Abstract

   The therapeutic efficacy of vaccines for treating cancers in clinics
   remains limited. Here, a rationally designed cancer vaccine by placing
   immunogenically differential and clinically approved aluminum (Al) or
   manganese (Mn) in a 2D nanosheet (NS) architecture together with
   antigens is reported. Structurally optimal NS with a high molar ratio
   of Mn to Al (MANS‐H) features distinctive immune modulation, markedly
   promoting the influx of heterogeneous innate immune cells at the
   injection site. Stimulation of multiple subsets of dendritic cells
   (DCs) significantly increases the levels, subtypes, and functionalities
   of antigen‐specific T cells. MANS‐H demonstrates even greater
   effectiveness in the production of antigen‐specific antibodies than the
   commercial adjuvant (Alhydrogel) by priming T helper (Th)2 cells rather
   than T follicular helper (Tfh) cells. Beyond humoral immunity, MANS‐H
   evokes high frequencies of antigen‐specific Th1 and CD8^+ cell
   immunity, which are comparable with Quil‐A that is widely used in
   veterinary vaccines. Immunized mice with MANS‐H adjuvanted vaccines
   exert strong potency in tumor regression by promoting effector T cells
   infiltrating at tumor and overcoming tumor resistance in multiple
   highly aggressive tumor models. The engineered immunogen with an
   intriguing NS architecture and safe immunopotentiators offers the next
   clinical advance in cancer immunotherapy.

   Keywords: 2D nanosheets, antigen‐specific immunity, cancer
   immunotherapy, cancer vaccines, material adjuvants
     __________________________________________________________________

   Cancer vaccines offer unprecedented advancements in cancer
   immunotherapy. Here, the impact of the heterogenicity of
   immunogenically differential aluminum or manganese presented in a 2D
   nanosheet architecture on the diversity and effectiveness of anti‐tumor
   immunity is reported. The optimal bimetallic adjuvant exhibits superior
   anti‐tumor performance than commercial adjuvants in aggressive melanoma
   and cervical cancer models, enhancing its appeal for clinical use.

   graphic file with name ADVS-11-2405729-g002.jpg

1. Introduction

   Breakthrough development of new vaccines could save millions of lives,
   of which adjuvants are essential ingredients in vaccine formulations,
   harnessing the host immune response against antigens with remarkably
   enhanced magnitude, longevity, and effectiveness.^[ [46]^1 ^] Despite
   over 100 years old history of the use of adjuvants (e.g., aluminum
   salts first used in 1926) in human vaccines, only a few adjuvants have
   been approved in licensed vaccines in clinics to date.^[ [47]^2 ^]
   Numerous innovative adjuvant platforms containing distinct
   immunostimulants are undergoing explosive exploration to complement the
   classical adjuvants that hardly trigger an appropriate immune
   response against a broad spectrum of diseases that are problematic to
   be prevented or cured, such as cancer, HIV (human immunodeficiency
   virus), and malaria.^[ [48]^1 , [49]^3 ^] The understanding of the
   mechanism of action of adjuvants is of great interest, forming the
   foundation for advances in adjuvant development and vaccinology.
   Aluminum‐containing adjuvants are known to produce powerful antibody
   responses and T helper 2 (Th2) cell responses, while having limited
   cellular immunity. Nonetheless, cellular immune responses, particularly
   CD8^+ T cells, are key mediators for the elimination of virus‐infected
   cells or mutated cancerous cells. To understand how the immune system
   senses and responds to aluminum‐containing adjuvants, several
   prevailing mechanisms have been proposed but contested. “Depot theory”
   proposed by Glenny and co‐workers in 1931 suggested aluminum salts
   mediated the sustained antigen release at the injection site,
   prolonging the stimulation of the immune system.^[ [50]^4 ^] Recent
   work highlights the crucial role of innate immune cells in initiating
   adaptive immune response, whereby the “danger hypothesis” was proposed
   by Polly Matzinger in 1994. It suggested that cell death from aluminum
   salt particulate‐caused tissue damage led to the release of danger
   signals, ultimately evoking the activation of innate immune cells and
   lymphocytes.^[ [51]^5 ^] More recently, inflammasome activation
   triggered by the phagocytosis of particulate aluminum salts was
   observed by Veit Hornung and colleagues, which regulated its
   immunogenicity.^[ [52]^6 ^] Huge efforts are still required to fully
   understand the mechanism of aluminum‐containing adjuvants at cellular
   and molecular levels.

   Another intensively explored chemical element in immune modulation is
   manganese.^[ [53]^7 ^] The pioneering work by Jiang and co‐workers
   first discovered that manganese ions were indispensable for the host
   sensing cytosolic double stranded DNA (dsDNA) via cyclic guanosine
   monophosphate‐adenosine monophosphate synthase (cGAS) and triggering
   the activation of downstream stimulator of interferon genes (STING)
   signaling against virus infection.^[ [54]^7 ^] Strikingly, manganese
   ions were found potent immunostimulants by directly activating
   STING‐type I Interferons (IFNs) pathway in a cGAS‐independent manner.^[
   [55]^8 ^] The adjuvant activity of manganese ions varies based on their
   physical forms. It was found that the colloidal form of manganese
   salts, rather than soluble free ions, effectively stimulates the NLR
   family pyrin domain containing 3 (NLRP3)‐apoptosis‐associated
   speck‐like protein containing a C‐terminal caspase recruitment domain
   (ASC) inflammation pathway. This activation enhances the adjuvant's
   potency, leading to a strong cellular and humoral immune response, in
   conjunction with manganese‐STING signaling.^[ [56]^9 ^] These findings
   are of remarkable significance in supporting the clinical studies of
   manganese salts in cancer immunotherapy,^[ [57]^8 ^] igniting enormous
   research interest in the development of advanced manganese‐based
   adjuvants.^[ [58]^10 ^] Beyond inorganic adjuvants, recent advances in
   small molecular adjuvants that target other pattern‐recognition
   receptors (PRRs), such as toll‐like receptor (TLR)7/8 agonist,^[
   [59]^11 ^] STING agonists of cyclic dinucleotide (CDN)^[ [60]^10 ,
   [61]^12 ^] and MSA‐2,^[ [62]^13 ^] have revealed the importance of the
   formulation of these immunostimulants in micro‐ or nano‐particulates,
   which dramatically augmented the immunogenicity, reduced the
   immunological toxicity, and altered their pharmaceutical
   biodistribution in tissue‐specific innate immune cells (e.g., draining
   lymph node (dLN) resident DCs^[ [63]^14 ^] and tumor‐infiltrating
   monocytes^[ [64]^15 ^]). Thin‐layered 2D nanosheet (NS) formulation of
   immunostimulants is technologically intriguing with unprecedented
   advancements in cancer immunotherapy^[ [65]^16 ^] by offering a high
   surface area to volume ratio and enhanced nano‐immune interactions.
   However, it remains unexplored how the heterogenicity of
   immunogenically differential aluminum or manganese placed in an NS
   architecture impacts the coordination of innate and adaptive immunity
   in cancer vaccination.

   Here, we reported the construction of 2D NS adjuvants with different
   molar ratios of aluminum to manganese (Figure [66] 1 top panel) and
   explored their adjuvant activity and mechanism of action in cancer
   vaccines. The results revealed that the bimetallic NS adjuvant with a
   high ratio of manganese to aluminum (MANS‐H) markedly triggered the
   recruitment of heterogenous subsets of innate immune cells at skin
   post‐injection, such as proinflammatory macrophages, monocytes, and
   neutrophils (Figure [67]1 bottom panel). MANS‐H exhibited superior
   performance in enhancing antigen uptake and activating distinct subsets
   of DCs, including type 1 conventional DCs (cDC1, CD103^+ migratory DCs,
   and CD8^+ resident DCs), type 2 conventional DCs (cDC2, CD11b^+ DCs)
   and Langerhans cells (LCs) via stimulation of STING‐type I IFN and
   Interleukin‐1 beta (IL‐1β) inflammation pathways. Mechanically, Mn
   showed remarkably greater effectiveness in priming antigen‐specific
   IL‐4^+CD4^+ Th2, IFN‐γ^+CD4^+ Th1, and multifunctional CD8^+ T cells,
   which further developed into memory phenotypes. By contrast, Al showed
   potency in evoking the production of CXCR5^+CD4^+ Tfh and antibodies.
   Mice vaccinated with MANS‐H adjuvanted vaccines demonstrated
   significant tumor suppression by promoting the frequency of
   tumor‐infiltrating CD8^+ cells with distinct activated phenotypes and
   cytotoxic natural killer (NK) cells while suppressing the suppressive
   innate immune cells and CD4^+Foxp3^+ Tregs at the tumor site. The
   potent therapeutic efficacy of MANS‐H combined with tumor‐associated or
   specific antigens was validated in aggressive murine cancer models,
   indicating the great potential of bimetallic oxides NS adjuvant cancer
   vaccine immunotherapy for enhancing clinical efficacy.

Figure 1.

   Figure 1
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   Schematic illustration of the preparation procedures of NSadjuvant with
   immunogenically differing Mn and Al and action mechanisms by which NS
   determines the modulation of innate immune cells and the subtypes of
   antigen‐specific immunity for enhanced anti‐tumor efficacy.

2. Results and Discussion

2.1. Preparation and Characterization of Several Nanosheet Adjuvants

   MANS adjuvants were prepared via a facile hard template method as
   illustrated in Figure [69] 2a. Briefly, NaCl microcrystals
   (Figure [70]S1, Supporting Information) were wetted with aluminum
   acetylacetonate toluene solution, which underwent hydrolytic
   polycondensation in a humidity chamber to form aluminum containing NS
   (ANS) after washing with water. ANS directly reacted with potassium
   permanganate (KMnO[4]) aqueous solution to yield MANS with low or high
   manganese to aluminum ratios (denoted as MANS‐L and MANS‐H).
   Transmission electron microscopy (TEM) images revealed a thin layered
   structure of ANS with a size ≈3–4 µm and a smooth surface
   (Figure [71]S2a, Supporting Information). Consistently, scanning
   electron microscope (SEM) images of ANS displayed micrometer‐sized
   layers (Figure [72]S2b, Supporting Information). Following
   ultrasonication, ANS was downsized to 200–500 nm (Figure [73]2b;
   Figure [74]S2, Supporting Information). After incorporating Mn, MANS‐L
   (Figure [75]2c) and MANS‐H (Figure [76]2d) maintained a nanosheet
   structure, and the elemental mapping images of MANS‐H (Figure [77]2e)
   implied that Mn was uniformly doped in ANS. Atomic force microscopy
   (AFM) image further confirmed the lamellar structure of ANS with a thin
   thickness of 7 nm (Figure [78]2f), the thickness of MANS‐L increased to
   11 nm (Figure [79]2g) and MANS‐H increased to 16 nm (Figure [80]2h),
   contributed by the doping layer of Mn. Inductively coupled plasma
   emission spectrometer (ICP) analysis (Figure [81]2i) showed that the
   quantified molar ratios of Mn to Al were 0.05 ± 0.01 in MANS‐L and 0.46
   ± 0.01 in MANS‐H.

