Abstract

   Apoptosis is crucial for maintaining internal homeostasis. Apoptotic
   vesicles (ApoVs) derived from mesenchymal stem/ stromal cells
   (MSCs‐ApoVs) as natural lipid nanoparticles are attractive candidates
   for the next generation of immunotherapies. However, the therapeutic
   potential of MSCs‐ApoVs in managing hypersensitivity reactions mediated
   by CD8^+ T cells remains elusive. This research utilized contact
   hypersensitivity and oral lichenoid reaction models, both of which
   represent type IV hypersensitivity reactions. ApoVs are shown that
   derived from stem cells from human exfoliated deciduous teeth
   (SHED‐ApoVs), a subtype of MSCs, directly fused with the plasma
   membrane of CD8^+ T cells, subsequently increasing membrane
   permeability through L‐type voltage‐gated Ca^2+ channels. This
   initiates a cascade of events including calcium overload, mitochondrial
   dysfunction, and the initiation of apoptosis in these cells. As known,
   this is the first study to characterize SHED‐ApoVs as immune
   microenvironment modulators, demonstrating their therapeutic potential
   and mechanism in these reactions. Moreover, analysis of blood samples
   from patients with oral lichenoid reactions verified the
   antihypersensitivity property of SHED‐ApoVs. This study sheds light on
   the therapeutic prospects of MSCs‐ApoVs and their underlying mechanisms
   in diseases mediated by CD8^+ T cells, contributing novel perspectives
   for the clinical application of ApoVs and nanovesicle‐based cell‐free
   therapies.

   Keywords: apoptotic vesicles, CD8^+ T cells, mesenchymal stem/stromal
   cells, mitochondrial dysfunction, type IV hypersensitivity responses
     __________________________________________________________________

   The therapeutic potential of ApoVs derived from stem cells from human
   exfoliated deciduous teeth (SHED‐ApoVs) in the treatment of CD8^+ T
   cell‐mediated hypersensitivity reactions is noteworthy. SHED‐ApoVs are
   capable of fusing with the plasma membrane of CD8^+ T cells, which
   subsequently triggers a series of events characterized by calcium
   overload, mitochondrial dysfunction, and the initiation of apoptosis in
   these cells.

   graphic file with name ADVS-12-2407446-g008.jpg

1. Introduction

   Apoptosis serves as the predominant form of programmed cell death,
   essential for preserving internal homeostasis.^[ [50]^1 ^] Apoptotic
   vesicles (ApoVs) are natural lipid nanoparticles that encapsulate
   apoptotic cell components during biogenesis, inheriting diverse and
   unique properties from their parent cells.^[ [51]^2 ^] However, the
   precise therapeutic role of ApoVs derived from various cell sources
   remains elusive. To date, numerous patients have undergone
   transplantation with culture‐expanded mesenchymal stem/ stromal cells
   (MSCs) for the treatment of various immune diseases, including systemic
   lupus erythematosus and rheumatoid arthritis.^[ [52]^3 ^] Mounting
   evidences indicate that the therapeutic efficacy of MSCs
   transplantation may be associated with the secretion of MSC‐derived
   ApoVs (MSC‐ApoVs). This is because transplanted MSCs are swiftly
   recognized and eliminated by the host immune system, while still
   retaining their immunoregulatory function.^[ [53]^4 ^] Compared to the
   hurdles faced in MSC therapy, such as ethical concerns and tumor
   formation, MSCs‐ApoVs offer a promising alternative. Their tissue
   repair‐promoting characteristics and immunoregulatory potential,
   inherited from their parent cells, make these natural lipid
   nanoparticles attractive candidates for advancing nanovesicle‐based
   cell‐free immunotherapies.

   Stem cells from human exfoliated deciduous teeth (SHED), a subtype of
   MSCs originating from the early neural crest stem cells, possess
   optimal immunomodulatory and regenerative properties.^[ [54]^5 ^]
   Studies have demonstrated that SHED exhibits a more favorable cytokine
   profile in relation to inflammatory, immunomodulatory, proliferative,
   and osteogenic potential compared to human dental pulp stem cells
   (hDPSCs).^[ [55]^6 ^] Moreover, SHED displays significant effects in
   inhibiting T helper 17 cells when compared to human bone marrow
   mesenchymal stem cells (hBMSCs).^[ [56]^7 ^] Previously, our team
   confirmed the ideal anti‐inflammatory and regenerative properties of
   SHED and successfully achieved tooth regeneration in both animal models
   and clinical trials.^[ [57]^5 , [58]^8 ^] Additionally, it has been
   reported that the immunomodulatory potential of SHED in ameliorating
   gland inflammation and dryness symptoms of Sjögren's syndrome via
   secreting soluble programmed cell death ligand 1 (PD‐L1).^[ [59]^9 ^]
   Furthermore, we verified that exosomes and ApoVs derived from SHED
   serve as modulators of the inflammatory microenvironment, thereby
   promoting the angiogenesis process during the regeneration of dental
   pulp tissue.^[ [60]^10 ^] However, whether SHED‐derived ApoVs
   (SHED‐ApoVs) are regulators of the immune microenvironment, possessing
   immunomodulatory function as their parent cells remains largely
   unclear. Investigating the immunomodulatory properties of SHED‐ApoVs
   and elucidating their impact on immune cells has the potential to
   broaden their clinical application and underscore their promise as
   immune therapeutics.

   The cytotoxic function of CD8^+ T cells against tumor cells plays a
   crucial role in immune surveillance against cancer. Nonetheless, when
   autoreactive responses occur and target the body's own cells, CD8^+ T
   cells can result in autoimmune disorders, graft rejection reactions,
   and type IV hypersensitivity reactions.^[ [61]^11 ^] Type IV
   hypersensitivity is involved in the pathogenesis of various diseases,
   including allergic contact dermatitis, oral lichenoid reactions (OLR),
   and multiple sclerosis.^[ [62]^12 ^] Given the intricate etiology and
   varied manifestations of type IV hypersensitivity, the primary
   treatment focus revolves around symptom management. Factors like
   medication effectiveness, optimal glucocorticoid timing, and patient
   tolerability are key determinants of treatment outcome.^[ [63]^13 ^]
   Thus, the advancement of tailored and potent immunotherapy strategies
   is essential in this research field. Type IV hypersensitivity, commonly
   referred to as delayed hypersensitivity, typically manifests 24–72 h
   after exposure and is distinguished by two distinct phases. The
   sensitization phase involves events following the initial exposure to
   the hapten, leading to the development of CD8^+ T cells. During the
   elicitation phase, re‐exposure of sensitized individuals to the hapten
   results in clinical manifestations.^[ [64]^14 ^] However, there is
   still insufficient knowledge regarding the therapeutic potential of
   ApoVs on hypersensitivity responses. Exploring this area will provide
   new insights into the physiology of ApoVs and offer a promising
   approach for the management of CD8^+ T cell‐related diseases, such as
   viral infections, tumors, and autoimmune diseases.

