

[0110] 2. Experimental Results

[0111] 2.1 Investigating the effect of glycosylation on the PD-1 binding ability of PD-L1 on DCs using HCR

[0112] The adjacent dual-label induced HCR strategy developed in Example 1 was employed. This strategy targets the non-functional epitope of the PD-L1 protein, enabling sensitive detection of its glycosylation level without interfering with the biological function of the target glycoprotein. This provides a powerful tool for functional studies of PD-L1 glycosylation on dendritic cells (DCs). To verify the applicability of this strategy, we first investigated whether metabologlycan labeling and HCR products affect the binding of PD-L1 to PD-1 protein on DCs. Figure 10As shown in Figure a, flow cytometry demonstrated that neither metabolized glycan labeling nor HCR assembly on DCs significantly altered the PD-1 protein binding capacity of PD-L1. This low interference may be due to the difference between the selected PD-L1 aptamer and the binding epitope of PD-1 on PD-L1, and the rigid structure of the double-stranded DNA product avoiding spatial obstruction of the PD-1 binding site. Next, we further explored whether the HCR strategy could sensitively monitor subtle changes in the PD-L1 glycosylation level on the cell surface, and further investigated the effect of glycosylation on the PD-1 binding capacity of PD-L1 on DCs. The results showed that with the increase of N-glycosylation inhibitor treatment concentration, the fluorescence intensity of HCR amplification gradually decreased, indicating a decrease in the PD-L1 glycosylation level. At the same time, the number of PD-1 proteins bound to PD-L1 on the DC surface also gradually decreased. Figure 10 These results indicate that the HCR assembly strategy can detect subtle changes in PD-L1 glycosylation levels on DCs, and that PD-L1 glycosylation on DCs can promote PD-L1 / PD-1 interactions. More importantly, PD-L1 glycosylation signals are positively correlated with PD-1 signals (Pearson correlation coefficient = 0.8918, P = 0.0169). Figure 10 (e) suggests the potential of HCR signaling to characterize the PD-1 binding capacity of PD-L1 on DCs. In summary, the HCR strategy enables sensitive monitoring of PD-L1 glycosylation levels on the cell surface and further confirms the crucial role of PD-L1 glycosylation on DCs in regulating PD-L1 / PD-1 binding.

[0113] 2.2 Investigating the effect of PD-L1 glycosylation on CD8 in DCs based on HCR + Regulation of T cell proliferation

[0114] Given that PD-L1 glycosylation on DCs is crucial for regulating PD-L1 / PD-1 interaction, we further investigated its role in DC-mediated immune function. First, mature BMDCs were obtained by inducing differentiation from C57BL / 6 mouse bone marrow mononuclear cells. Figure 11 a). Microscopic observation showed that, compared with immature BMDCs, mature BMDCs exhibited reduced cell colonies, more elongated dendrites, and outward expansion. Figure 11 b). Meanwhile, PD-L1 expression was significantly upregulated during BMDC maturation ( Figure 11 c); Notably, significant PD-L1 glycosylation signals were detected in mature BMDCs using HCR combined with flow cytometry. Figure 11 d). Next, we investigate whether PD-L1 glycosylation on DCs affects CD8. +T cell proliferation. As core antigen-presenting cells in the tumor microenvironment, dendritic cells (DCs) activate CD8+ proliferation by capturing and presenting tumor-associated antigens. + Cytotoxic T lymphocyte-mediated antitumor immune responses. Previous studies have shown that dendritic cells (DCs) loaded with tumor-cell lysates (TCLs) (i.e., tumor antigen-specific BMDCs) can effectively induce CD8+. + Cytotoxic T lymphocyte-dependent antitumor immunity. Therefore, we loaded mature BMDCs with TCL derived from the mouse leukemia cell line C1498, treated them with PNGase F, and then combined them with CD8 derived from mouse spleen. + T cells were co-cultured, and then flow cytometry was used to observe the PD-L1 glycosylation level and CD8 levels on the surface of BMDCs. + Changes in T cell proliferation capacity Figure 11 e). The results showed that PNGase F treatment significantly reduced the HCR fluorescence intensity, confirming that the HCR strategy can sensitively detect subtle changes in PD-L1 glycosylation levels on BMDCs. Figure 11 f). Importantly, CD8+ co-cultured with PNGase F-pretreated BMDCs (dg-BMDCs) + T cells showed significantly higher expression of the proliferation marker Ki67 compared to the group co-cultured with untreated BMDCs (g-BMDCs). This result suggests that PD-L1 glycosylation on BMDCs may affect CD8+ expression. + T cell proliferation has an inhibitory effect ( Figure 11 g). In summary, the above results indicate that PD-L1 glycosylation on DCs may play a key role in immunosuppression in the tumor microenvironment, expanding our understanding of PD-L1 glycosylation on antigen-presenting cells as a novel checkpoint regulatory mechanism of tumor immunity, and highlighting its important value as a potential immunotherapeutic target.