Figure 2.

   Figure 2
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   Characterization of nanosheet adjuvants. a) Schematic illustration of
   the preparation of ANS, MASN‐L, and MANS‐H. b–d) TEM images of b) ANS,
   c) MANS‐L, and d) MANS‐H. e) The element mapping images of MANS‐H. f–h)
   AFM topography images and height profiles of f) ANS, g) MANS‐L, and h)
   MANS‐H. Scar bar: 200 nm. i) The atomic ratios of Mn/Al in MANS‐L and
   MANS‐H were determined by ICP analysis (n = 3). j) FTIR spectra of ANS,
   MANS‐L, and MANS‐H. k‐l) XPS spectra of k) MANS‐L and l) MANS‐H. m)
   Zeta potential values of ANS, MANS‐L, and MANS‐H in PBS solution (n =
   3). Data are presented as mean ± SD (i and m).

   Fourier transform infrared (FTIR) spectroscopy analysis was performed
   to characterize the chemical composition of ANS, MANS‐L, and MANS‐H.
   FTIR spectrum of ANS (Figure [83]2j) exhibited characteristic
   absorption peaks at 1294 cm^−1 (C–O stretching), 1396 cm^−1(O–H
   bending), 2924 cm^−1 (C–H stretching), and 2957 cm^−1 (C–H stretching),
   suggesting partially unhydrolyzed C‐O, O‐H and C–H groups derived from
   the Al precursors. The typical peaks at 1028 cm^−1 (δ[s](Al‐O‐H)) and
   3463 cm^−1 (ν[as](Al–O–H)) implied the formation of AlOOH.^[ [84]^17 ^]
   By contrast, the typical peaks of C–O, O–H, and C–H groups in MANS‐L
   and MANS‐H were less distinguished compared to those in ANS, indicating
   the enhanced hydrolysis of Al precursor residual during the
   oxidation‐reduction reaction with KMnO[4] solution. X‐ray photoelectron
   spectroscopy (XPS) was used to assess the valence states of Mn in
   MANS‐L and MANS‐H. The XPS survey of MANS‐L and MANS‐H (Figure [85]S3,
   Supporting Information) displayed characteristic peaks of Mn and Al
   with an atomic ratio of 0.14 and 0.81, respectively, which were 2–3
   times those values with ICP analysis, implying that Mn is predominately
   decorated on the surface of ANS. XPS spectra of Mn 2p[3/2] from both
   MANS‐L and MANS‐H exhibited characteristic peaks at 654.6 eV for Mn^2+,
   653.0 eV and 641.5 eV for Mn^3+, and 643.0 eV for Mn^4+
   (Figure [86]2k,l), suggesting mixed valence states of Mn. Additionally,
   the zeta potential of three NS adjuvants was negative in phosphate
   buffered solution (PBS) (Figure [87]2m). Collectively, several NS
   adjuvants with varied Mn/Al ratios were successfully fabricated.

2.2. MANS‐H Adjuvant Promotes the Maturation of Antigen‐presenting Cells
(APCs) via Activating cGAS‐STING and Inflammasome Signaling Pathway

   DCs are a crucial subset of APCs, which are speciliazed in antigen
   capture and presentation to prime adaptive immune cells.^[ [88]^18 ^]
   Bone marrow‐derived DCs (BMDCs) were co‐cultured with ANS, MANS‐L, or
   MANS‐H to assess their adjuvant activities (Figure [89] 3a). MANS
   robustly promotes the proportion of CD80^+ CD86^+ DCs
   (Figure [90]3b,c). The flow cytometry results displayed that ANS
   slightly upregulated the expression of co‐stimulatory markers of CD80
   and CD86. By contrast, BMDCs co‐cultured with MANS‐L or MANS‐H
   expressed a significantly higher level of CD80 (Figure [91]3d) and CD86
   (Figure [92]3e), compared with control and ANS groups, particularly
   MANS‐H. These results suggest that the maturation of BMDCs profoundly
   depends on the dose of Mn contained in the NS adjuvants. Previous
   reports showed that Al‐containing rods were able to effectively
   activate BMDCs into a mature state under a greatly higher concentration
   (100 versus 15 µg mL^−1).^[ [93]^17 ^] Regarding BMDC stimulation, Mn
   exhibited superior adjuvanticity over Al.

Figure 3.

   Figure 3
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   MANS‐H adjuvant potentially triggers BMDC maturation, ROS production,
   and activation of the inflammasome and STING‐IFN‐β pathway. a)
   Illustration shows the procedure to induce the BMDC maturation (created
   with permission by BioRender). b) Flow cytometry shows the proportion
   of CD80^+ CD86^+ DCs (in the MHC II^+ CD11c^+ DCs) treated with
   different NS adjuvants (n = 3). c) Representative counterplot of CD80^+
   CD86^+ DCs. d‐e) Representative histogram overlays and median
   fluorescence intensity (MFI) of the expression of d) CD80 and e) CD86
   on BMDCs after incubation with ANS, MANS‐L or MANS‐H (n = 3). f)
   Confocal microscopy images and g) flow cytometry analysis shows
   intracellular ROS levels in BMDCs induced by ANS, MANS‐L, or MANS‐H (n
   = 3). h) ELISA analysis summarizes IL‐1β secretion levels in BMDMs
   treated with PBS or NS adjuvants for 48 h (n = 4). i) Western blot
   analysis shows the expression levels of pTBK1, p‐p65 and ‐IRF3 in BMDCs
   treated with PBS or NS adjuvants for 20 h (n = 3). j)ELISA analysis of
   IFN‐β secretion levels in the supernatants of BMDCs (n = 4). Data are
   presented as means ± SD. P values were determined by a one‐way ANOVA
   test. ns, p > 0.05, **p < 0.01, ****p < 0.0001.

   Reactive oxygen species (ROS) are key stimulants triggering the
   activation of inflammation signals and the release of pro‐inflammatory
   cytokines, which are essential in adjuvant activity.^[ [95]^19 ^] To
   explore the cellular mechanism of MANS‐H adjuvant effect, we next
   examined whether metal ions were able to induce intracellular ROS.
   BMDCs were treated with ANS, MANS‐L, or MANS‐H and then co‐cultured
   with ROS probe dichlorofluorescin diacetate (DCF‐DA). The confocal
   microscopy images showed that MANS‐H but not ANS or MANS‐L exhibited
   strong green fluorescence signals, implying that MANS‐H markedly
   promoted the production of intracellular ROS (Figure [96]3f). The
   quantified results from flow cytometry (Figure [97]3g) confirmed a
   consistent trend in ROS generation, which was mainly contributed by the
   Mn‐Fenton reaction.^[ [98]^20 ^] We next performed western blot (WB)
   and enzyme‐linked immunosorbent assay (ELISA) analysis to test whether
   ROS mediates the activation of IL‐1β inflammation signal pathway in
   BM‐derived macrophages (BMDMs) that are appropriate in inflammation
   assessment.^[ [99]^9 , [100]^21 ^] The results demonstrated that MANS‐H
   but not ANS or MANS‐L significantly promoted the secretion of
   proinflammatory cytokine IL‐1β (Figure [101]3h) in lipopolysaccharide
   (LPS)‐primed BMDMs, suggesting that MANS‐H potently activated
   inflammasome‐IL‐1β signaling pathway.

   The activity of STING agonist Mn^2+ was reported to be dependent on its
   dose and physical forms,^[ [102]^9 ^] thus, we explored the capability
   of NS containing Al/Mn in STING activation. MANS‐H but not ANS
   significantly promoted downstream signaling of STING pathway by
   apparently increasing the expression of phosphorylated tank‐binding
   kinase 1 (TBK1), interferon regulatory factor 3 (IRF3), and p65
   (Figure [103]3i; Figure [104]S4, Supporting Information), thereby
   significantly stimulating the production of type I IFN‐β in the
   supernatants (Figure [105]3j). Of note, MANS‐L moderately upregulated
   the expression of STING downstream signaling molecules, which
   inadequately triggered the secretion of IFN‐β (Figure [106]3i,j). The
   calculated dose of Mn derived from MANS‐H was 0.037 mm, which was much
   lower than the effective dose in Mn salts (0.2 mm),^[ [107]^9 ^]
   implying that the superior potency of MANS‐H might be contributed by
   the NS structure. We previously observed that Mn‐based nanoparticles
   with a high oxidative status of Mn^4+ showed pH‐buffering capability in
   the acidic endo‐lysosomal compartments,^[ [108]^21 ^] which might slow
   down the degradation of STING.^[ [109]^22 ^] It therefore could be the
   reason why MANS‐H showed significantly enhanced downstream activity of
   STING activation and IFN‐β secretion than soluble Mn^2+ ions. In
   addition, Mn^2+ released from the nanosheet structure provided local
   concentrated STING agonists, which potentially led to multivalent
   interactions with STING.

