DOI: 10.5061/dryad.zpc866tk7
title: Data from: A nanovaccine for immune activation and prophylactic protection of atherosclerosis in mouse models

description: The raw flow cytometry files uploaded contain the activation of bone
 marrow derived dendritic cells (BMDCs), the proliferation profile after
 co-culture of BMDCs and T lymphcytes. We also analyzed splenic and
 lymphatic cells from C56BL/6J and ApoE knockout (KO) mice after
 vaccination of our nanovaccines (D+P, SP-D1P1, SP-D1+P1, and SP-P).
description: For in vitro experiments such as to check the surface expression
 of different markers, the collected cells were stained with antibodies
 before staining with Zombie Aqua fixable viability dye  Finally, the cells
 were resuspended in 200 µL FACS buffer (5 mM EDTA and 1% FBS in PBS) for
 flow cytometry analysis by BD FACSCantoII or a BD FACSCeleste cell
 analyzer. Data were analyzed by the FlowJo software (Tree Star) or the FCS
 Express 7 software (Reachsoft). For in vivo experiments
 at 10 weeks and 26 weeks of age, the immunized
 ApoE<sup>-/-</sup> mice were anesthetized for blood collection
 and euthanized as indicated above. The spleens and dLNs were excised,
 minced, and filtered (70 μm; BD Biosciences) to generate a single-cell
 suspension and centrifuged at 4 °C at 1800 rpm for 10 min. After lysis of
 red blood cells, the pelleted splenocytes were resuspended in FACS buffer
 together with antibodies. The dLNs were extracted and passed through a 70
 μm cell strainer, stained with antibodies. The cells were then stained
 with Zombie Aqua Fixable Viability Kit and resuspended in 200 µL of FACS
 buffer for flow cytometry analysis by a flow cytometer (BD FACSCantoII).
 Data were analyzed by the FlowJo software (Tree Star).
description: # Data from: A potent athero-protective nanovaccine cocktail by
 two-pronged intracellular delivery of antigen and adjuvant for dendritic
 cell activation
 [https://doi.org/10.5061/dryad.zpc866tk7](https://doi.org/10.5061/dryad.zpc866tk7) ## Description of the data and file structure The samples were named after the type of nanovaccines, antigens, and/or adjuvants administrated. The BMDCs were directly incubated with them while the animals were injected by subcutaneous injections at the ages of 7, 9, and 12 weeks. Untreated mean negative control; PEG-SP indicates the superparamagnetic nanoparticles coated with PEG (nanoparticle control); p210 is the antigen used for managing atherosclerosis; CpG-ODN is the adjuvant. D+P indicates the physical mixture of CpG-ODN and p210; Sp-D indicates PEG-SP coated with only CpG-ODN; SP-P indicates PEG-SP coated with only p210; SP-DxPy indicates the PEG-SP co-delivering both CpG-ODN and p210, where x and y are the molar ratios of them; SP-Dx+Py indicates the mixture of SP-D and SP-P at the molar ratio of x and y.  FITC, APC, PE, BV786, ECD, PE-Cy5, BV421, BV650 were the names of the fluorophores conjugated to the antibodies indicated. The samples named with them are the single stains for the compensation of each panel. 1. Fig. 4 D80-FITC CD86-PE This set of experiment was used to assess the optimised nanovaccine composition in promoting the co-signal expression of BMDCs after 24 h. 2. Fig. 4 MHC-I-PE and MHC-II-FITC This set of experiment was used to assess the optimised nanovaccine composition in promoting the co-signal expression of BMDCs after 24 h. 3.  Fig. 5 Functional assay of activated BMDC: The activated BMDCs by nanovaccines were co-cultured with splenocytes, and the effector CD4+ and CD8+ T cells were analyzed to understand how they promote cellular immunity ex vivo. In the folder the file named as "compensation file. compensation. compensation" is the compensation file used for the panel. 4. Fig. 6 FITC-positive dendritic cells: at 24 h after one single dose injection of fluorescnetly labeled nanovaccines, the dLN of C57BL/6 mice were collected, grounded to single suspensions and analysed for the draining and retention of nanoparticles in the DCs in dLN. 5. Fig. 8 Effector cell analysis-ApoE 13 wks: To study the effector cells after vaccination in ApoE mice at 13 wks (shortly after full vaccination) by analyzing the splenic and lymphatic cells. The first four subfolders included were the different panels used to evaluate the effector CD4+ and CD8+ T cells, including by checking the expression of CD62 and CD44 as memory T cell markers, or IL10L and CD25 as regulatory or IL-10-secreting subpopulations. Panel 5 include the markers to evaluate the CD86+ dendritic cells. 6. Fig. 8i Coculture of BMDC and splenocytes: To study the cytolytic properties of CD8+ T cells from vaccinated mice to antigen-experienced BMDCs. 7. Fig. 9d CD 4+ T cell proliferation: Three weeks after the second booster (week 15), spleen T lymphocytes from C57BL/6 mice were labeled with CSFE and stimulated with p210 for 5 days, before being subjected to flow cytometric analysis of CD4+ T cells. 8. Fig. 9e CD 8+ T cell proliferation: Three weeks after the second booster (week 15), spleen T lymphocytes from C57BL/6 mice were labeled with CSFE and stimulated with p210 for 5 days, before being subjected to flow cytometric analysis of CD8+ T cells. 9. Supplementary Fig. 4-6: This set of experiment was used to assess the optimised nanovaccine composition in promoting the co-signal expression of BMDCs after 72 h. It contains four subfolders as below. (1) Sup Fig. 4b D80-FITC: the CD80 expression was assessed. (2) Sup Fig. 5 CD86-PE: the CD86 expression was assessed. (3) Sup Fig. 6a MHC I-PE:  the MHC I expression was assessed. (4) Sup Fig. 6b MHC II-FITC: the MHC II expression was assessed. 10. Supplementary Fig. 7a-b: To assess the optimised nanovaccine composition (the ration between antigen and adjuvant) in promoting the co-signal expression of BMDCs after incubation for 24 h.  The subfolders contain two different panels to separately evaluate the co-signal expression of activated DCs, which were CD 80 and CD86 (the first subfolder), and MHC-I and MHC II (the second subfolder). 11. Supplementary Fig. 28-29 effector cell analysis-ApoE 26 wks: To study the effector cells after vaccination in ApoE mice at 26 wks by analyzing the splenic and lymphatic cells. The first four subfolders included were the different panels used to evaluate the effector CD4+ and CD8+ T cells, including by checking the expression of CD62 and CD44 as memory T cell markers, or IL10L and CD25 as regulatory or IL-10-secreting subpopulations. Panel 5 include the markers to evaluate the CD86+ dendritic cells. ### Files #### File: 20250128\_NC\_paper\_upload.zip **Description:**  FACS data of the flow cytometric analysis in the manuscript. ## Code/software FlowJo or FCS Express may be used to view the files of .fcs files. ### Version changes 24-Feb-2025: Added more raw data for cytometric analysis, including the folder named "Supplementary Fig. 4-6" and its four subfolders. These data describe the optimised nanovaccine composition in promoting the co-signal expression of BMDCs after 72 h.