   This research aims to explore the potential of ApoVs as nano‐mediators
   in the immune microenvironment for modulating type IV hypersensitivity.
   We characterized SHED‐ApoVs and employed contact hypersensitivity (CHS)
   and oral lichenoid reactions (OLR) models, both of which represent type
   IV hypersensitivity reactions, to investigate the influence of ApoVs on
   CD8^+ T cells. Interestingly, we uncovered the therapeutic role of
   ApoVs in type IV hypersensitivity reactions and first revealed a
   significant anti‐hypersensitivity function of SHED‐ApoVs on CD8^+ T
   cells, which was confirmed by patients with type IV hypersensitivity.
   Mechanically, ApoVs interact with CD8^+ T cells via membrane fusion,
   resulting in upregulation of L‐type voltage‐gated Ca^2+ channels. This
   triggers a cascade of reactions including calcium overload,
   mitochondrial dysfunction, BAX translocation, and eventual apoptosis in
   CD8^+ T cells (Scheme [65] 1 ). The findings provide a foundation for
   further preclinical research on MSCs‐ApoVs and endorse the application
   of nanovesicle‐based cell‐free therapy for diseases mediated by CD8^+ T
   cells.

Scheme 1.

   Scheme 1
   [66]Open in a new tab

   The schematic diagram demonstrates the ApoVs interact with CD8^+ T
   cells via membrane fusion, triggering cascade reactions including
   calcium overload, mitochondrial dysfunction, BAX translocation, and
   eventual apoptosis in CD8^+ T cells.

2. Results

2.1. Isolation and Characterization of MSCs and MSCs‐ApoVs

   SHED as a kind of MSCs was used in this study, which possessed general
   MSCs characteristics including the capacity for self‐renewal and
   multipotent differentiation (Figure [67]S1A, Supporting Information).
   Furthermore, these cells expressed MSCs surface molecules including
   CD90, CD105, CD73, and CD29, while showing negative expression of
   leukocyte and hematopoietic progenitor cell surface markers CD14,
   HLA‐DR, CD45, CD34, and CD79a (Figure [68]S1B, Supporting Information).
   Apoptosis in SHED was induced using Staurosporine (STS) for 16 h, and
   ApoVs were isolated through an established sequential certification
   process (Figure [69]S1C, Supporting Information). The characteristic
   morphology changes, such as swelling and spherification in apoptosis of
   SHED were observed by microscope (Figure [70]S1D, Supporting
   Information). Flow cytometry analysis confirmed an increased percentage
   of apoptotic SHED using Annexin V/PI double staining (Figure [71]S1E,
   Supporting Information). Transmission electron microscopy (TEM)
   demonstrated that ApoVs displayed a classical double‐membrane spherical
   structure (Figure [72]S1F, Supporting Information). Nanoparticle
   tracking analysis (NTA) indicated that the size of ApoVs ranged from
   400 to 600 nm (Figure [73]S1G, Supporting Information). ApoVs expressed
   common extracellular vesicles (EVs) markers CD63, CD9, and TSG101, but
   failed to express exosomes‐specific marker GM130. Compared to uninduced
   SHED, ApoVs expressed the specific apoptosis‐associated marker
   cleaved‐caspase3 (Figure [74]S1H, Supporting Information). These
   findings verified that the ApoVs derived from SHED in this study
   exhibit the specific characteristics of high‐purity apoptotic vesicles
   originating from MSCs.

2.2. MSCs‐ApoVs Treatment Attenuated CD8^+ T Cells‐mediated Contact
Hypersensitivity

   In this study, a mouse CHS model was established using oxazolone (OXA)
   as a hapten, following a previously described protocol.^[ [75]^14 ,
   [76]^15 ^] Briefly, mice were sensitized epicutaneously on the shaved
   abdomen with 100 µL of 3.0% OXA on day 0. Five days later, OXA was
   challenged on both ears. After 20 min of OXA challenge, ApoVs were
   intradermally injected into one ear while the other ear served as the
   control group which was injected with phosphate‐buffered saline (PBS)
   (Figure [77] 1A). The varying degree of swelling between the left and
   right ears showed that ApoVs significantly inhibited ear swelling in a
   dose‐dependent manner compared to the control group. A concentration of
   20 mg mL^−1 ApoVs was selected for subsequent in vivo studies (Figure
   [78]S2A, Supporting Information). To confirm the presence of ApoVs at
   the lesion site, the membrane of ApoVs was labeled with PKH26.
   Following injection of labeled ApoVs into the lesioned ears of mice,
   the ears were collected after 6 h and examined via immunofluorescence
   staining. Immunofluorescence staining revealed the PKH26‐positive cells
   within the lesion area, indicating successful injection of ApoVs
   (Figure [79]S2B, Supporting Information). Dermoscopic analysis revealed
   that the control group displayed elicited reactions such as slight
   local skin hyperemia and redness compared with the normal group, while
   the ApoVs injection group showed attenuated reactions (Figure [80]1B).
   Hematoxylin and eosin (H&E) staining revealed the presence of
   spongiosis and capillary dilation in the control group, which differed
   from the observed characteristics in both normal ears and the group
   that received ApoVs injection (Figure [81]1C). Ear thickness
   measurements using an Engineer's micrometer caliper before and after
   the challenge showed a notable suppression of ear swelling in the ApoVs
   injection group (Figure [82]1D; Figure [83]S2C, Supporting
   Information).

Figure 1.

   Figure 1
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   MSCs‐ApoVs Treatment Attenuated CD8^+ T Cells‐mediated Contact
   Hypersensitivity. A) Schematic illustration of contact hypersensitivity
   experimental design. B) Representative phenotype of ears captured by
   dermoscopy. C) Hematoxylin‐eosin (H&E) staining of ear samples
   collected 24 h after the challenge. The lower panel magnifies the boxed
   area in the top panel. Scale bar: 150 µm for the upper panel and 50 µm
   for the lower panel. Black arrowheads indicate dilated capillaries. D)
   Ear thickness was measured 24 h after the elicitation in three groups
   (n = 15). E) Volcano plots displayed the profile of differentially
   expressed genes of ear samples collected form the Control and ApoVs
   injection groups (n = 3). The 222 upregulated DEGs (red) and 198
   downregulated DEGs (blue) were shown in plots. F) Heatmap exhibited the
   profiles of upregulated and downregulated DEGs in the ApoVs injection
   group compared to the Control group (n = 3). G‐H) Gene Ontology (GO) of
   biological processes enrichment analysis related to the downregulated
   and upregulated DEGs in ear samples. The top 10 enriched terms were
   presented as bubble charts. I‐J) Representative images and
   quantification of CD8^+ T cells in the Control and ApoVs injection
   groups (n = 5). Scale bar: 50 µm. ** p < 0.01, ***p < 0.001.

   To identify the gene expression profile of ear tissue treated with
   ApoVs, RNA transcriptomic sequencing was performed on ear samples from
   the control and ApoVs injection group. Volcano maps illustrated the
   magnitude of fold change for all genes with false discovery rate (FDR),
   in the ApoVs injection group compared to the control group. The
   profiles of 222 differentially upregulated and 198 downregulated genes
   (DEGs) were shown in volcano plots and heatmap (Figure [85]1E,F).
   Moreover, Gene ontology (GO) analysis of biological processes exhibited
   the top 10 gene enrichment pathways for upregulated and downregulated
   DEGs. The downregulated genes were notably associated with inflammatory
   and immune response, implying that ear hypersensitivity leads to tissue
   inflammatory and immune response. The upregulated genes were primarily
   linked to metabolic and apoptotic processes, implying that ApoVs
   treatment accelerated local tissue metabolic and apoptotic processes
   (Figure [86]1G,H). qRT‐PCR analysis of ear tissues after ApoVs
   treatment confirmed a decreased level of inflammation, with lower
   expression of Ifng (IFN‐γ), Il1b (IL‐1β), and Il6 (IL‐6), and reduced
   expression of T cell‐attracting chemokines like Cxcl9, Cxcl10, and
   Cxcl11 (Figure [87]S2D,E, Supporting Information). Immunostaining of
   endothelial markers CD31 in ear lesions displayed a decrease of
   capillary dilation in the ApoVs injection group, consistent with H&E
   finding (Figure [88]S2F,G, Supporting Information). Simultaneously,
   following ApoVs treatment, immunostaining showed decreased expression
   of pro‐inflammatory TNF‐α^+ cells and increased anti‐inflammatory
   TGF‐β^+ cells in the lesion site compared to the control group (Figure
   [89]S2H,I, Supporting Information). In addition, there was a reduction
   in the percentage of CD8^+ T cells at lesional sites after 24 h of
   ApoVs injection (Figure [90]1I,J). The data presented implicate that
   treatment with SHED‐ApoVs effectively ameliorates
   hypersensitivity‐induced inflammatory and immune responses by
   potentially decreasing the percentage of CD8^+ T cells in the affected
   area.