   To further investigate the immune modulation mechanism of MANS‐H, we
   used transcriptomics RNA sequencing to analyze BMDCs treated with
   MANS‐H. A total 23 899 genes were investigated, of which 3222 genes
   were significantly upregulated in BMDCs treated with MANS‐H compared to
   the control group (Figure [110]S5, Supporting Information). The top
   upregulated genes mainly include interferon‐stimulated genes (Isg15,
   Isg20, and Ifi205) and chemokines (Ccl4 and Ccl5), which strongly
   suggested the stimulation of IFN and inflammation signals (Figure [111]
   4a). Interestingly, genes related to M2 anti‐inflammatory phenotypic
   macrophages (Apoe and Fn1) were observed to be remarkably downregulated
   (Figure [112]4a). The representative significantly upregulated genes
   associated with STING downstream signals of ISGs and IFNs were
   visualized in heatmaps (Figure [113]4b), which were consistent with the
   WB analysis. In addition, sets of significantly upregulated genes were
   found related to antigen presentation (H2‐Q6, H2‐Q7, and Tap1/2
   involved in antigen‐MHC class I complexes) and DC maturation,
   chemokines (Ccl2/3 and Cxcl5/9) for inflammasome, and cytokines (Il6,
   Il33 and Il18) for T cell proliferation and differentiation. We found
   that top‐regulated genes were significantly enriched in cellular
   response to interferon beta and antigen processing and presentation via
   major histocompatibility complex (MHC) class I pathways
   (Figure [114]4c). Gene ontology (GO) enrichment analysis and Kyoto
   encyclopedia of genes and genomes (KEGG) pathway enrichment analysis
   were performed. Compared to the control group, immune‐associated
   signaling pathways were found prominent in the chemokine signaling
   pathway, Th1 and Th2 cell differentiation and regulation of
   interleukin‐1 beta production and cytokine‐cytokine receptor
   interaction, contributing to the potent adjuvant functions of MANS‐H
   (Figure [115]4d,e; Figure [116]S6, Supporting Information).

Figure 4.

   Figure 4
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   Bulk RNA sequencing analysis of BMDCs treated with PBS or MANS‐H. a)
   Volcano plot shows the differentially expressed genes that are
   significantly or non‐significantly upregulated or downregulated in
   BMDCs treated with MANS‐H by comparing with the PBS group. b). Heatmaps
   summarize the selected genes related to the STING‐IFN‐β pathway (ISGs
   and IFNs), cytokines for T cell proliferation and differentiation,
   chemokines for inflammasome, antigen presentation, and DC maturation.
   c) Gene set enrichment analysis (GSEA), d) KEGG, and e) GO enrichment
   analysis of differentially expressed genes associated with innate and
   adaptive immune response.

2.3. Differential Immunogenicity of Distinct NS Adjuvants in Recruitment of
Heterogenous Subsets of Innate Immune Cells at Injection Site and Activation
of Distinct Subsets of DCs In vivo

   Adjuvant function in large measure by creating the local
   pro‐inflammatory environment and triggering the activation of DCs,
   which is a prerequisite for initiating antigen‐specific adaptive immune
   responses.^[ [118]^23 ^] To compare the adjuvant activity of NSs with
   immunogenically different ratios of Mn to Al elements in vivo,
   immunocompetent C57BL/6J mice were subcutaneously injected with ANS,
   MANS‐L, and MANS‐H, and then flow cytometry analysis was performed to
   characterize the cellular influx at the injection site (Figure [119]
   5a). At 48 h post‐injection, immunization of MANS‐H but not ANS or
   MANS‐L resulted in a significant decrease in the frequency of CD11c^+
   DCs in the skin (Figure [120]5b; Figure [121]S7a, Supporting
   Information). In line with the observation in CD11c^+ DCs, EpCAM^+MHC
   II^+ Langerhans cells (LCs) dramatically declined in all NS‐vaccinated
   skin (Figure [122]5c), indicating the migration of DCs and LCs into
   dLNs following activation. Differently, we observed a dramatic increase
   in CD11c^− non‐DC population in MANS‐H but not ANS or MANS‐L immunized
   skin (Figure [123]5d). Further analysis was performed on the subsets of
   CD11c^− non‐DCs, which revealed that CD11b^+ myeloid cells
   predominately contributed to the substantial increase in cellular
   influx in all NS‐vaccinated skin, particularly MANS‐H (Figure [124]5e).
   Additionally, MANS‐H markedly promoted the infiltration of
   proinflammatory CD11b^+Ly6G^+ neutrophils (Figure [125]5f,g), which was
   likely mediated by MANS‐Hinduced ROS.^[ [126]^24 ^] However, skin T
   cells markedly decreased (Figure [127]5h), implying the motility of T
   cells within the lymphatic capillaries in response to NS‐induced
   inflammation.^[ [128]^25 ^] A substantial decline was also observed in
   the percentage of CD11b^+Ly6G^− monocytes (Figure [129]5i) in the skin
   immunized with MANS‐H, which was possibly caused by their
   differentiation into skin‐specific macrophages or DCs.^[ [130]^26 ^]
   Interestingly, the immunization of ANS or MANS‐L but not MANS‐H
   enhanced the influx of the sub‐population of CD11b^+Ly6G^−Ly6C^−
   myeloid‐derived cells (Figure [131]S7b, Supporting Information), the
   function of which has been rarely explored with a possibility of acting
   as an immune suppressor.^[ [132]^27 ^] A similar trend was observed in
   macrophages comprising distinct subsets of F4/80^+MHC II^+ macrophages
   (Figure [133]5j) and F4/80^+MHC II^− macrophages (Figure [134]5k).

Figure 5.

   Figure 5
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   MANS‐H adjuvant dramatically promotes the infiltration of heterogeneous
   subsets of innate immune cells at the injection site. a) The
   experimental timeline: C57BL/6J mice subcutaneously received 300 µg of
   ANS, MANS‐L or MANS‐H or PBS (Ctrl) at the injection site and skin
   tissues (1 cm^2) were harvested at 48 h for flow cytometry analysis
   (created with permission by BioRender). b‐k) The percentages of b)
   CD11c^+ cells, c) LCs, d) CD11c^− cells, e) CD11b^+ myeloids, f)
   neutrophils, h) T cells, i) monocytes, j) F4/80^+ MHC II^+ macrophages,
   k) F4/80^+ MHC II^− macrophages (n = 4). g) Representative dot plot of
   neutrophils in the injection site with different treatment. Data are
   presented as means ± SD. P values were determined by a one‐way ANOVA
   test. ns, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p <
   0.0001.

   DCs comprise highly heterogenous subgroups, of which CD11b^+ cDC2 and
   CD103^+ (CD8a^+) cDC1s are exceedingly specialized in MHC class II and
   MHC class I antigen presentation for priming CD4^+ T cells and CD8^+ T
   cells, respectively.^[ [136]^28 ^] The discriminated functions of DCs
   determine or shape the subtypes of adaptive immunity in vaccination.^[
   [137]^29 ^] The effectiveness of T cell priming heavily relies on the
   level of DC maturation, such as the upregulation of co‐stimulatory
   molecules and secretion of cytokines that support T cell proliferation
   and differentiation.^[ [138]^30 ^] To understand the influence of ANS,
   MANS‐L, and MANS‐H on DCs, we analyzed the phenotypes of DCs in skin
   dLNs following immunization (Figure [139] 6a). The results demonstrated
   that MANS‐H and MANS‐L but not ANS immunized groups displayed an
   obvious increase in dLN size (Figure [140]6b), which was likely caused
   by the rapid recruitment of APCs and other sub‐populations of innate
   immune cells from the skin, such as CD11c^+ DCs and LCs
   (Figure [141]5b,c). Flow cytometry analysis on the cellularity of skin
   dLNs showed that vaccination with MANS‐H led to a significant decrease
   in the frequency of migratory DCs (MigDCs, MHC II^highCD11c^+,
   Figures [142]S8 and [143]S9a, Supporting Information).^[ [144]^31 ^]
   MigDCs exhibited marked upregulation of CD80 and CD86 molecules,
   suggesting potent DC activation. In comparison with MANS‐H, MANS‐L
   showed a similar level of MigDC maturation, but ANS induced
   semi‐maturation of MigDCs with promoted expression of co‐stimulatory
   markers that were statistically non‐significant (Figure [145]6c,d). The
   trend of distinct NSs in DC maturation was consistent with the
   observations in BMDCs. In further phenotyping analysis, MigDCs were
   subdivided into subsets of LCs, CD11b^+ cDCs (cDC2), CD11b^− DCs and
   migratory CD103^+ DCs (cDC1) (Figure [146]6e). Importantly, MANS‐H but
   not ANS or MANS‐L significantly promoted the frequencies of CD103^+DCs
   (Figure [147]6f) and immature LCs (Figure [148]6g; Figure [149]S9b,
   Supporting Information). The observation of downregulation of CD80 and
   CD86 on CD103^+DCs (Figure [150]S9c, Supporting Information) suggested
   a tolerogenic phenotype, which might have contributed to the absence of
   antigen presentation (will be further discussed in the next section).
   All NS adjuvants significantly enhanced the recruitment and maturation
   of CD11b^+DCs (Figure [151]6h–j). The immunization of MANS‐H and MANS‐L
   but not ANS resulted in a decrease in the number of mature CD11b^− DCs
   (Figure [152]6k–m). These results implied that the decrease in MigDCs
   was mainly caused by a marked decrease of CD11b^− DCs. MANS‐H with a
   high ratio of Mn/Al exhibited superior performance in the effective
   activation of multiple subsets of MigDCs.

Figure 6.

   Figure 6
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   MANS‐H adjuvant remarkably triggers the maturation of distinct
   phenotypes of MigDCs and ResDCs. a) The experimental timeline: C57BL/6J
   mice subcutaneously received 300 µg of ANS, MANS‐L or MANS‐H, or PBS
   (Ctrl), and dLNs were harvested at 48 h for flow cytometry analysis
   (created with permission by BioRender). b) Representative images of
   inguinal LNs post different treatments. c,d) Representative histogram
   overlays c) and MFI of CD80 and CD86 d) on MigDCs derived from inguinal
   LNs (n = 4). e) The proportion sum of MigDC subsets and the percentages
   of each subset. f–n) Flow cytometry analysis shows the percentage of
   CD103^+ DCs f), LCs g), CD11b^+ DCs h) and CD11b^− DCs k), histogram
   plots of CD80 and CD86 of CD11b^+ DCs i) and CD11b^− DCs l) and MFI of
   CD80 and CD86 of CD11b^+ DCs j) and CD11b^− DCs m) (n = 4). n) The
   proportion sum of ResDC subsets and the percentages of each subset.
   o‐r) Flow cytometry analysis shows the percentage of o) CD11b^+CD8a^int
   DCs, q) CD8a^+ DCs, representative dot plots of p) CD11b^+CD8a^int DCs,
   MFI of CD80 and CD86 of r) CD11b^+CD8a^int DCs (n = 4). Data are
   presented as means ± SD. P values were determined by a one‐way ANOVA
   test. ns, p > 0.05, *p < 0.05, **p < 0.01.

