Laponite Lights Calcium Flickers by Reprogramming Lysosomes to Steer DC Migration for An Effective Antiviral CD8+ T‐Cell Response

Abstract Immunotherapy using dendritic cell (DC)‐based vaccination is an established approach for treating cancer and infectious diseases; however, its efficacy is limited. Therefore, targeting the restricted migratory capacity of the DCs may enhance their therapeutic efficacy. In this study, the effect of laponite (Lap) on DCs, which can be internalized into lysosomes and induce cytoskeletal reorganization via the lysosomal reprogramming–calcium flicker axis, is evaluated, and it is found that Lap dramatically improves the in vivo homing ability of these DCs to lymphoid tissues. In addition, Lap improves antigen cross‐presentation by DCs and increases DC‐T‐cell synapse formation, resulting in enhanced antigen‐specific CD8+ T‐cell activation. Furthermore, a Lap‐modified cocktail (Lap@cytokine cocktail [C‐C]) is constructed based on the gold standard, C‐C, as an adjuvant for DC vaccines. Lap@C‐C‐adjuvanted DCs initiated a robust cytotoxic T‐cell immune response against hepatitis B infection, resulting in > 99.6% clearance of viral DNA and successful hepatitis B surface antigen seroconversion. These findings highlight the potential value of Lap as a DC vaccine adjuvant that can regulate DC homing, and provide a basis for the development of effective DC vaccines.

DCs (1 × 10 6 cells/mL) were incubated with different doses of Lap (25 μg/mL, 50 μg/mL, and 100 μg/mL) for 12 h in RPMI-1640 complete medium in 6-well culture plates.Flow cytometry was used to detect the expression of costimulatory molecules (CD40, CD80, CD86, and MHC II) and chemokine receptors (CCR5, CCR7, and CXCR4) in the DCs. 1 × 10 6 cells were harvested by centrifugation at 400 rcf for 5 min, and then stained with the corresponding antibodies for 20 min at 4 °C.The reaction was stopped by washing twice with PBS and the cells were subjected to FACS analysis in an 8-color FACS Calibur (BD Biosciences, Mountain View, CA).For apoptosis detection, cells were stained with Annex Ⅴ (BD PharMingen).To detect antigen-presenting ability, DCs were stained with anti-H-2Kb-SIINFEKL after coincubation with OVA257-264 (1 µg/mL) for 12 h.Thereafter, DCs were collected and washed twice to remove the free antigens before use.Total cell populations were gated by CD11c for all the analyses.Data were collected and analyzed using FlowJo V10 software.The isotype contrast for each antibody was used as the gate control.

Cytokine Secretion Assay
For cytokine secretion assays, the medium supernatant were harvested and centrifuged at 500 rcf for 5 min at 4 °C.Supernatants were used to measure pro-inflammatory cytokines IL-6, IL-12p70, IL-1β, and TNF-α secretion with commercially available ELISA kits (Dakewe, Shenzhen, China) following the producer's protocol.The optical density of each well was determined using a microplate reader (Molecular Devices, USA) at 450 nm.

In vitro migration analysis of DCs
DCs derived from tdTomato transgenic mice were cocultured with different doses of Lap (25 μg/mL, 50 μg/mL, and 100 μg/mL). 2 × 10 5 DCs were seeded in 8-well Nunc™ Lab-Tek™ Chambered Coverglass (Thermo Scientific, USA), and the velocity and migration distance per unit time were monitored using a live cell time-lapse imaging system (PerkinElmer, USA).
Statistical analysis was performed using Volocity software.

Immunofluorescence staining and confocal microscopy
Cytoskeletal rearrangement of DCs was measured using an immunofluorescence assay after different treatments. 1 × 10 6 DCs subjected to different treatments were adhered to a 35mm confocal dishe at 37 °C for 2 h, fixed with 4% paraformaldehyde (Merck, Schwalbach, Germany) for 30 min, and permeabilized with 0.15% Triton X-100 in PBS (PBST) for 15 min at room temperature.The cells were labeled with 10 μg/mL rabbit anti-β-tubulin antibody (Abcam, ab179513) in 2% BSA in PBS at 4°C overnight, and then washed twice with PBS for 5 min each.After washing, the cells were incubated with 2% BSA in PBS for 30 min at room temperature.After blocking, DCs were incubated with AF594-conjugated phalloidin (Invitrogen, A12381), DAPI and 10 μg/mL AF488-conjugated goat anti-rabbit IgG (Abcam, ab150077) to visualize F-actin and β-tubulin.After incubation, cells were washed three times in PBS for 10 min each, and then dishes were mounted with the Prolong Gold Antifade reagent (Invitrogen) and observed under a laser confocal microscope (PerkinElmer, USA).