2.3. ApoVs‐afforded Antihypersensitivity Effects by Promoting the Apoptosis
of CD8^+ T Cells

   Next, to validate whether ApoVs alleviated hypersensitivity reactions
   by specifically targeting CD8^+ T cells during the elicitation phase,
   an adoptive transfer experiment was carried out. Specifically, cells
   were collected from the inguinal lymph nodes of OXA‐sensitized mice 5
   days after sensitization and then enriched for sensitized CD8^+ T
   cells. The sorted sensitized CD8^+ T cells were treated with PBS or
   ApoVs and adoptively transferred to unsensitized mice via tail vein
   injection (Figure [91] 2A). Flow cytometric analysis confirmed the
   purity of sorted CD8^+ T cells (CD3^+ CD8^+ T cells) (Figure [92]S3A,
   Supporting Information). The optimal concentration of ApoVs (10 µg/mL)
   was determined based on in vitro analysis of IFN‐γ production, a marker
   of CD8^+ T cell function (Figure [93]S3B, Supporting Information).
   Subsequent ELISA analysis revealed a decrease in the expression of
   CD8^+ T cells effector cytokines, including IFN‐γ, TNF‐α, perforin‐1,
   and granzyme B, after treatment with ApoVs (Figure [94]S3C–F,
   Supporting Information). Following exposure to OXA, the ear response of
   CD8^+ T cells treated with PBS displayed signs of redness and
   hyperemia, indicating the success of the adoptive transfer experiment.
   Furthermore, H&E staining and micrometer caliper measurements showed
   the recipient mice injected with ApoVs‐treated CD8^+ T cells had
   significantly decreased ear redness and swelling compared to those
   injected with PBS‐treated CD8^+ T cells (Figure [95]2B–D).
   Additionally, IHC staining of ear lesions revealed a significant
   reduction in enlarged blood vessels and the presence of
   pro‐inflammatory TNF‐α positive cells following treatment with ApoVs
   (Figure [96]S4A,B, Supporting Information). In this study, it was
   observed that the ear hypersensitivity response in recipient mice was
   dependent on the sensitized CD8^+ T cells that were injected. These
   findings imply a direct inhibitory effect of ApoVs on CD8^+ T cells
   during the elicitation phase of hypersensitivity reactions.

Figure 2.

   Figure 2
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   ApoVs‐afforded Anti‐hypersensitivity Effects by Promoting the Apoptosis
   of CD8^+ T Cells. A) Schemes of the adoptive transfer experimental
   design. CD8^+ T cells from the draining lymph nodes of
   oxazolone‐sensitized WT mice were isolated and cultured with or without
   ApoVs. Naïve WT mice then received adoptive transfer of these CD8^+ T
   cells, followed by treatment on the ears of recipient mice with OXA 2 h
   post‐transfer. B) Representative ear lesions visualized by dermoscopy.
   C) H&E staining of ear samples collected 24 h after the challenge. The
   lower panel magnifies the boxed area in the top panel. Scale bar:
   150 µm for the upper panel and 50 µm for the lower panel. Black
   arrowheads indicate dilated capillaries. D) Ear thickness was measured
   24 h after the elicitation in groups receiving intravenous infusion of
   CD8^+ T cells treated with PBS or ApoVs (n = 10). E) Bubble diagram
   displayed the top 10 KEGG pathways enriched terms of upregulated DEGs
   in ApoVs‐treated CD8^+ T cells compared to PBS‐treated CD8^+ T cells
   (n = 3). F) The bar chart showed the top 12 Reactome enrichment
   pathways of upregulated DEGs in ApoVs‐treated CD8^+ T cells compared to
   PBS‐treated CD8^+ T cells (n = 3). G) Annexin V/PI double staining
   showed that ApoVs induced CD8^+ T cell apoptosis (n = 3). H) Westen
   blot displayed the expression level of classical apoptosis protein
   cleaved‐Caspase 9 and cleaved‐Caspase 3 in CD8^+ T cells after ApoVs
   treatment. * p < 0.05, ***p < 0.001, MACS: Magnetic‐Activated Cell
   Sorting.

   To further understand the mechanisms underlying the effects of ApoVs on
   CD8^+ T cells, RNA transcriptomic sequencing was performed on
   ApoVs‐treated CD8^+ T cells, with PBS‐treated CD8^+ T cells as a
   control. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway
   analysis exhibited that the upregulated genes in the ApoVs‐treated
   group were significantly enriched in terms related to
   apoptosis‐relative signaling (Figure [98]2E). Similarly, Reactome
   pathway enrichment analysis showed that the upregulated genes were
   enriched in terms involving cell death‐related signaling
   (Figure [99]2F). Hence, we examined the apoptosis levels of CD8^+ T
   cells subsequent to ApoVs administration. Flow cytometric analysis
   revealed an increase in the percentage of Annexin V‐positive apoptotic
   CD8^+ T cells following ApoVs treatment (Figure [100]2G). ApoVs
   treatment markedly induced CD8^+ T cells apoptosis with upregulated
   expression of specific apoptosis‐associated markers cleaved caspase‐9
   and cleaved caspase‐3 (Figure [101]2H). The co‐localization of
   apoptotic marker TUNEL and CD8^+ T cells at lesion sites 6 h post‐ApoVs
   injection further confirmed the apoptosis of CD8^+ T cells after ApoVs
   treatment in vivo (Figure [102]S4C, Supporting Information). The
   findings revealed that ApoVs not only repressed the function of CD8^+ T
   cells in vivo but also triggered their apoptosis. The presented
   findings indicate that treatment with ApoVs notably ameliorated
   hypersensitivity responses via attenuating the function and inducing
   apoptosis of CD8^+ T cells.

2.4. ApoVs Induced Apoptosis in CD8^+ T cells via Affecting Mitochondrial
Morphology and Function

   To delve deeper into the potential molecular mechanisms of ApoVs,
   proteomic analysis of ApoVs was performed. Considering that ApoVs
   represent a specific subtype of extracellular vesicles (EVs) released
   by cells under particular cellular conditions, we utilized EVs
   originating from the same cellular source as the control group to
   ensure comparability. GO of molecular function exhibited the top 10
   enrichment pathways of the upregulated proteins significantly enriched
   in terms related to mitochondrial function, such as ATP hydrolysis
   activity and electron transfer activity (Figure [103] 3A). Similarly,
   Reactome pathway enrichment analysis showed enrichment of upregulated
   proteins in mitochondrial‐related terms, including mitochondrial
   translation termination and mitochondrial protein degradation
   (Figure [104]3B). The enrichment analysis consistently pointed toward
   mitochondrial‐related pathways, implying ApoVs harness CD8^+ T cells
   apoptosis via affecting mitochondrial pathways.