   Cooperation between MigDCs and resident DCs (ResDCs, MHC II^intCD11c^+)
   in skin‐dLNs allows optimization of DC capability responding to
   stimulatory vaccines or infection,^[ [154]^29 , [155]^31 ^] thus we
   next assessed the impact of NS adjuvants on ResDCs, which were found in
   an unexpanded and immature state (Figures [156]S8 and [157]S10a,
   Supporting Information). ResDCs were then subdivided into three
   phenotypes: CD8a^+ DCs, CD11b^+CD8a^int DCs, and CD11b^− DCs
   (Figure [158]6n). MANS‐L and MANS‐H but not ANS induced a significant
   increase in CD11b^+CD8a^int DCs (Figure [159]6o,p) rather than the
   other two subsets (Figure [160]6q; Figure [161]S10b, Supporting
   Information). Interestingly, only MANS‐H was efficient to induce a
   significant upregulation of CD80 and moderate promotion of CD86
   (Figure [162]6r; Figure [163]S10c, Supporting Information) expressed on
   CD11b^+CD8a^int DCs, but not on CD11b^− DCs (Figure [164]S10b,
   Supporting Information) or CD8a^+ DCs (Figure [165]S10d, Supporting
   Information). This effect would potentially contribute to the
   cross‐presentation capability of MANS‐H adjuvanted vaccines.

   Collectively, we answered the question of how the differential
   immunogenicity of distinct NS adjuvants regulated the maturational
   states of dLN‐DCs with highly phenotypic and spatial heterogeneity.
   MANS‐H is the most pronounced adjuvant for adequately stimulating the
   activation of MigDCs and ResDCs, which contain both cDC1 and cDC2 for
   broad T‐cell priming.

2.4. Enhanced Antigen Delivery to DCs and Antigen Cross Presentation Mediated
by Distinct NS Adjuvants In vitro and In vivo

   To evaluate the antigen delivery capability of ANS, MANS‐L, and MANS‐H,
   the negatively charged NS adjuvants (≈55 mV) were coated with positive
   poly(allylamine hydrochloride) (PAH),^[ [166]^32 ^] which turned to be
   positively charged (≈ +60–80 mV, Figure [167] 7a) to promote the
   adsorption of ovalbumin (OVA, a model antigen) via electrostatic
   interactions. All NS@OVA samples exhibited a negative surface charge
   (≈10 mV, Figure [168]7a), indicating successful loading of OVA.
   Absorption rates of OVA for all NS exceed 70% (Figure [169]7b),
   corresponding to a loading capability of 70 µg OVA per 1 mg NSs
   (Figure [170]S11a, Supporting Information). To track antigens in vitro
   and in vivo, OVA was conjugated with Cy5.5 (OVA‐Cy5.5), which displayed
   a slow antigen release from NSs with only ≈20% release up to 96 h
   (Figure [171]7c), implying stable vaccine formulations. It is vital to
   avoid the induction of self‐tolerance by free antigens.^[ [172]^21 ^]

Figure 7.

   Figure 7
   [173]Open in a new tab

   Dynamic cellular biodistribution of NS adjuvanted OVA‐Cy5.5 vaccines.
   a) Zeta potential values of three NS adjuvants before and after forming
   complexes with PAH or OVA (n = 3). b) OVA absorption efficiency and c)
   release profiles of three NS adjuvants (n = 3). d) Representative
   confocal microscopy images of BMDCs after incubation with NS@OVA‐Cy5.5
   (red) and staining with DAPI (nucleus, blue) and iFluorTM 488
   phalloidin (F‐actin in membrane, green). Scar bar: 40 µm. e) Bar charts
   and f) representative histograms from flow cytometry analysis show the
   proportions of OVA‐Cy5.5^+ BMDCs after incubation with NS@OVA‐Cy5.5 for
   6 h (n = 3). g‐j) Flow cytometry analysis shows the proportions of g)
   OVA‐Cy5.5^+DCs, i) OVA‐Cy5.5^+MigDCs, and j) OVA‐Cy5.5^+CD103^−DCs in
   dLN‐DCs, corresponding MFI heatmaps, and h) representative dot plots of
   OVA‐Cy5.5^+MigDCs at different timepoints (n = 3 or 4). Data are
   presented as means ± SD. P values were determined by a one‐way ANOVA
   test. ns, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p <
   0.0001.

   To assess intracellular antigen delivery in vitro, BMDCs were
   co‐cultured with distinct NS@OVA‐Cy5.5 and then analyzed using confocal
   microscopy and flow cytometry. The confocal microscopy images revealed
   that OVA‐Cy5.5 was readily delivered into the cytoplasm of BMDCs
   (Figure [174]7d). The quantified data by flow cytometry analysis
   demonstrated that OVA‐Cy5.5^+ BMDCs in all NS@OVA‐Cy5.5 groups reached
   ≈50%, which was significantly higher than that of free OVA‐Cy5.5
   (Figure [175]7e,f). However, ANS@OVA‐Cy5.5 displayed a markedly higher
   Cy5.5 MFI value than MANS‐L@OVA‐Cy5.5 and MANS‐H@OVA‐Cy5.5
   (Figure [176]S11b, Supporting Information), indicating enhanced
   cellular uptake of thinner ANS in vitro.

   We next determined the dynamic cellular biodistribution of OVA‐Cy5.5 in
   skin dLN DCs in C57BL/6J mice (Figure [177]S12, Supporting
   Information). All OVA‐Cy5.5 groups showed a steady and significant
   increase in the proportions of OVA‐Cy5.5^+CD11c^+MHC II^+ DCs in skin
   dLNs from 24 to 48 h post‐injection (Figure [178]7g left) but a
   dramatic decline at 72 h. Of note, the frequency of OVA‐Cy5.5^+ DCs and
   MFI value (Figure [179]7g right; Figure [180]S13a, Supporting
   Information) at 48 h was found prominent in MANS‐L@OVA group, which was
   probably caused by the combined effect of thin thickness‐mediated
   cellular uptake and Mn‐facilitated DC maturation and migration. A
   similar trend was observed in the subsets of MigDCs (Figure [181]7h,i;
   Figure [182]S13b, Supporting Information) and ResDCs (Figure [183]S13c,
   Supporting Information). However, OVA‐Cy5.5 positive populations in
   MigDCs were round 2–3 times those in ResDCs across experimental time,
   suggesting that MigDCs were the primary contributor to sampling and
   presenting antigens. In addition, the observed significantly decreased
   antigen uptake from 48 to 72 h was likely caused by antigen digestion
   and presentation of MigDCs to ResDCs and T/B cells.^[ [184]^33 ^]

   MigDCs were subdivided into CD103^+ cDC1 and CD11b^+ cDC2. The dynamic
   changes of OVA‐Cy5.5 positive populations in cDC2s (Figure [185]7j;
   Figure [186]S13d, Supporting Information) and cDC1s (Figure [187]S13e,
   Supporting Information) exhibited a similar trend as that in MigDCs.
   However, OVA‐Cy5.5 positive populations in cDC2s were evidently higher
   than those in cDC1s (≈3 times), indicating that cDC2s were the primary
   antigen transporters. Beyond DCs, CD11c^−CD11b^+F4/80^+ macrophages
   (Figure [188]S13f, Supporting Information) demonstrated a similar
   capability in antigen capture and transport with cDC1s.

   Effective antigen cross‐presentation of DCs is necessary for priming
   CD8^+ T cells against tumors. As a result, we next evaluated the
   dynamics of OVA cross‐presentation by DCs in skin dLNs using
   H‐2Kb/SIINFEKL (MHC I restricted OVA peptide) antibodies
   (Figure [189]S14, Supporting Information). Following immunization of
   MANS‐H@OVA, total DCs, MigDCs, and ResDCs all demonstrated the optimal
   cross‐presentation capability at 48 h with ≈2% MHC I‐OVA^+ DCs, ≈20%
   MHC I‐OVA^+ MigDCs and ≈4% MHC I‐OVA^+ ResDCs, respectively
   (Figure [190]S15a–d, Supporting Information), which were in agreement
   with OVA capture trend. Interestingly, the cDC2 in MigDCs exhibited a
   similar trend as that in MigDCs (Figure [191]S15e, Supporting
   Information), however, the frequency of cDC1 presenting MHC I‐SIINFEKL
   remained increasing over time with the highest population (≈4%) at 72 h
   (Figure [192]S15f, Supporting Information). The activated DCs were
   found effectively cross‐presenting SIINFEKL on the surface of CD8^+ T
   cells maximally at 48 h (Figure [193]S15g, Supporting Information).

   Taken together, MigDCs, particularly cDC2 of MigDCs, effectively
   sampled antigens delivered by NSs in cooperation with macrophages and
   transported the captured antigens into dLNs to present ResDCs, which
   offered promising prerequisite conditions for antigen
   cross‐presentation and evoking adaptive immune response at the initial
   stage. Activated DCs are short‐lived, thus cDC1 that cross‐presented
   antigens at a later stage might complement the capability of activated
   cDC2 in priming CD8^+ T cells.