Lysosomal membrane permeabilization assay
Acridine orange (AO) (sigma, USA) was performed to assess the Lysosomal membrane permeabilization (LMP). 1 × 10 6 DCs subjected to different treatments were adhered to a 35mm confocal dishe at 37 °C for 2 h.DCs were incubated by AO (5 μg/mL) for 20 min at 37°C.After washing, DCs were visualized using a Zeiss LSM-900.The excitation wavelength was 488 nm, while the data were obtained at two separate emission wavelengths (505-560 nm, 590-690 nm).
The decreased red fluorescence and enhanced green fluorescence were the sign of LMP.
For galectin puncta assay, 1 × 10 6 DCs subjected to different treatments were replated on a well in a 35mm confocal dishe at 37 °C for 2 h.After replating, DCs were fixed with 4% paraformaldehyde (Merck, Schwalbach, Germany) for 30 min, permeabilized with 0.15% Triton X-100 in PBS (PBST) for 15 min at room temperature, and then washed twice with PBS for 5 min each.After washing, the cells were incubated with 2% BSA in PBS for 30 min at room temperature.After blocking, cells were incubated with the mixture of 10 μg/mL rabbit anti-LAMP1 pAb and 10 μg/mL mouse anti-galectin-3/LGALS3 mAb (Abclonal, A13506) in 2% BSA in PBS at 4°C overnight.The cells were washed three times in PBS for 10 min each, and then incubated with the mixture of DAPI, 10 μg/mL Alexa Fluor 647 anti-mouse IgG and 10 μg/mL Alexa Fluor 488 anti-rabbit IgG (Abclonal, AS053) in 2% BSA in PBS for 2 h at room temperature.After incubation, cells were washed three times in PBS for 10 min each, and then visualized using a Zeiss LSM-900.

Cytosolic Ca 2+ measurements
For cytosolic Ca 2+ imaging, DCs were replated on a well in a 35mm confocal dishe and washed 3 times with HBSS (Ca 2+ free) . 2 × 10 5 DCs were loaded with 1 μM Fluo-4 AM (Dojindo Laboratories, F311) in HBSS for 30 min at 37°C.After incubation, cells were washed 3 times with HBSS to adequately remove residual Fluo 4-AM working solution, and then add 400μL HBSS to cover the cells.After DCs were treated by Lap (50 μg/mL) for 9 mins, imaging was performed at 20 ms per frame using a Zeiss LSM-900.To explore the role of Lap in DC-T-cell interactions, FITC-labeled Lap was added to the coculture system at a dose of 25 µg/mL.Thereafter, the attachment of Lap to DCs and T cells was observed using a live cell imaging system and measured using Imaris 9.0.1 software.
To detect in vitro activation of T-cells, Lap-pretreated and OVA257-264-pulsed DCs were cocultured with CD8 + T-cells isolated from OT-I transgenic mice at a ratio of 1:5 (DCs:T-cells) for 72 h.T-cell activation was detected by costaining for the markers CD8α with respective CD25, CD69, and CD107α.All samples were tested by FACS and analyzed by FlowJo V10, and the CD8α + cell population was gated as T-cells.

Label-free quantitative proteomics analysis
Lap-treated and control DCs were collected with urea (300 μL of 8 M) and protease inhibitor (10% of lysate) added.After centrifugation at 14,000 rcf for 20 min, the supernatant was collected and the protein concentration was determined by Bradford's method, and a 100 µg aliquot of sample was taken for LC-MS/MS measurement.Label-free mass spectra were analyzed using MaxQuant software, and protein data were screened by the Beijing QLBio Company using the UniProt database.

Bioluminescence imaging of DC homing in vivo
Fluc + DCs (2 × 10 6 ) were injected into the footpads of wild-type mice and monitored every 24 h using an IVIS (PerkinElmer, USA).The migration rate was calculated by the ratio of fluorescence signal in the popliteal lymph nodes and inguinal lymph nodes to the signal on the footpads.Representative images and SI were captured and measured using Living Image 4.5.2software.

Adenovirus challenge mouse model and in vivo T-cell priming
OVA257-264-pulsed DCs were incubated with Lap and successively injected into the footpads of mice on days 1 and 7. On day 14, mice were challenged with Ad-OVAp-Fluc.Viral clearance was monitored every 24 h using IVIS.Mice were euthanized on day 16 and LLNs were collected for lymphocyte fractionation and pathological analysis.

Anti-HBV evaluation based on HBV mouse model
On day 1, a plasmid carrying HBV overfull-length 1.2 DNA and Fluc + (pGL3/Fluc-HBV1.2) was hydrodynamically injected into mice to establish a model of HBV infection.DC vaccines were administered via footpads on days 7 and 14.HBV clearance was monitored at 1 week intervals using IVIS, and the mice were euthanized 7 days after the secondary vaccination for virological, serological, and toxicological analyses.

Analysis of Lap degradation products
The lysosomes of DCs were extracted using the Lysosome Enrichment Kit (Thermo Scientific, USA) and the lysates were titrated into the Lap dispersion.The resulting product was freeze-dried, analyzed, and tested by the Shanghai WEIPU Testing Technology Group Ltd.