Figure 3.

   Figure 3
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   ApoVs Induced Apoptosis in CD8^+ T cells via Affecting Mitochondrial
   Morphology and Function. A) Proteomic analysis of ApoVs comparing with
   EVs showed that GO top 10 enrichment terms of upregulated DEPs,
   categorized into “Molecular Function” (n = 3). B) The bar chart showed
   the top 10 Reactome enrichment pathways of upregulated DEPs in ApoVs,
   compared to EVs (n = 3). C) Doubling the resolution of structured
   illumination microscopy (SIM^2) showed the fragmentation of
   mitochondrial morphology in CD8^+ T cells after ApoVs treatment. Scale
   bar: 2 µm. D‐E) The mean perimeter and mean area of mitochondria in
   ApoVs treated CD8^+ T cells significantly decreased (n = 40). F)
   Transmission electron microscopy images revealed the mitochondrial
   morphology. The lower panel magnifies the boxed area in the top panel.
   Scale bar: 1 µm for the upper panel and 0.5 µm for the lower panel. G)
   SIM^2 exhibited the mitochondrial permeability increased in ApoVs
   treated CD8^+ T group, represented by decreasing of relative
   fluorescence intensity of Calcein AM (n = 5). Scale bar: 2 µm. H) Flow
   cytometry analysis exhibited the relative fluorescence intensity of
   Calcein AM (n = 3). I) Mitochondrial membrane potential (∆Ψm) was
   analyzed by the relative ration of JC‐1 aggregates (OD = 525) and
   monomer (OD = 490) (n = 5). J) Relative mitochondrial ROS level of two
   groups (n = 5). ** p < 0.01, ***p < 0.001.

   Thus, we used the MitoTracker kit to trace mitochondrial morphology.
   Lattice doubling the resolution of structured illumination microscopy
   (SIM^2) revealed that ApoVs treatment resulted in a distinct alteration
   in the mitochondrial structure of CD8^+ T cells, characterized by
   punctate and fragmented morphology. This was accompanied by a
   significant reduction in both the average perimeter and area of
   individual mitochondria. (Figure [106]3C–E). TEM further showed changes
   in mitochondrial shape from tubular to spherical with translucent
   morphology following ApoVs treatment (Figure [107]3F). Furthermore, a
   mitochondrial permeability transition pore (MPTP) assay kit was
   employed to assess mitochondrial permeability. A decrease in
   fluorescence intensity of Calcein AM was indicative of increased
   permeability of the mitochondria. Both SIM^2 and flow cytometric
   analysis revealed a reduction in Calcein AM fluorescence intensity in
   CD8^+ T cells following ApoVs treatment, indicating that ApoVs
   treatment results in heightened permeability of mitochondria in CD8^+ T
   cells (Figure [108]3G,H). Then, a mitochondrial membrane potential
   assay kit with JC‐1 was applied. The data revealed a dramatic decline
   of mitochondrial membrane potential in CD8^+ T cells after ApoVs
   treatment, implying the depolarization of the mitochondrial membrane
   potential and early onset of apoptosis of CD8^+ T cells subsequent to
   ApoVs treatment (Figure [109]3I). Moreover, the assessment of
   mitochondrial reactive oxygen species (ROS) levels indicated a rise in
   mitoROS accumulation in CD8^+ T cells exposed to ApoVs
   (Figure [110]3J). In this study, the findings demonstrated that
   treatment with ApoVs triggered apoptosis in CD8^+ T cells by affecting
   the morphology and function of mitochondria.

2.5. ApoVs Evoked Calcium Influx through Membrane Fusion with CD8^+ T Cells

   To further investigate the specific molecular mechanism of how ApoVs
   induce apoptosis of CD8^+ T cells, a time‐lapse study was designed to
   observe the interaction pattern between ApoVs and CD8^+ T cells. The
   membrane of CD8^+ T cells was labeled with green PKH67, while the ApoVs
   membrane was labeled with red PKH26. Strikingly, SIM^2 imaging
   demonstrated a time‐dependent fusion of ApoVs membranes with CD8^+ T
   cell membranes, with a significant increase in red PKH26 fluorescence
   intensity on CD8^+ T cells observed after 6 h of ApoVs exposure. This
   time point was selected for further investigation post‐ApoVs treatment
   (Figure [111] 4A,B). Furthermore, co‐culture experiments involving
   ApoVs and CD8^+ T cells were conducted, demonstrating a time‐dependent
   accumulation of ApoVs (red) on the membrane of CD8^+ T cells (green)
   over a 7‐hour period (Figure [112]S5A,B, Supporting Information). To
   determine the percentages of ApoVs that would fuse with CD8^+ T cells
   membrane, the ratio of intracellular and cytomembrane fluorescence
   intensity of PKH26 (ApoVs) were counted. The finding showed that the
   fluorescence intensity of intracellular significantly increased after
   6 h, implying the membrane of ApoVs was gradually devoured by CD8^+ T
   cells after 6 h (Figure [113]S5C, Supporting Information). To further
   investigate the interaction between ApoVs and CD8^+ T cells, we
   conducted a sequential observation of CD8^+ T cells after ApoVs
   treatment using scanning electron microscopy (SEM). The data showed
   that the presence of ApoVs on the CD8^+ T cell membrane increased in a
   time‐dependent manner. Remarkable membrane fusion was observed 4 h
   after ApoVs treatment (Figure [114]4C). Collectively, the data
   presented indicate that SHED‐ApoVs induced apoptosis in CD8^+ T cells
   mainly via membrane fusion, leading to mitochondria dysfunction.

Figure 4.

   Figure 4
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   ApoVs Evoked Calcium Influx through Membrane Fusion with CD8^+ T Cells.
   A) SIM^2 showed the ApoVs (red) fused with the membrane of CD8^+ T
   cells (green) in a time manner. Scale bar: 2 µm. B) Relative
   fluorescence intensity of PKH26 (ApoVs) enhanced after 6 h in CD8^+ T
   cells treated with ApoVs (n = 3). C) SEM showed a sequential
   observation of ApoVs contact with CD8^+ T cells. D) Cytosolic Ca^2+
   levels in CD8^+ T cells after being treated with ApoVs or PBS in 6 min
   (n = 5). E) Quantification of the mean maximal [Ca^2+] rises (Δ[Ca^2+])
   in CD8^+ T cells when treated with ApoVs or PBS in 6 min (n = 60). F)
   Cytosolic Ca^2+ levels in CD8^+ T cells treated with ApoVs or PBS after
   6 h (n = 5). G) Quantification of the mean maximal [Ca^2+] rises
   (Δ[Ca^2+]) in CD8^+ T cells after being treated with ApoVs or PBS after
   6 h (n = 47). H) SIM^2 displayed the ApoVs (red) promote calcium probe
   Fluo‐8 AM (green) expression in CD8^+ T cells. Scale bar: 2 µm. I)
   Quantification of the relative expression of Fluo‐8 AM (n = 5). J)
   Western blot showed the expression level of L‐type voltage‐gated
   calcium channel protein in the membrane and cytoplasm respectively. K)
   SIM^2 showed the Ca[v]1.2 (red) and calcium probe Fluo‐8 AM (green)
   expression in CD8^+ T cells. Scale bar: 2 µm. L‐M) Quantification of
   the relative expression of Ca[v]1.2 and Fluo‐8 AM (n = 5). N) SIM^2
   showed the Cavβ1 (red) and calcium probe Fluo‐8 AM (green) expression
   in CD8^+ T cells. Scale bar: 2 µm. O‐P) Quantification of the relative
   expression of Ca[v]β1 and Fluo‐8 AM (n = 5). * p < 0.05, ***p < 0.001.