2.5. The Potency and Subtypes of Antigen‐Specific Humoral and Cellular Immune
Response Determined by the Immunogenically Distinct NS Adjuvants

   Given the striking differences between NS adjuvants in
   immunopotentiation and antigen delivery, we next analyzed the humoral
   and cellular response using OVA as immunogens. The commercially
   available adjuvants, Alhydrogel (Th2 bias) and Quil‐A (saponin‐based
   adjuvants, Th1 bias), were selected for comparison. C57BL/6J mice were
   subcutaneously injected with OVA combined with adjuvants as indicated
   in a standard prime‐boost regimen (Figure [194] 8a). Serum samples were
   collected following boost vaccination for humoral immune response
   evaluation. All immunized mice demonstrated detectable OVA‐specific IgG
   at day 21 post‐immunization (Figure [195]8b; Figure [196]S16a,
   Supporting Information). MANS‐H indued the highest titer of anti‐OVA
   IgG, which was comparable with Alhydrogel at day 28. The subclass of
   IgG1‐type antibodies reflected the involvement of the Th2‐type humoral
   immune response.^[ [197]^34 ^] At day 21, MANS‐H and Alhydrogel induced
   higher tiers of anti‐OVA IgG1 antibodies than the other three adjuvants
   (Figure [198]8c; Figure [199]S16b, Supporting Information). However,
   MANS‐H outperformed other adjuvants on day 28, suggesting a
   long‐lasting Th2‐type humoral immune response. Collectively, MANS‐H was
   the most effective adjuvant in the production of durable humoral immune
   response, particularly biased Th2‐subtype, followed by Alhydrogel and
   then ANS/MANS‐L/Quil‐A.

Figure 8.

   Figure 8
   [200]Open in a new tab

   MANS‐H adjuvanted vaccine induces durable and potent humoral and
   cellular immunity. a) The experiment timeline: C57BL/6 mice were
   vaccinated twice on day 0 and day 7. Blood samples were collected on
   day 21 and day 28, and the spleens were harvested at the endpoint for
   further analysis (created with permission by BioRender). b–c) Serum IgG
   b) and IgG1c) antibody titers against OVA in vaccinated or control mice
   (n = 5). d–f) The number of OVA[323‐339]specific IL‐4^+CD4^+ T cells in
   500 000 cells d), the fractions of OVA[323‐339]‐specific CXCR5^+CD4^+ T
   cells e) and OVA[323‐339]specific IFN‐γ^+ CD4^+ T cells f) in spleens
   derived from vaccinated or control mice (n = 4 or 5). g‐h)
   Representative digital photos show the ELISPOT spots of
   OVA[257‐264]‐specific CD8^+ T cells secreting IFN‐γ^+ g) and numbers of
   OVA‐specific IFN‐γ^+CD8^+ T cells per 10^6 cells h) (n = 5). i‐k, m)
   Flow cytometry analysis shows the proportions of OVA[257‐264]specific
   IFN‐γ^+CD8^+ T cells i), perforin^+CD8^+T cells j), and granzyme
   B^+CD8^+ T cells k), and representative dot plots of perforin^+CD8^+T
   cells m) in spleens from vaccinated or control mice (n = 4 or 5). l, n)
   Flow cytometry analysis shows the fractions of CD44^+CD62L^+CD8^+ T
   cells (TCM, l), and the proportion sums of TCM, TEM, and naïve T cells
   presented in pie charts in spleens from vaccinated or control mice (n =
   5). o) A schematic illustration shows the favored subtypes of adaptive
   immune response mediated by the increased ratio of Mn to Al (created
   with permission by BioRender). Data are presented as means ± SD. P
   values were determined by a one‐way ANOVA test. ns, p > 0.05, *p <
   0.05, ***p < 0.001, ****p < 0.0001.

   To analyze OVA‐specific T cell immune response, splenocytes derived
   from immunized mice were re‐stimulated by SIINFEKL (OVA[257‐264], MHC
   class I restricted OVA peptide) and ISQAVHAAHAEINEAGR (OVA[323‐339],
   MHC class II‐restricted OVA peptide) and analyzed by enzyme‐linked
   immune absorbent spot (ELISPOT) assay and flow cytometry analysis.
   MANS‐H and Alhydrogel significantly enhanced the frequencies of
   OVA‐specific CD4^+ T cells producing IL‐4 cytokine (Figure [201]8d;
   Figure [202]S17a, Supporting Information), implying a high production
   of Th2‐ cells. By contrast, ANS, MANS‐L, and Quil‐A adjuvants showed
   relatively low frequencies of OVA‐specific Th2 cells. Interestingly,
   ANS and Alhydrogel drove dramatically enhanced differentiation of Th
   cells into CXCR5^+CD4^+ Tfh in comparison to MASN‐L, MASN‐H, and Quil‐A
   adjuvants (Figure [203]8e), indicating that Al element favored
   Tfh‐biased immunity. By contrast, MANS‐H and Quil‐A exhibited
   significantly higher proportions of OVA‐specific IFN‐γ^+CD4^+ Th1
   subtype of T cell immunity than other adjuvants (Figure [204]8f),
   implying that Mn element and saponin supported Th1 T cell immune
   response. MANS‐H and Quil‐A adjuvanted OVA vaccines elicited markedly
   higher OVA‐specific CD8^+ T cells producing IFN‐γ than the other
   groups, evidenced by the significant spots in the IFN‐γ ELISPOT assay
   (Figure [205]8g,h).

   To assess the cytolytic activity of OVA‐specific effector CD8^+ T
   cells, the secretion levels of cytotoxic molecules, including IFN‐γ
   cytokine, granzyme B, and pore‐forming perforin in lytic granules,^[
   [206]^10 , [207]^35 ^] were analyzed upon restimulation. The flow
   cytometry results revealed that the frequencies of OVA‐specific CD8^+ T
   cells producing IFN‐γ were prominent in MANS‐H and Quil‐A adjuvanted
   vaccines (Figure [208]8i; Figure [209]S17b, Supporting Information),
   which were consistent with ELISPOT analysis. MANS‐H adjuvant displayed
   the highest proportion of OVA‐specific CD8^+ T cells secreting perforin
   (Figure [210]8j,m). However, a moderate increase was observed in the
   population of granzyme B^+CD8^+ T cells in MANS‐H adjuvanted vaccine
   (Figure [211]8k). We also examined memory phenotypes of T cells in the
   spleen. Vaccination of MANS‐H@OVA but not other vaccines resulted in
   markedly increased CD44^+CD62L^+CD8^+ T cells (T central memory, TCM,
   Figure [212]8l). The frequencies of other phenotypes of
   CD44^+CD62L^−CD8^+ T cells (T effector memory, TEM) and
   CD44^−CD62L^+CD8^+ T cells (naïve T cells) were not obviously altered
   in all groups (Figure [213]8n; Figure [214]S18a,b, Supporting
   Information).

   In summary, the increase of Mn/Al ratio favored the induction of
   durable Th2‐type humoral immune response and heterogeneous subsets of
   the antigen‐specific cellular immune response, including Th2, Th1, and
   multiple functional CD8^+ T cells, and long‐lived CD8^+ TCM, but
   suppressed the production of Tfh subtype of T cell immunity. MANS‐H
   adjuvanted vaccine induced multifaceted, effective, and long‐lasting
   humoral and cellular immunity, particularly polyfunctional CD8^+ T
   cells, predicting superior therapeutic effect in eliminating cancer by
   comparing to other NS adjuvants (Figure [215]8o).

2.6. Murine Melanoma Regression by MANS‐H Adjuvanted Vaccine Therapy

   To examine the immunotherapeutic potency of NS‐adjuvanted OVA vaccines,
   we established a therapeutic regimen in an OVA‐expressing murine
   melanoma B16F10 (B16‐OVA, Figure [216]S19a, Supporting Information)
   model, in which C57BL/6J mice were inoculated with B16‐OVA cells on day
   0 and then received prime‐boost vaccination at day 5 and day 12
   respectively (Figure [217] 9a). All vaccinated groups exhibited
   significant suppression of B16‐OVA tumor growth (Figure [218]9b–e),
   while the suppression effect of MANS‐H@OVA and Quil‐A@OVA was
   comparable and the most pronounced. During the treatment, there was no
   obvious weight loss among all groups of mice (Figure [219]S19b,
   Supporting Information), suggesting that vaccination treatment was well
   tolerated. We next characterized the magnitude and functionality of
   antigen‐specific CD8^+ T cells in the spleen, which are key mediators
   for tumor elimination.^[ [220]^36 ^] To this end, splenocytes were
   re‐stimulated by SIINFEKL and analyzed by ELISPOT assay and flow
   cytometry analysis. In comparison with unvaccinated or vaccinated mice
   with Alhydrogel@OVA, MANS‐L@OVA, or ANS@OVA, mice vaccinated with
   MANS‐H@OVA elicited significantly higher OVA‐specific CD8^+ T cells
   producing IFN‐γ, proved by the substantial spots in the IFN‐γ ELISPOT
   assay (Figure [221]9f,g). A similar magnitude of OVA‐specific CD8^+ T
   cells was observed in the Quil‐A@OVA group. The flow cytometry results
   revealed a similar trend in the frequency of OVA‐specific IFN‐γ^+CD8^+
   T cells (Figure [222]9h,k; Figure [223]S20a, Supporting Information).
   Beyond the production of IFN‐γ, OVA‐specific CD8^+ T cells elicited by
   MANS‐H@OVA and Quil‐A@OVA were capable of secreting markedly higher
   levels of perforin (Figure [224]9i; Figure [225]S20b, Supporting
   Information) and cytolytic pore‐forming proteins of granzyme B
   (Figure [226]9j) than other groups. Altogether, these results suggested
   that the antigen‐specificity, high number, and multifaceted effector
   functionality of vaccination‐induced CD8^+ T cells supported the
   observed potent anti‐tumor effect of MANS‐H@OVA and Quil‐A@OVA.

Figure 9.