[0115] Example 3: Verification of Method Universality

[0116] To verify the cellular universality of this HCR detection method, human lung adenocarcinoma cells A549 (tumor cell model) and mouse macrophages RAW264.7 (immune cell model) were further selected for experiments. The results are as follows: Figure 12 As shown, both cell lines with intact HCRs exhibited a significant rightward shift of FAM fluorescence signal, while the background signal in all control groups remained at a low level. Figure 12 (a) This demonstrates that the method can be specifically triggered in PD-L1 positive cells from different sources, and is not limited to a single cell type.

[0117] To further validate the applicability of this method for dynamic glycosylation analysis, RAW264.7 cells were used as a model to examine the effect of PNGase F treatment at different times on HCR assembly efficiency. The results showed that with prolonged deglycosylation treatment time, the FAM fluorescence intensity of HCR decreased significantly in a time-dependent manner. Figure 12 (b) indicates that this method can effectively quantitatively reflect the dynamic changes in PD-L1 glycosylation levels on the cell surface and is suitable for glycosylation function studies in various cell types.

[0118] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A method for in-situ visualization detection of cell surface PD-L1 glycosylation based on hybridization chain reaction, characterized in that: Includes the following steps: (1) Acetylated azide-modified mannosamine derivatives were used to label metabolic glycans in cells; (2) The DBCO-T1 probe and Aptamer-T2 probe were bound to the glycan and PD-L1 protein on the cell surface, respectively, and unbound probes were washed away. (3) Add Connector probe, H1 hairpin probe and H2 hairpin probe to make DBCO-T1 and Aptamer-T2 adjacent to each other, thereby triggering the cascade hybridization chain reaction of H1 and H2 hairpin probes to achieve signal amplification; (4) PD-L1 glycosylation was visualized in situ by flow cytometry or laser confocal fluorescence microscopy.

2. The method for in-situ visualization detection of cell surface PD-L1 glycosylation based on hybridization chain reaction according to claim 1, characterized in that: The sequence of the Aptamer-T2 probe is shown in SEQ ID NO: 1; the sequence of the DBCO-T1 probe is shown in SEQ ID NO: 2; the sequence of the Connector probe is shown in SEQ ID NO: 3; the sequence of the H1 hairpin probe is shown in SEQ ID NO: 4; and the sequence of the H2 hairpin probe is shown in SEQ ID NO:

5.

3. The method for in-situ visualization detection of cell surface PD-L1 glycosylation based on hybridization chain reaction according to claim 1, characterized in that: The cascade hybridization reaction is carried out in a two-step process. First, it is incubated with DBCO-T1 and Aptamer-T2, and then washed before adding Connector, H1 and H2 to carry out the reaction.

4. The method for in-situ visualization detection of cell surface PD-L1 glycosylation based on hybridization chain reaction according to claim 3, characterized in that: The second step reaction conditions are: incubation time 20~60 min, incubation temperature 4~37 ℃; final concentration of connector 500 nM~2.5 μM, and final concentration of H1 and H2 100 nM~1 μM.

5. The method for in-situ visualization detection of cell surface PD-L1 glycosylation based on hybridization chain reaction according to claim 1, characterized in that: The cells include human melanoma cell line A375, human lung adenocarcinoma cell line A549, mouse dendritic cell line DC2.4, mouse bone marrow-derived dendritic cells BMDCs, and mouse macrophage cell line RAW264.

7.

6. The application of the detection method as described in any one of claims 1-5 in the preparation of cellular PD-L1 glycosylation detection reagents or tumor immune checkpoint function research kits.

7. An application of the detection method according to any one of claims 1-5 in studying the biological function of PD-L1 glycosylation on the surface of dendritic cells, characterized in that: Including the following applications: (1) Using the cascade hybridization chain reaction detection system, compare the binding ability of PD-L1 and PD-1 protein on the surface of dendritic cells in different glycosylation states; (2) Using the aforementioned cascade hybridization chain reaction detection system, assess the effect of PD-L1 glycosylation level on the surface of dendritic cells on CD8 cells co-cultured with them. + Regulatory role of T cell proliferation.

8. The method according to claim 7, characterized in that: The assembly of the cascade hybridization chain reaction detection system does not affect the binding ability of PD-L1 protein on the surface of dendritic cells; after treatment with PNGase F enzyme to downregulate PD-L1 glycosylation, the binding ability of PD-L1 to PD-1 was significantly reduced.

9. The method according to claim 7, characterized in that: Bone marrow-derived dendritic cells from mice with downregulated PD-L1 glycosylation treated with PNGase F enzyme and CD8 + T cells were co-cultured, and the detection method described above was used to verify that downregulation of PD-L1 glycosylation levels could enhance dendritic cells' resistance to CD8+. + The proliferative effect of T cells.

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