   Considering the impact of calcium on modulating mitochondrial function,
   apoptotic signaling, as well as T cells activity, we hypothesized that
   membrane fusion with CD8^+ T cells triggers calcium influx, ultimately
   leading to cell apoptosis. Fluo‐8‐AM calcium influx assay was
   performed, revealing that ApoVs treatment instantaneously increased
   intracellular calcium ions in CD8^+ T cells, persisting for up to 6 h.
   This suggests that ApoVs induced a robust and sustained elevation of
   calcium levels in CD8^+ T cells (Figure [116]4D–G). SIM^2 further
   confirmed the heightened expression of calcium ions in CD8^+ T cells
   following 6 h of ApoVs treatment (Figure [117]4H,I). Then, we applied
   Western blot to examine the levels of membrane and cytoplasmic L‐type
   Ca^2+ channel proteins respectively, including Ca[v]1.1, Ca[v]1.2, and
   regulatory Ca[v]β1. The finding showed that ApoVs treatment enhanced
   the membrane expression of Ca[v]1.2 and Ca[v]β1, while no significant
   change was observed in Ca[v]1.1 (Figure [118]4J). Subsequent
   immunofluorescence staining displayed that the expression levels of
   Ca[v]1.2 and Ca[v]β1 increased with calcium influx in CD8^+ T cells
   after ApoVs treatment (Figure [119]4K–P). In this study, the findings
   suggested that ApoVs evoked calcium influx by fusing with CD8^+ T
   cells, leading to the upregulated of L‐type voltage‐gated Ca^2+
   channels and subsequent calcium overload.

2.6. Harnessing Calcium Influx in CD8^+ T Cells Weaken the Efficacy of ApoVs

   To confirm the impact of calcium influx on CD8^+ T cells, we employed
   verapamil, an inhibitor of L‐type Ca^2+ channels. Fluo‐8‐AM calcium
   influx assay showed that ApoVs‐mediated intracellular calcium influx
   was reduced by verapamil treatment (Figure [120] 5A,B), suggesting that
   ApoVs‐induced intracellular calcium overload is partly dependent on the
   L‐type calcium channels. Calcein AM assay displayed increased
   fluorescence intensity after verapamil treatment, indicating that
   inhibiting calcium influx alleviated the mitochondrial membrane
   permeability (Figure [121]5C,D). Additionally, compared to the ApoVs
   group, the mitochondrial membrane potential increased and mitochondrial
   ROS levels decreased after verapamil treatment, implying that
   inhibiting calcium influx alleviated mitochondrial function (Figure
   [122]S6A,B, Supporting Information). Histomorphology observations and
   measurements of relative ear thickness further confirmed that blocking
   calcium influx diminished the efficacy of ApoVs in treating CHS mice
   (Figure [123]5E,F).

Figure 5.

   Figure 5
   [124]Open in a new tab

   Harnessing Calcium Influx in CD8^+ T Cells Weaken the Efficacy of
   ApoVs. A) Cytosolic Ca^2+ levels and the mean maximal [Ca^2+] rises
   (Δ[Ca^2+]) in CD8^+ T cells when stimulated with PBS, ApoVs, and ApoVs
   pretreatment with 1 µM verapamil groups in 6 min (n = 5). B)
   Quantification of the mean maximal [Ca^2+] rises (Δ[Ca^2+]) in CD8^+ T
   cells after being treated with PBS, ApoVs, and ApoVs pretreatment with
   verapamil groups in 6 min (n = 60). C) SIM^2 showed the Calcein AM
   expression in CD8^+ T cells treated with PBS, ApoVs, and ApoVs
   pretreatment with verapamil respectively. Scale bar: 5 µm. D)
   Quantification of relative fluorescence intensity of Calcein AM in
   three groups (n = 5). E) H&E staining of ear samples collected 24 h
   after the challenge. Scale bar: 150 µm. Black arrowheads indicate
   dilated capillaries. F) Ear thickness was measured 24 h after the
   elicitation in groups of intravenous infusion of CD8^+ T cells (n = 5).
   G) SIM^2 showed the co‐localization of BAX (red) and Mitotracker
   (green) in three groups respectively. Scale bar: 2 µm. Boxed scale bar:
   0.5 µm. H) Quantification of percentage of BAX and Mitotracker
   co‐localized Area (n = 5). I) Western blot displayed the expression
   level of BAX in isolated mitochondria from CD8^+ T cells in three
   groups. J) Western blot displayed the expression level of classical
   apoptosis protein cleaved‐Caspase 9 and cleaved‐Caspase 3 in three
   groups. * p < 0.05, ** p < 0.01, ***p < 0.001.

   To clarify whether ApoVs triggered the classical mitochondrial
   apoptotic cascades via L‐type Ca^2+ channels, we conducted
   co‐localization staining and mitochondria isolation assay in the
   aforementioned experimental groups. The findings revealed a notable
   rise in the co‐localization of BAX and mitochondria (Mitotracker) in
   CD8^+ T cells following ApoVs treatment. Furthermore, the
   co‐localization of BAX and Mitotracker was substantially diminished
   upon the inhibition of L‐type calcium channels using verapamil
   (Figure [125]5G,H). After mitochondria isolation, Western blot analysis
   confirmed an elevated level of BAX expression in mitochondria
   (Figure [126]5I). Meanwhile, Western blot analysis showed the
   inhibition of the calcium channel alleviated CD8^+ T cells apoptosis
   with lower expression of specific apoptosis‐associated markers cleaved
   caspase‐9 and cleaved caspase‐3 (Figure [127]5J). Suggesting the
   translocation of BAX from cytoplasm to mitochondria, thereby initiating
   apoptosis in CD8^+ T cells. These findings collectively suggest that
   ApoVs facilitate calcium overload through L‐type calcium channels,
   inducing mitochondria dysfunction, initiating apoptosis of CD8^+ T
   cells, and ameliorating CHS responses.

2.7. ApoVs Treatment Ameliorated Oral Lichenoid Reaction

   To further validate the therapeutic efficacy of ApoVs in managing type
   IV hypersensitivity responses, we constructed an additional OLR murine
   model, which represents a type IV hypersensitivity response in
   pathology. During the elicitation phase, reticular stripes can be
   observed after the application of OXA to the buccal mucosa of mice
   (Figure [128] 6A). H&E staining revealed liquefaction degeneration of
   the basal keratinocytes and band‐like lymphocytic infiltration in the
   submucosal layer, closely resembling the features of oral lichen planus
   (OLP) (Figure [129]6B). However, in mice treated with ApoVs injection,
   these inflammatory manifestations were significantly alleviated
   (Figure [130]6A–C). Immunostaining of lesions displayed ApoVs injection
   alleviated the vascular remodeling and decreased expression of TNF‐α
   compared to PBS injection (Figure [131]6D, Figure [132]S7A,B,
   Supporting Information). The percentage of CD8^+ T cells also reduced
   after ApoVs injection, consistent with the histological findings
   (Figure [133]6E). More importantly, peripheral blood samples were
   collected from three healthy volunteers and three patients with OLR.
   CD8^+ T cells were enriched and activated by CD3 and CD28 microbeads.
   Then, the CD8^+ T cells from the patients with OLR were treated with
   ApoVs or PBS. The results indicated that in vitro treatment with ApoVs
   significantly reduced the levels of perforin‐1, granzyme B, TNF‐α, and
   IL‐1β secreted by the patient's CD8^+ T cells (Figure [134]6F).
   Additionally, after being treated with ApoVs, the MPTP assay showed
   increased mitochondrial permeability in CD8^+ T cells from patients
   (Figure [135]S7C, Supporting Information), aligning with the CHS model.
   Therefore, our data suggested that ApoVs from MSCs can induce CD8^+ T
   cell apoptosis and ameliorate CD8^+ T cells mediated type IV
   hypersensitivity responses, shedding new light on preclinical and
   clinical research of MSCs‐derived ApoVs.