   Figure 9
   [227]Open in a new tab

   MANS‐H adjuvanted OVA vaccine significantly suppresses B16‐OVA tumor
   growth by boosting polyfunctional T cell immune response. a) The
   experimental timeline of the therapeutic regimen of the B16‐OVA
   melanoma model: C57BL/6J mice were subcutaneously inoculated with 5 ×
   10^5 B16‐OVA cells on day 0 and then vaccinated twice with weekly
   intervals on day 5 and day 12 (created with permission by BioRender).
   b) Average and c) individual B16‐OVA tumor growth volume curves of
   immunized and unimmunized mice (n = 5). d) Digital photos and e)
   weights of tumor samples harvested at the endpoint (n = 5). f) ELISPOT
   analysis shows the numbers of OVA‐specific IFN‐γ^+CD8^+ T cells per
   10^6 splenocytes and g) the representative digital photos of CD8^+ T
   cells secreting IFN‐γ dots in the spleen after restimulation with
   OVA[257‐264] peptides (n = 4). h–k) Flow cytometry analysis shows the
   proportions of h) OVA‐specific IFN‐γ^+CD8^+ T cells, i) perforin^+CD8^+
   T cells and j) granzyme B^+ CD8^+ T cells, and representative dot plots
   of k) IFN‐γ^+CD8^+ T cells in spleens (n = 4). l–p) Flow cytometry
   analysis summarizes the percentages of B16‐OVA tumor‐infiltrating l)
   CD25^+CD8^+ T cells, m) CD69^+CD8^+ T cells, n) PD‐1^+CD8^+ T cells, o)
   Foxp3^+CD4^+ Tregs and q) NK cells, and p) representative dot plots of
   Foxp3^+CD4^+ T cells (n = 5). r) A schematic illustration shows the
   cytolytic functions of polyfunctional effector CD8^+ T cells and NK
   cells mediate potent anti‐tumor effect (created with permission by
   BioRender). Data are presented as means ± SEM (B) or SD (e,f, h–j, l–o,
   and P) (n = 5). P values were determined by two‐way ANOVA, Tukey's
   multiple‐comparison test (b), or one‐way ANOVA, Tukey's
   multiple‐comparison test (e,f, h–j, l–o, and p). n.s., not significant;
   ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001; ^**** p < 0.0001.

   We next examined the frequency of immune cells and phenotypes of
   tumor‐infiltrating lymphocytes (TILs) to explore the immuno‐features of
   the tumor microenvironment (Figure [228]S21, Supporting Information). A
   substantial increase in tumor‐infiltrating CD45^+ immune cells was
   observed in mice vaccinated with MANS‐H@OVA and Quil‐A@OVA compared
   with unimmunized mice (Figure [229]S22a, Supporting Information), and a
   trend toward increased CD45^+ immune cells in other immunized groups.
   MANS‐H@OVA and Quil‐A@OVA significantly promoted the frequency of CD8^+
   TILs (Figure [230]S22b, Supporting Information), while suppressing
   CD4^+ TILs (Figure [231]S22c, Supporting Information), resulting in
   enhanced ratios of CD8^+/CD4^+ TILs (Figure [232]S21d, Supporting
   Information). MANS‐H@OVA and Quil‐A@OVA exhibited dramatically enhanced
   distinct subpopulations of CD8^+ TILs with activation markers of CD25
   (Figure [233]9l; Figure [234]S23a, Supporting Information), CD69
   (Figure [235]9m; Figure [236]S23b, Supporting Information) and PD‐1
   (Figure [237]9n; Figure [238]S23c, Supporting Information). CD25^+ TILs
   and PD‐1^+ TILs that are terminally differentiated are also defined as
   exhausted and dysfunctional phenotypes of TILs.^[ [239]^37 ^] In
   addition, the infiltration of regulatory T cells (Tregs, Foxp3^+CD4^+ T
   cells) was greatly suppressed in tumors when mice received MANS‐H@OVA
   or Quil‐A@OVA, but not other immunization treatments
   (Figure [240]9o,p). Interestingly, the proportions of
   tumor‐infiltrating NK cells were strikingly raised in the MANS‐H@OVA
   and Quil‐A@OVA group (Figure [241]9q; Figure [242]S23d, Supporting
   Information), which also contributed to their outperformed
   tumor‐killing effect. Tumor tissue staining images revealed decreased
   proliferative activity and increased apoptosis and necrosis
   (Figure [243]S24, Supporting Information) in tumor samples from mice
   vaccinated with MANS‐H@OVA or Quil‐A@OVA.

   Collectively, MANS‐H displayed comparable potency compared with the
   commercial cellular response adjuvant Quil‐A in triggering the
   production of antigen‐specific CD8^+ T cells with multifaceted
   functionality, which resulted in dramatically increased cytolytic
   effector CD8^+ TILs and NK cells while suppressed Tregs infiltrated in
   tumor for superior anti‐tumor efficacy (Figure [244]9r).

2.7. Tumor Suppression in Preclinical Murine Human Papillomavirus (HPV)
Positive Tumor Models

   Instead of model antigens, we incorporated our MANS‐H adjuvant with an
   HPV16‐E7 antigen (RAHYNIVTF) and evaluated the therapeutic efficacy in
   a preclinical model of HPV16‐positive tumor TC‐1 in C57BL/6J mice. In a
   therapeutic regimen, C57BL/6J mice were subcutaneously injected with
   TC‐1 cells on day 0 and received two doses of therapeutic adjuvanted E7
   vaccines on day 11 and 18 respectively (Figure [245] 10a). Compared
   with the groups of Ctrl, ANS@E7, and MANS‐L@E7, MANS‐H@E7 vaccine
   significantly inhibited TC‐1 tumor growth (Figure [246]10b–d;
   Figure [247]S25a, Supporting Information). There was no obvious weight
   loss during the experimental period cross all groups (Figure [248]S25b,
   Supporting Information), suggesting the well‐tolerated safety profiles
   of these NS‐adjuvanted vaccines.

Figure 10.

   Figure 10
   [249]Open in a new tab

   MANS‐H adjuvanted E7 vaccine remarkably regresses the HPV E7‐expressing
   tumor growth. a) A scheme shows the experimental timeline: C57BL/6J
   mice were subcutaneously inoculated with 2 × 10^5 TC‐1 cells on day 0,
   and then vaccinated with diverse E7 vaccine formulations on day 11 and
   day 18 (created with permission by BioRender). b) Average and c)
   individual TC‐1 tumor growth curves of unimmunized and immunized mice
   (n = 6). d) Digital photos of tumor samples harvested at the endpoint
   on day 23 (n = 6). e,f) The splenocytes were re‐stimulated with E7
   peptide for the detection of E7‐specific IFN‐γ^+ CD8^+ T cells. e) Flow
   cytometry analysis shows the percentages and f) the representative dot
   plots of E7‐specific IFN‐γ^+CD8^+ T cells (n = 5). g‐o) Flow cytometry
   analysis shows the proportions of g) CD8^+ T cells, h) CD4^+ T cells
   and the ratios of i) CD8^+/CD4^+ T cells in CD45^+ cells, the
   representative dot plots and bar charts presenting the proportions of
   j,k) CD25^+CD8^+ T cells, l,m) TIM‐3^+CD8^+ T cells and n,o)
   PD‐1^+CD8^+ T cells pre‐gated on CD8^+ T cells in TC‐1 tumors derived
   from unimmunized and immunized mice (n = 5). p,q) Representative
   immunofluorescence images of tumor tissue sections stained with p)
   TUNEL or q) Ki‐67. Scale bars: 100 µm. Data are presented as means ±
   SEM (b) or SD (e, g–i, k, m and o). P values were determined by two‐way
   ANOVA, Tukey's multiple‐comparison test (b), or one‐way ANOVA, Tukey's
   multiple‐comparison test (e, g–i, k, m and o). n.s., not significant,
   ^* p < 0.05; ^** p < 0.01; ^**** p < 0.0001.

   We next sought to understand the anti‐tumor activity of the MANS‐H@E7
   vaccine by examining the E7‐specific CD8^+ T cell immune response
   induced by vaccination. The flow cytometry results demonstrated that
   the MANS‐H@E7 vaccine induced the highest proportion of E7‐specific
   IFN‐γ^+CD8^+ T cells among all groups (Figure [250]10e,f;
   Figure [251]S26, Supporting Information), implying a potent cytotoxic
   function against the tumor. We next assessed the subsets of TILs in
   TC‐1 tumor. As expected, the MANS‐H@E7 vaccine group substantially
   increased the fraction of CD8^+ TILs (Figure [252]10g; Figure [253]S27,
   Supporting Information) and reduced the proportion of CD4^+ TILs
   (Figure [254]10h; Figure [255]S27, Supporting Information), leading to
   a higher ratio of CD8^+/CD4^+ TILs (Figure [256]10i) than other three
   groups. We next analyzed the phenotypes of CD8^+ TILs (Figure [257]S28,
   Supporting Information). The results displayed a remarkable rise in the
   effector functional CD25^+CD8^+ TILs in mice vaccinated with MANS‐H@E7
   but not ANS@E7 or MANS‐L@E7 (Figure [258]10j,k). T cell immunoglobulin
   mucin receptor 3 (TIM‐3) and PD‐1 are usually defined as inhibitor
   markers, but also termed effector‐like markers when TILs are
   non‐terminally exhausted.^[ [259]^37 ^] Interestingly, the MANS‐H@E7
   vaccine dramatically promoted the frequencies of TIM‐3^+CD8^+ TILs and
   PD‐1^+CD8^+ TILs (Figure [260]10l–o). Further analysis of the
   proliferative activity of these subsets of CD8^+ TILs would define
   their effector‐like or exhausted phenotypes, thereby determining the
   necessity of the combination of anti‐PD‐1 or anti‐TIM‐3 monoclonal
   antibodies for enhanced cancer treatment. In addition, the analysis of
   the proliferation (Figure [261]10p), apoptosis and necrosis
   (Figure [262]10q; Figure [263]S29, Supporting Information) of TC‐1
   tumor samples suggested the strongest anti‐tumor efficacy of MANS‐H@E7
   vaccine, which was in line with the trend of tumor growth.
   Immunofluorescent staining results revealed a substantially increased
   number of CD8‐positive T cells infiltrated in tumor sites from mice
   receiving vaccination with MANS‐H@E7, which was consistent with the
   flow cytometry results.

2.8. In Vivo Assessments of Biodistribution and Biosafety Profiles of MANS‐H

   To explore the biodegradability of MANS‐H in vivo, mice were
   subcutaneously injected with MANS‐H@OVA‐Cy5.5 and then imaged by the in
   vivo imaging systems (IVIS) at different timepoints. The results showed
   that fluorescence signals of OVA‐Cy5.5 gradually declined over the
   monitored period while there were still some signals at day 14
   (Figure [264]S30, Supporting Information), suggesting that the vaccine
   formulation was readily degraded and retained at the injection site for
   more than 14 days. The biosafety of MANS‐H was further evaluated over a
   period of 14 days. The blood routine test and hepatorenal function
   indices (Figure [265]S31a,b, Supporting Information) revealed that
   there was no significant difference between the control and MANS‐H
   immunized groups. The histology studies demonstrated that there was no
   evident lesion toxicity in the major organs and no apparent
   inflammation was observed at the injection site either at day 7 or 14
   post‐vaccination (Figure [266]S31c, Supporting Information). These
   results demonstrated satisfactory biosafety of MANS‐H in vivo.