For
image analysis, F0 was defined as the background-subtracted fluorescence intensity at the initial time point, F denoted the background-subtracted fluorescence intensity at each time point, and ΔF / F0 = (F -F0) / F0 reflected the changes in cytosolic Ca 2+ concentration of DCs during Lap internalization.DC-T-cell interaction and in vitro T-cell primingDCs derived from tdTomato transgenic mice were incubated with OVA257-264 for 12 h and then incubated for another 12 h with different doses of Lap.CD8 + T-cells were isolated and purified with mouse CD8 + T Cell Isolation Kit (Miltenyi Biotec, 130-104-075) from OT-I transgenic mice following the producer's protocol.Then CD8 + T-cells labeled with CellTracker™ Deep Red (Thermo Scientific, C34565, 5μM).Thereafter, the DCs were cocultured with CD8 + T-cells at a 1:5 ratio (DCs: T-cells).Interactions between DCs and Tcells were monitored using a live cell imaging system (PerkinElmer, Massachusetts, USA), and colocalized areas visualized in dynamic images were analyzed using Imaris 9.0.1 software to calculate the DC-T-cell contact volume and duration.

Figure S3 .
Figure S3.GO enrichment analysis for up-regulated DEPs.The bar color indicates the GO categories, and top 20 GO terms are shown.

Figure S5 .
Figure S5.DC homing to popliteal lymph nodes (PLNs) detected by tissue staining.GFP expressing-DCs were treated with different doses of Lap and then were injected into the footpads of mice.After 72 h, mice were euthanized, and the PLNs were harvested for tissue section.Green: DCs; Blue: nuclei.

Figure S6 .
Figure S6.GO enrichment analysis for up-regulated DEPs involving vesicular transport and Ca 2+ homeostasis.The bar color indicates the GO categories.

Figure S7 .
Figure S7.Cytoskeletal rearrangement of DCs after co-incubation with HBSS, Lap, TG+Lap and BAPTA-AM+Lap.a) Representative images of cytoskeletal rearrangement of DCs.Red: F-Actin; Green: β-Tubulin; Blue: Nuclei.The mean fluorescence intensity of F-actin and βtubulin was measured and displayed as b).Data represent mean ± SD (n =10 statistics are shown

Figure S9 .
Figure S9.LPO accumulation in DCs after Lap co-incubation.Left panel: CLSM images of DCs treated with Lap for 1 h.Green: LPO indicator; Red: LysoTracker; Blue: rhodaminelabeled Lap.Right panel: fluorescence intensity statistics along the green arrows.

Figure S12 .
Figure S12.Dynamic monitoring of Cyto-Ca 2+ in DCs.a) Representative Cyto-Ca 2+ transients frames.b) Cyto-Ca 2+ transients amplitudes were quantified by measuring the change in maximum fluorescence intensity of Fluo-4A.Statistics are shown as mean ± SD (n = 21, "n" represents the number of cells observed per experimental group).).*: P < 0.05 compared with Lap group.

Figure S14 .
Figure S14.The statistical data of the homing percentage of tissue-resident DCs homing to lymph nodes.Data showed as mean ± SD (n = 3).*: P < 0.05 compared to PBS group.

Figure S15 .
Figure S15.Dynamic monitoring of Cyto-Ca 2+ in DCs.DCs were challenged with Lap ± Nacetylcysteine (NAC, ROS inhibitor, 5 mM).a) Representative tracking of the Cyto-Ca 2+ dynamics of DCs labeled with Fluo-4AM.b) Cyto-Ca 2+ transient amplitudes were quantified by measuring the change in maximum fluorescence intensity of Fluo-4AM.Statistics are shown as mean ± SD (n = 21, "n" represents the number of cells observed per experimental group).c) Percentage of DCs exhibiting Cyto-Ca 2+ transients after different pretreatment.*P < 0.05 between the two groups indicated.

Figure S17 .
Figure S17.SEM images of DC-T cell contact.Green arrows: DCs; Red arrows: CD8 + T cells derived from OT-I TCR transgenic mice.

Figure S18 .
Figure S18.BAPTA-AM pretreatment impaired Lap enhanced DC-T cell IS formation.DCs from tdTomato transgenic mice were incubated with Lap (50 µg/mL).CD8 + T cells from OT-I transgenic mice were purified by CD8 + T Cell Isolation Kit (MiltenyiBiotec, Germany) and were labeled with the CSFE (Thermo Scientific, USA).Thereafter, DCs treated differently were cocultured with the labeled T cells at the ratio of 1:5 (DC: T cell).The interactions between DCs and T cells were monitored using a live cell imaging system (PerkinElmer, Massachusetts, USA).a) The colocalized areas of DC-T cells were analyzed using Volocity software.Red: DCs from tdTomato transgenic mice; Green: CD8 + T cells from OT-I transgenic mice.b) The percentage of DC-T cell colocalization volume / DC volume.Data are mean±s.d.; n = 24 fields of view are shown; *P < 0.05 between the two groups indicated.

Table S2 .
The whole blood count of mice.