Figure 6.

   Figure 6
   [136]Open in a new tab

   ApoVs Treatment Ameliorated Oral Lichenoid Reaction. A) Intraoral photo
   showed the lesion of oral lichenoid reaction with short white stripes
   in mice. The right panel magnifies the boxed area in the left panel.
   Black arrows indicate dilated blood vessels and white arrows indicate
   reticular white patches of oral mucosa. B) H&E staining of mice oral
   mucosa samples. The right panel magnifies the boxed area in the left
   panel. Scale bar: 150 µm for the left panel and 50 µm for the right
   panel. C) Quantification of H&E staining exhibited decreased
   inflammatory cells in lesion sites (n = 5). D) Immunostaining of mucosa
   lesions for CD31 (green) and DAPI (blue) showed the alleviated of
   dermal blood dilatation. Scale bar: 50 µm. Quantification of blood
   vessel area per section (n = 10). E) Representative images and
   quantification of CD8^+ T cells in PBS and ApoVs injection groups
   (n = 5). Scale bar: 100 µm. F) The proinflammatory cytokines produced
   by CD8^+ T cells in patients with OLR, as well as in the co‐cultured
   group of ApoVs and the control group, were assessed using ELSIA
   (n = 15). * p < 0.05, ** p < 0.01, ***p < 0.001.

3. Discussion

   In the present study, we dissect the novel function of SHED‐ApoVs in
   type IV hypersensitivity reactions for induction apoptosis of CD8^+ T
   cells. MSCs‐ApoVs possess unique therapeutic functions in various
   diseases, including immune disorders, osteoporosis, skin injuries, and
   tumors. However, it remains elusive whether SHED‐ApoVs, acting as
   immune microenvironment modulators, possess immunomodulatory properties
   and interact with CD8^+ T cells. This research utilized two models of
   type IV hypersensitivity reactions and identified for the first time
   the therapeutic role of SHED‐ApoVs in these reactions. Specifically,
   ApoVs promote membrane expression of L‐type voltage‐gated Ca^2+
   channels through membrane fusion in CD8^+ T cells, leading to
   cytoplasmic calcium overload and fragmentation of mitochondria,
   subsequently induction of apoptosis in these cells. This study
   highlights the role of SHED‐ApoVs in inducing CD8^+ T cell apoptosis
   and suggests their potential for treating CD8^+ T cells‐mediated
   diseases, providing experimental evidence for nanovesicle‐based
   cell‐free therapies.

   Apoptosis triggered by MSCs transplantation produces a specific
   quantity of ApoVs, a notable population of extracellular vesicles
   (EVs), which modulate the immune microenvironment.^[ [137]^16 ^] Based
   on their size, ApoVs can be categorized into three main subtypes:
   larger apoptotic bodies (1–5 µm), smaller apoptotic microvesicles
   (0.1–1 µm), and even more diminutive apoptotic exosomes (smaller than
   150 nm).^[ [138]^17 ^] SHED as a type of MSCs possesses optimal
   immunomodulatory properties, engaging in immune remodeling and tissue
   regeneration processes.^[ [139]^1 , [140]^18 ^] It has been reported
   that SHED expresses PDL‐1 to ameliorate gland inflammation and dryness
   symptoms of Sjögren's syndrome by directly upregulating the ratio of
   Treg/Th17 via cell‐cell contact.^[ [141]^9 ^] Our team has previously
   demonstrated successful tooth regeneration in both animal models and
   clinical trials through the use of SHED, confirming their ideal
   anti‐inflammatory and regenerative properties.^[ [142]^5 , [143]^8 ,
   [144]^19 ^] ^[ [145]^2 ^] However, there existed several challenges for
   stem cell therapy, which hindered the clinical practice, such as
   ethical considerations, tumor formation, immune rejection, substantial
   heterogeneity between donors, as well as batch‐to‐batch variation.
   ApoVs originating from MSCs serve as the modulator within the
   microenvironment and putatively mirror the biological function of their
   parent cells.^[ [146]^2 ^] For instance, SHED‐ApoVs enhanced the
   endothelial cells' angiogenic function and accelerated
   revascularization under the inflammatory microenvironment, promoting
   the efficient regeneration of dental pulp.^[ [147]^10b ^] ApoVs derived
   from bone marrow mesenchymal stromal cells prevented Th17
   differentiation and memory formation in the immune microenvironment,
   ameliorating joint erosion in murine arthritis.^[ [148]^4 , [149]^20 ^]
   MSCs‐ApoVs ameliorated the multiple myeloma in the tumor
   microenvironment by inducing multiple myeloma cell apoptosis.^[
   [150]^21 ^] Due to the diverse and unique properties inherited from
   their parent cells, the exact biological characteristics of ApoVs from
   various sources and the mechanisms driving their physiological and
   therapeutic impacts remain incompletely elucidated. As for SHED‐ApoVs,
   their role as immune microenvironment modulators and their effects on
   immune cells are still largely unclear. Exploring the ApoV profile from
   diverse sources is beneficial for comprehending the significance of
   apoptosis in maintaining homeostasis and facilitates the advancement of
   cell‐free therapy employing nanovesicles. In this study, we first
   isolated and characterized SHED and SHED‐ApoVs as previously reported
   (Figure [151]S1, Supporting Information). The findings confirmed the
   ApoVs obtained for this study are MSCs‐derived ApoVs.