   According to the guidelines provided by the World Health Organization,
   pharmacokinetic studies of vaccines are not considered a prerequisite
   for their clinical approval. However, there are a few reports exploring
   the pharmacokinetics, half‐life, and clearance mechanism of inorganic
   adjuvants.^[ [267]^34 , [268]^38 ^] These results suggested that the
   half‐life of Al ions released from intramuscularly injected Alum
   adjuvants was ≈28 days in rabbits,^[ [269]^38 ^] while the silica
   adjuvants subcutaneously injected were possibly excreted via the
   kidneys.^[ [270]^34 ^] Our MANS‐H adjuvant might follow similar
   features with these inorganic adjuvants, which would be validated in
   our future work.

3. Conclusion

   Immunotherapy aims to train the host to generate potent and durable
   immunity against cancer, which has revolutionized cancer treatments,
   particularly antigen‐specific cancer immunotherapy.^[ [271]^39 ^]
   Despite the great success of immune checkpoint blockade (ICB) therapy
   in the clinic, only a small fraction of cancer patients can benefit
   from ICB (≈10–30%). Therapeutic vaccines are an alternative strategy to
   efficiently eradicate malignant cells by orchestrating antigen‐specific
   T cells.^[ [272]^40 ^] DCs are the largest and most crucial subsets of
   APCs, which are responsible for the capture, process, and presentation
   of various antigens. In the context of cancer immunotherapy, priming
   antigen‐specific CD8^+ T cells by cross‐presenting DCs is the key to
   regressing tumors. The cytolytic functions and numbers of CD8^+ T cells
   primarily rely on the positive signals from DCs stimulated by adjuvants
   and help signals of CD4^+ Th cells. The activity of adjuvants
   determines the maturation levels of DCs and subtypes of T‐cell
   immunity. MANS‐H constructed in this work demonstrated sufficient
   stimulation of cDC2 and cross‐presenting CD103^+/CD8^+ cDC1, which
   supported the observation of a markedly high magnitude of effector and
   memory phenotypes of CD8^+ T cells and durable humoral immunity,
   significantly suppressing tumor growth. In addition, accumulated
   evidence has revealed the significance of incorporating appropriate
   immunostimulants and antigens in structuring nano‐vaccines for
   sufficient co‐transport of antigens and stimulators, and augmented
   effectiveness and functions of CD8^+ T cells.^[ [273]^9 ^] In addition
   to chemical components, physical architecture is also a key determining
   factor, driving the immune response to rational structuring vaccines.^[
   [274]^1 , [275]^41 ^] Current emerging strategies mainly focus on zero‐
   or three‐dimensional architectures to regulate vaccine biodistribution
   and immune modulation, with few reports on 2D particulate vaccines.^[
   [276]^16 ^] Our results revealed that Al either in the form of NS or
   salts drove the production of antigen‐specific Tfh immune response,
   while the incorporation of Mn into ANS shaped the immune response to
   Th1 and CD8^+ T immunity and long‐lasting antibodies, which were
   consistent with the observations in the comparison between Alum and Mn
   salts.^[ [277]^9 ^] Differently, ANS containing high contents of Mn
   showed a similar level of Th2 immune response compared to Alum, which
   might be contributed by the hybrid components of Al and Mn.
   Additionally, the potent therapeutic effectiveness of MANS adjuvanted
   cancer vaccines was evidenced in the therapeutic regimens that were not
   examined in the previously reported Mn salt adjuvanted vaccine.^[
   [278]^9 ^]

   It has been reported that the hyperactive DCs stimulated long‐lived
   T‐cell responses, which are critically important for the complete
   eradication of established tumors.^[ [279]^42 ^] Therefore, to take the
   encouraging results from the preclinical model forward
   (Figure [280]10), it necessitates assessing T cell memory immune
   responses of MANS‐H adjuvanted HPV16‐E7 vaccine in long‐term
   survivability (more than 150 days in murine therapeutic settings) and
   tumor rechallenge models. These in‐depth preclinical studies will
   uncover additional factors that might impact the therapeutic efficacy
   of our adjuvant and cancer vaccines.

   In conclusion, we have uncovered the immune‐potentiating qualities of
   2D NS containing immunologically different MnAl. MANS‐H has shown
   remarkable proficiency in stimulating various subsets of innate immune
   cells and triggering a highly heterogeneous and functional
   tumor‐specific immunity. This led to superior tumor regression when
   combined with model or viral antigens in aggressive malignant melanoma
   and preclinical HPV^+ murine cancer models. These results highlight its
   potential for clinical use in developing therapeutic cancer vaccines.

4. Experimental Section

Chemicals and Biological Agents

   Aluminum acetylacetonate was purchased from J&K Scientific Ltd.
   KMnO[4], toluene, and sodium chloride were purchased from Sinopharm
   Chemical Reagent Co., Ltd. IFN‐β and IL‐1β ELISA kits were purchased
   from MULTISCIENCES (LIANKE) Biotech, Co., Ltd. DNase I was purchased
   from Nanjing Keygen Biotech. Collagenase D was purchased from Roche.
   OVA, OVA[257‐264] (SIINFEKL), OVA[323‐339] (ISQAVHAAHAEINEAGR), Quil‐A,
   and Alhydrogel were purchased from Invivogen. OVA‐Cy5.5 was purchased
   from XiAn QIYUE Biotech, Co Ltd. HPV16 E7 peptide (RAHYNIVTF) was
   purchased from GL Biochem, Ltd., Shanghai.

Mice and Cell Lines

   Six to eight weeks aged C57BL/6J mice were purchased from Shanghai
   Model Organisms Centre Inc. All experimental procedures were performed
   under the policies of the National Ministry of Health with the approval
   of the Shanghai University Animal Ethics Committee (approved ID:
   YS‐2023‐134). B16‐OVA and TC‐1 cells were maintained in a humidified
   atmosphere (37 °C, 5% CO[2]).

Synthesis of NaCl Crystals

   Typically, the equivalent volume of ethanol was added into the
   saturated aqueous NaCl solution (37 g NaCl per 100 mL deionized H[2]O)
   and kept at static conditions for 30 min for recrystallization. The
   slurry‐like sediment was harvested by filtration through filter papers
   and washed with absolute ethanol. The wet NaCl templates were filled
   into 2 mL centrifuge tubes for condensing under 3000 rpm for 5 min,
   followed by a drying process at 70 °C oven for 4 h.

Synthesis of ANS, MANS‐L, and MANS‐H

   ANS was synthesized by a hard template method. Briefly, aluminum
   acetylacetonate (768 mg) was dissolved in toluene (40 mL) for 30 min
   under sonication. The precursor supernatant was collected with
   centrifugation (9000 rpm, 10 min), and then was added to hard NaCl
   templates with a ratio of 30% (V/W, 300 µL precursor solution per gram
   of templates). Standing for 10 min allowed the precursor solution to
   wet through the surface of NaCl templates filled in centrifuge tubes,
   which were kept in a humidity chamber (Shanghai Yiheng Technology
   Instrument Co., Ltd.) with 85% relative humidity and 25 °C temperature
   for 48 h. Finally, ANS was obtained by washing with water 3 times to
   remove NaCl templates and washing with absolute ethanol once to remove
   incomplete reacted precursors.

   MANS‐L and MANS‐H were prepared by doping Mn on ANS. In particular, ANS
   (5 mg) was suspended in water (5 mL) to prepare the solution with a
   concentration of 1 mg mL^−1. Then, 4 mL KMnO[4] solution with a
   concentration of 1.3 or 0.1625 mg mL^−1 was added in ANS (5 mL, 1 mg
   mL^−1) under stirring (300 rpm h^−1, in dark) for 1 h for the synthesis
   of MANS‐L and MANS‐H, respectively. MANS‐L and MANS‐H samples were
   obtained with centrifugation (15 000 rpm, 10 min) and washed 3 times
   with water.

Characterization of Micro‐nanosheets

   NSs were suspended in anhydrous ethanol for a simple ultrasound
   followed by dropping on the carbon‐covered Cu grid, which was used for
   the TEM imaging or energy dispersive X‐ray spectroscopy (EDS) analysis
   (JEOL 1400 or JEOL 2100F). Additionally, NSs were dried at 70 °C in the
   drying oven for 6 h for the XPS analysis (Escalab 250Xi, Thermo
   Scientific) and Fourier transformed infrared (FTIR) analysis (Nicolet
   iS50, Thermo Scientific). NSs were suspended in PBS and measured by
   Zetasizer Nano ZSE for the detection of zeta potential (25 °C). Few NSs
   were soluble in aqua regia for 24 h which was used for the analysis of
   ICP (PERKINE 7300DV, Perkin Elmer).

BMDC and Macrophage Culture

   The extraction and culture of BMDCs were according to the previously
   reported method.^[ [281]^43 ^] In brief, bone marrow cells were
   collected from the femur and tibia of naive C57BL/6J mice and seeded in
   a 100 mm culture dish with RPMI1640 complete medium supplemented with
   100 ng mL^−1 IL‐4 and 200 ng mL^−1 GM‐CSF. On day 3 and day 6, the
   culture medium was replaced with a fresh BMDC medium. On day 7, BMDCs
   were harvested for subsequent experiments. For bone marrow macrophages,
   bone marrow cells were cultured in DMEM complete medium with 200 ng
   mL^−1 M‐CSF, while the remaining procedures remained unchanged.

BMDC Activation

   Briefly, BMDCs (5 × 10^5 cells per tube, 1 mL) were seeded in a flow
   cytometry caped tube (BD Falcon), followed by the treatment of PBS or
   NSs (ANS, MANS‐L or MANS‐H) (15 µL, 1 mg mL^−1). After 24 h incubation
   in a cell incubator, the cell pellets were washed with cold PBS
   followed by collection by centrifugation (350 g, 4 °C, 5 min) for the
   further staining of flow cytometry analysis.