   CD8^+ T cells mainly participate in combating viral infections and
   eliminating malignant cells in cancer.^[ [152]^22 ^] Nonetheless, when
   autoreactive responses occur and target the body's own cells, excessive
   activation of CD8^+ T cells leads to autoimmune disorders and type IV
   hypersensitivity reactions.^[ [153]^23 ^] Type IV hypersensitivity is a
   class of diseases mediated by CD8^+ T cells, including ACD^[ [154]^13
   ^] and oral lichen planus.^[ [155]^24 ^] Type IV hypersensitivity also
   called delayed type hypersensitivity which primarily driven by CD8^+ T
   cells, making it an ideal model for investigating the effects of ApoVs
   on these cells due to its distinct sensitization and elicitation
   phases. However, whether SHED‐ApoVs contribute to antihypersensitivity
   therapy has not been investigated. In this study, two type IV
   hypersensitivity disease models, CHS and OLR, were established and
   verified the therapeutic properties of SHED‐ApoVs on OXA‐induced tissue
   hypersensitivity responses (Figures [156]1A–D and [157]6A–D). ACD is a
   common skin disorder caused by external irritation that causes the skin
   to appear red, pimples, blisters, and pain.^[ [158]^15 ^] Alongside
   ACD, OLR represents type IV hypersensitivity responses associated with
   precancerous lesions, closely resembling the features of oral lichen
   planus.^[ [159]^12b ^] Although several symptom‐based treatments are
   currently available to alleviate symptoms and manage the condition,
   there is a lack of established first‐line targeted treatment drugs.
   Developing effective immunotherapy and the realization of targeted
   therapy are the hot research issues for hypersensitivity responses. The
   function of CD8^+ T cells was usually marked by the secretions of
   interferon γ (IFN‐γ), tumor necrosis factor α (TNF‐α), granzyme B, and
   perforin‐1.^[ [160]^25 ^] Additionally, it has been shown that CD8^+
   effector T cells produce IFN‐γ which boosts the production of IL‐1β and
   IL‐6 in allergen‐exposed sites and IFN‐γ also induces the production of
   chemokines like CXCL9, CXCL10 and CXCL11.^[ [161]^11 ^] This study
   showed that ApoVs treatment decreased the expression of
   pro‐inflammatory cytokines, suppressing tissue edema and hemangiectasis
   (Figure [162]S2, Supporting Information). RNA sequencing of mice ear
   tissues implied the anti‐inflammatory and immunomodulatory function of
   SHED‐ApoVs, which is putatively related to apoptosis and metabolic
   process. Importantly, the local percentage of CD8^+ T cells in tissue
   reduced after ApoVs treatment (Figures [163]1E–J and [164]6E). To
   further confirm the therapeutical effect of ApoVs, we collected a blood
   sample from a patient with oral lichen planus. The results suggested
   that ApoVs treatment significantly reduced the expression of CD8^+ T
   cells function‐related cytokines, consisting with CHS findings
   (Figure [165]6F; Figures [166]S3C–F and [167]S7, Supporting
   Information). Thus, the results showed an anti‐inflammatory and
   immunomodulatory profile of ApoVs for alleviating CHS and OLR,
   implicating ApoVs serve as the immune microenvironment modulators in
   effectively attenuating the function and count of local CD8^+ T cells.

   Interest in EVs has progressively grown due to their potent role as
   microenvironment mediators locally and systemically.^[ [168]^26 ^] In
   addition, they can modulate the immune cells in a diversity paradigm
   including promoting macrophage M2 polarization,^[ [169]^27 ^]
   modulating Th17 cells,^[ [170]^20 ^] and generating neutrophil
   extracellular traps (NETs) formation.^[ [171]^28 ^] However, whether
   SHED‐ApoVs can modulate the function of CD8^+ T cells remains to be
   elucidated. Our previous study utilized the adoptive transfer method to
   verify that the CHS were alleviated in WT mice which transferred with
   draining lymph node cells from OXA‐sensitized Sema4D KO mice.^[
   [172]^14 ^] In the present study, we applied an adoptive transfer assay
   to transfer sensitized CD8^+ T cells into unsensitized mice
   (Figure [173]2A–D). After 24 h of OXA treatment, there was no notable
   inflammation or ear thickening detected in wildtype recipient mice that
   did not undergo CD8^+ T cell transfer. This observation indicated that
   the endogenous CD8^+ T cells present in the wildtype recipient mice are
   incapable of mediating the local inflammation elicited by OXA. However,
   current research suggests that CD8^+ T cells that are not specific for
   the antigen can be activated by cytokines without cognate antigens, a
   phenomenon termed bystander T cell activation.^[ [174]^29 ^] It is
   possible that during the OXA‐induced inflammation, bystander CD8^+ T
   cells from wildtype recipient mice might also participate in the
   process, which will be explored in future studies. Collectively, the
   results uncovered that ApoVs treatment directly attenuated CHS response
   via promoting apoptosis of CD8^+ T cells (Figure [175]2).

   ApoVs can promote the apoptosis of several types of immune cells and
   maintain or remodel immune homeostasis through various mechanisms.^[
   [176]^30 ^] Exploring the mechanism of ApoVs on the immune system is
   helpful to understand the significance of apoptosis in tissues and
   organs under the homeostasis condition, and is conducive to the
   construction of cell‐free therapy based on nanovesicles. Recently,
   ApoVs, microvesicles (MVs), small EVs, and exosomes have been
   identified as carriers of both damaged and intact mitochondria, as well
   as mitochondrial components, facilitating intercellular mitochondrial
   transfer.^[ [177]^31 ^] This transfer can lead to significant effects
   on the recipient cells, such as mitophagy, metabolic reprogramming, and
   changes in mitochondrial dynamics.^[ [178]^32 ^] The study has shown
   that co‐culturing bone marrow stromal cells (BMSCs) with CD8^+ T cells
   enhances the antitumor efficacy of the latter through tunneling
   nanotubes‐mediated mitochondrial transfer from BMSCs. However, when
   CD8^+ T cells were co‐cultured with free‐floating mitochondria derived
   from BMSCs, no significant mitochondrial uptake was observed.^[
   [179]^33 ^] In this study, proteomic sequencing results indicate that
   SHED‐ApoVs contain mitochondrial components (Figure [180]3A,B), yet it
   is unclear whether these vesicles harbor intact, healthy, and
   functional mitochondria. Given that ApoVs encompass a wide variety of
   substances, such as nucleic acids, proteins, and lipids, it is
   essential to employ MicroRNA sequencing, lipidomics, and metabolomics
   to thoroughly analyze their compositions and elucidate the specific
   mechanisms. Previous proteomic sequencing of MSC‐derived ApoVs
   identified apoptotic signatures from parent cells, including Fas, SOD1,
   and Caspase family members, which induce apoptosis in recipient
   cells.^[ [181]^34 ^] During apoptosis, the loss of mitochondrial
   membrane potential and caspase expression can occur in the early stage,
   while the loss of membrane integrity occurs in the late stage.^[
   [182]^35 ^] Thus, the depolarization of mitochondrial membrane
   potential is a landmark event during the initial phase of apoptosis.
   Fragmentation of mitochondria is a multifaceted manifestation of
   various forms of mitochondrial dysfunction. It reflects disturbances in
   mitochondrial dynamics, membrane potential, energy metabolism,
   oxidative stress, and calcium homeostasis.^[ [183]^36 ^] Notably,
   mitochondrial dysfunction in apoptotic cells typically manifests
   through alterations in morphology, ROS accumulation, and permeability
   enhancement.^[ [184]^37 ^] In this study, we showed that the
   fragmentation of mitochondria with elevated mitochondrial permeability,
   declined mitochondrial membrane potential and ROS accumulation,
   implying the dysfunction of mitochondria (Figure [185]3C–J). The
   findings above suggest that ApoVs induced apoptosis of CD8^+ T cells by
   impairing the function of mitochondria.