Intracellular ROS Detection

   BMDCs (5 × 10^5 cells per tube, 0.5 mL) were seeded in a flow cytometry
   caped tube and then treated with PBS or NSs (15 µL, 1 mg mL^−1). After
   incubation for 10 h, the 0.5 mL DCF‐DA solution (40 µm, Sigma) was
   added for 20 min incubation. Then cell pellets were washed with cold
   PBS followed by collection by centrifugation (350 g, 4 °C, 5 min) for
   the further staining of flow cytometry analysis. In another experiment,
   BMDCs (5 × 10^5 per well, 0.5 mL) were seeded in a 24‐well plate
   containing round coverslips and cultured for 24 h. After a 10 h
   co‐culturation with PBS or NSs (15 µL, 1 mg mL^−1), the 0.5 mL DCF‐DA
   solution (40 µm) was added for 20 min incubation (in dark). Following
   two times wash with cold PBS, 4,6‐diamidino‐2‐phenylindole (DAPI,
   Beyotime) was dropped on coverslips for intracellular ROS detection by
   confocal microscopy (LSM710, Zeiss).

OVA Antigen Loading Capacity

   The loading capacity of ANS, MANS‐L, and MANS‐H NSs with OVA‐Cy5.5 was
   estimated by detecting fluorescence intensity using a microplate
   reader. In detail, NS (1 mL, 6 mg mL^−1) was mixed with poly
   (allylamine hydrochloride)^[ [282]^32 ^] (2 mL, 20 mg mL^−1) for
   stirring at 600 rpm for 1 h at room temperature. After that, the NS@PAH
   was collected by centrifugation (14 000 rpm, 10 min) and re‐suspended
   in 1 mL PBS. OVA‐Cy5.5 (1 mL, 600 µg mL^−1) was added in NS@PAH
   solution (1 mL, 6 mg mL^−1) under stirring at 600 rpm for 0.5 h at 4 °C
   (in dark). The supernatant containing excess OVA‐Cy5.5 was collected by
   centrifugation (15 000 rpm, 20 min). The fluorescence intensity of 100
   µL of supernatant or standard solutions placed in a 96‐well plate was
   measured by a microplate reader (fluorescence excitation peak at 656 nm
   and emission peak at 700 nm. VARIOSKAN LUX, Thermo Scientific).

Cellular Uptake of NS Adjuvanted OVA‐Cy5.5 Vaccines in BMDCs

   Briefly, OVA‐Cy5.5 was loaded with three NSs as previously described.
   BMDCs (5 × 10^5 cells per tube, 1 mL) were seeded in a flow cytometry
   capped tube. Then PBS, free OVA‐Cy5.5, and three NSs adjuvanted
   OVA‐Cy5.5 vaccines (15 µL, 1 mg mL^−1) were added respectively and
   further cultured for 6 h. Following the wash with cold PBS, the cell
   pellets were obtained for flow cytometry staining and analysis or
   staining with iFluorTM 488 phalloidin and DAPI for confocal imaging
   (STELLARIS 8, Leica).

In vivo Biodistribution of OVA‐Cy5.5 and Cross Presentation of OVA Mediated
by NS Adjuvants

   As mentioned above, mice were vaccinated with PBS, free OVA‐Cy5.5 or
   three NSs (300 µg) adjuvanted OVA‐Cy5.5 or OVA vaccines. After 24, 48,
   or 72 h, the skin‐dLNs were harvested respectively and were digested
   according to the established method (see below in single cell
   preparation) followed by staining for flow cytometry analysis. To
   investigate the degradation of NS, C57BL/6J mice were subcutaneously
   injected with MANS‐H adjuvanted OVA‐Cy5.5 vaccine and visualized by
   IVIS spectrum system at different time points (day 0, day 7, and day
   14).

Biosafety Evaluation

   The biosafety evaluation was performed in C57BL/6J mice subcutaneously
   injected with 300 µg MANS‐H. The blood samples, major organs, and skin
   samples at the injection site were harvested on days 7 and 14
   post‐injection. A blood routine test was performed to analyze the
   levels of red blood cells (RBC), white blood cells (WBC), platelets
   (PLT), and hemoglobin (HGB). After centrifugation at 12 000 rpm for
   20 min, the serum samples were obtained for biochemical analysis of the
   levels of alanine aminotransferase (ALT), aspartate aminotransferase
   (AST), creatinine (CREA), and UREA. The tissue samples were stained
   with hematoxylin and eosin for histology studies.

Tumor Cell Culture and Development

   Briefly, B16‐OVA and TC‐1 cells were injected subcutaneously into the
   abdomen of the mice with the indicated cell concentration in the
   Figures. Tumor size was measured every two days and tumor volume was
   estimated as length × width^2/2. When tumor volume reached 1500 mm^3,
   animals were sacrificed.

Single‐cell Suspension Preparation for Flow Cytometry Analysis

   Acquired skin samples (−1 cm^2, in the administration site), skin‐dLNs,
   and tumor tissues were chopped thoroughly and incubated with a solution
   containing DNase I (0.2 mg mL^−1) and collagenase D (1 mg mL^−1) for
   1.5 h,1 h, and 0.5 h (37 °C) respectively. All cells were filtered
   through the 70 µm cell strainers (Biosharp) followed by being
   re‐suspended for the next flow cytometry staining. Spleens were
   harvested at the indicated point in Figures, which were mechanically
   disrupted through 70 µm cell strainers. Following washed by FACS buffer
   and lysed with ACK lysis buffer, splenocytes were re‐suspended for
   further flow cytometry staining. For antigen‐specific T cell detection,
   splenocytes were seeded in 96‐well plates followed by the addition of
   OVA[257‐264], OVA[323‐339], or E7 peptides (10 µg mL^−1) for 2 h
   stimulation. The solution included GolgiPlug (1:1000, BD Biosciences)
   was added for another 4 h stimulation. Then, splenocytes were collected
   and washed for flow cytometry staining.

ELISPOT Analysis of Antigen‐specific CD8^+ T Cells

   Splenocytes (2.5 × 10^5 cells per well, 50 µL) were seed in a definite
   96‐well plate (Millipore, Merck), which was cultured with anti‐IFN‐γ
   antibody (14‐7313‐85, eBioscience) overnight at 4 °C in advance. Then
   the OVA[257‐264] or E7 peptides (10 µg mL^−1) were added for another
   20 h incubation in a cell incubator. After removal of cells,
   biotinylated Ab against IFN‐γ (13‐7312‐85, eBioscience) was added for
   4 h incubation at room temperature in the dark followed by the addition
   of HRP‐conjugated streptavidin (A3151, Sigma‐Aldrich) for incubation 45
   mins. After washing several times, PBS solution containing UREA and DAB
   tablets (D0426, Sigma‐Aldrich) was added to the holes of the plate.
   Lastly, spots were recorded and counted by an ELISPOT plate reader (AID
   EliSpot Reader System, Germany).

Cells Staining of Flow Cytometry Analysis

   To avoid non‐specific binding of antigens and to separate dead‐live
   cells, all cell suspensions were stained first with anti‐CD16/32 and
   LIVE/DEAD Fixable Aqua (Invitrogen). The skin cells were stained with a
   mixture of antibodies containing anti‐F4/80 (FITC), anti‐TCR‐β (FITC),
   anti‐CD45 (Percp‐Cy5.5), anti‐CD11b (Pacific blue), anti‐EpCAM (APC),
   anti‐MHC II (APC‐Cy7), anti‐CD103 (PE), anti‐CD11c (PE‐Cy7), anti‐Ly6G
   (Alexa Fluor 700), and anti‐Ly6C (Brilliant Violet 510). The dLNs cells
   were stained with a mixture of antibodies containing
   anti‐CD80/anti‐CD19/anti‐TCR‐β/anti‐CD3 (FITC), anti‐CD8a
   (PerCP‐Cy5.5), anti‐EpCAM/anti‐CD11b/anti‐CD8a (APC), anti‐MHC II/anti‐
   H2Kb to SIINFEKL (APC‐Cy7), anti‐CD103 (PE), anti‐CD11c (PE‐Cy7),
   anti‐CD11b (Alexa Fluor 700) and anti‐CD86/anti‐F4/80/anti‐MHC II
   (Pacific blue). The B16‐OVA or TC‐1 tumor cells were stained with a
   mixture of antibodies containing anti‐CD45 (Percp‐Cy5.5), anti‐TCR‐β
   (FITC), anti‐CD4 (AF700), anti‐CD25 (Brilliant Violet 421),
   anti‐CD69/anti‐NK 1.1/CD8a (PE‐Cy7), anti‐CD8a/anti‐CD3/anti‐TIM‐3 (PE)
   and anti‐PD‐1 (APC‐Cy7). Following Fixation/Permeabilization (Thermo
   Fisher) for 45 mins, tumor‐infiltrating lymphocytes were stained with
   anti‐Foxp3 (Alexa Flour 647) or isotype antibody. The splenocytes were
   stained with a mixture of antibodies containing anti‐TCR‐β (Pacific
   blue), anti‐CD8a (PCP‐Cy5.5), anti‐CD4 (PE‐Cy7), anti‐CD62L (PE),
   anti‐CXCR5 (APC‐Cy7) and anti‐CD44/anti‐CD4 (Alexa Fluor 700).
   Following Fixation/Permeabilization for 45 mins, splenocytes were
   stained with a master mix of antibodies containing anti‐IFN‐γ (FITC),
   anti‐Perforin/anti‐IL‐4 (APC), and anti‐Granzyme B (APC‐Cy7) or
   corresponding isotype antibodies for intracellularly cytokines
   detection. At last, all stained cells were resuspended in FACS buffer
   which was analyzed by flow cytometer (BD LSRFortessa). All detailed
   information on antibodies can be found in Table [283]S1 (Supporting
   Information). Flow cytometry data were analyzed by FlowJo 10.8.1 or
   Kaluza software.

Statistical Analysis

   Statistical data were analyzed by GraphPad Prism 9.5.1. and detailed
   analysis methods were outlined in the indicated Figures.

Conflict of Interest

   The authors declare no conflict of interest.

Supporting information

   Supporting Information
   [284]ADVS-11-2405729-s001.pdf^ (3.7MB, pdf)

Acknowledgements