   EVs can transmit information by acting at the cell surface, without
   content delivery. For example, it has been reported that apoEVs
   directly contact multiple myeloma cells to facilitate Fas trafficking
   from the cytoplasm to the cell membrane, leading to the initiation of
   apoptosis.^[ [186]^21 ^] Given the limited phagocytic capacity of T
   cells, their interaction patterns and mechanisms with EVs differ from
   those observed with macrophages and MSCs, which typically internalize
   EVs through endocytosis or phagocytosis.^[ [187]^38 ^] It has been
   reported that EVs derived from dendritic cells can bind to clusters of
   CD8 molecules on the surface of CD8^+ T cells, thereby boosting the
   responses of effector CD8^+ T cells during viral infections.^[ [188]^39
   ^] However, studies clarifying how MSCs derived‐ApoVs influence T
   cells, especially CD8^+ T cells, are still lacking. In this study, we
   unexpectedly found that ApoVs interact with CD8^+ T cells in a membrane
   fusion manner (Figure [189]4A–C; Figure [190]S5, Supporting
   Information). To our knowledge, this kind of membrane‐based
   communication manner has rarely been reported in ApoVs influence on T
   cells. Proper cellular functions rely on the precise regulation of
   intracellular Ca^2+ levels.^[ [191]^40 ^] It has been reported that
   ApoVs directly contact the membrane of multiple myeloma cells, evoking
   Ca^2+ influx and elevating cytosolic calcium levels.^[ [192]^21 ^]
   Therefore, we analyzed the levels of calcium after ApoVs interacted
   with CD8^+ T cells in the short and long term, suggesting that ApoVs
   induced persistent calcium overload of CD8^+ T cells with cell membrane
   fusion (Figure [193]4D–G), shedding new lights on membrane‐based
   communication between ApoVs and T cells. In future studies, immunogold
   staining, recognized as the gold standard for demonstrating the
   presence of SHED‐ApoVs—distinct from EVs secreted by CD8^+ T cells—on
   the surface of CD8^+ T cells, should be employed to enhance the
   credibility of findings.

   L‐type voltage‐gated Ca^2+ channels, such as Ca[v]1.1, Ca[v]1.2, and
   Ca[v]β1, have been shown to modulate Ca^2+ signaling and contribute to
   T cell function.^[ [194]^41 , [195]^42 ^] Previous research has
   highlighted the critical role of Ca[v]1.2 channels in the Ca^2+
   response and cytokine production of human Th2 lymphocytes stimulated by
   the T cell receptor.^[ [196]^43 ^] Additionally, Ca[v]β1 has been
   proved as a critical regulator in T cell activities including expansion
   and apoptosis.^[ [197]^44 ^] Calcium homeostasis is crucial for
   maintaining, and disruptions in calcium balance, particularly calcium
   overload, can induce morphological changes such as mitochondrial
   swelling and fragmentation.^[ [198]^40 , [199]^45 ^] In this study, we
   showed that the increased expression of L‐type Ca^2+ channels proteins
   on the membrane of CD8^+ T cells with calcium overloaded, resulting to
   the initiation of apoptosis (Figure [200]4H–P). More importantly,
   verapamil treatment, which inhibits L‐type voltage‐gated Ca^2+
   channels, has been shown to partially restore mitochondrial
   permeability, harness calcium influx, and diminish the efficacy of
   ApoVs on the CHS response (Figure [201]5A–F; Figure [202]S6, Supporting
   Information). Studies have indicated that mitochondrial dysfunction
   initiates classical apoptotic biochemical cascades by mitochondrial
   permeability transition and translocation of pro‐apoptotic BAX to
   mitochondria. This leads to the release of cytochrome C from
   mitochondria to the cytosol, activating the apoptogenic molecules such
   as cleaved‐caspase 9 and cleaved‐caspase 3.^[ [203]^46 ^] Therefore, we
   examined and confirmed the translocation of BAX from cytoplasm to
   mitochondria, initiating apoptosis CD8^+ T cells which were rescued by
   inhibitor of L‐type voltage‐gated Ca^2+ channels (Figure [204]5G–J).
   These findings implicated that membrane fusion elevated the membrane
   permeability of CD8^+ T cells via L‐type voltage‐gated Ca^2+ channels,
   providing a novel role of L‐type voltage‐gated Ca^2+ channels in CD8^+
   T cells. Additionally, the targeted elimination of endogenously
   generated ApoVs may offer therapeutic potential for treating diseases
   associated with reduced calcium influx, such as cardiovascular
   diseases, neurodegenerative diseases, diabetes, and others. However,
   whether ApoVs have similar functions in other cell types requires
   further exploration.

   Autoreactive CD8^+ T cells have been implicated in the pathogenesis of
   multiple autoimmune diseases, including Crohn's disease, rheumatoid
   arthritis, and multiple sclerosis. Pathogenic CD8^+ T cells express
   high levels of cytotoxic effector molecules such as IFN‐γ, TNF,
   granzyme B, and perforin.^[ [205]^47 ^] However, during chronic viral
   infections or cancer, CD8^+ T cells are in a dysfunctional state which
   is referred to as CD8^+ T cell exhaustion. It is characterized by a
   gradual loss of effector functions and an inability to effectively
   respond to antigens, leading to compromised immune surveillance and
   host defense.^[ [206]^48 ^] In this study, we demonstrated ApoVs
   treatment inducing apoptosis of CD8^+ T cells, suggesting that timely
   clearance of ApoVs could be beneficial for CD8^+ T cells in anti‐viral
   and anti‐tumor therapy. Furthermore, the significant role of L‐type
   voltage‐gated Ca^2+ channels during the interaction of ApoVs with CD8^+
   T cells indicates their potential as drug targets for exhausted CD8^+ T
   cells in antiviral and antitumor therapy.

4. Conclusion

   Apoptosis is a vital process in maintaining the homeostasis of
   multicellular organisms. Despite limited understanding of the precise
   functions of ApoVs in various pathological conditions and cell types,
   our research has revealed that MSCs‐ApoVs can induce apoptosis in CD8^+
   T cells, thereby ameliorating CD8^+ T cell‐driven type IV
   hypersensitivity reactions such as CHS and OLR. Notably, analysis of
   blood samples from a patient with OLR confirmed the
   anti‐hypersensitivity properties of ApoVs by modulating CD8^+ T cell
   activity. Upon interaction with CD8^+ T cells, ApoVs were observed to
   merge their membrane with that of the CD8^+ T cells, triggering calcium
   influx and mitochondrial dysfunction, ultimately leading to CD8^+ T
   cell apoptosis. These findings offered a new insight into the
   interaction of MSCs‐ApoVs and CD8^+ T cells, demonstrating that
   MSC‐ApoVs, as nano‐mediators in the immune microenvironment, have the
   potential to treat CD8^+ T cell‐mediated hypersensitivity. Our findings
   indicated that products from apoptotic cells putatively impede
   anti‐tumor or anti‐viral treatment, providing a promising avenue for
   developing therapeutic interventions targeting CD8^+ T cells
   participated diseases.

5. Experimental Section

Oxazolone‐induced Contact Hypersensitivity and Oral Lichenoid Reactions

   Mice models were established following the guidelines of the Ethical
   Committee of the School of Stomatology, Fourth Military Medical
   University (license number: kq‐2024‐016). were sensitized through
   topical application of 100 µL of 3.0% oxazolone (OXA, dissolved in
   anhydrous ethanol, MedChemExpress, USA) on the shaved abdomen. After a
   period of five days, mice were subjected to contact hypersensitivity by
   being challenged with 10 µL of 1% OXA or ethanol on both sides of one
   ear. For oral lichenoid reactions, 50 µL of 1% OXA or ethanol was
   applied to the buccal mucosa. The ear thickness before and after
   elicitation was measured using an engineer's micrometer caliper. The
   difference in the increase in thickness between the left and right ears
   was analyzed. The macroscopic and dermatoscopic appearances of the
   lesions were recorded using a dermatoscope (Illuco IDS 1100; South
   Korea).

Histology Assay

   Briefly, ear or mucosal tissues were collected, fixed in 4%
   paraformaldehyde for 12 h, dehydrated with graded ethanol, and embedded
   in paraffin. The samples were sectioned at a thickness of 4 µm
   thickness and stained with hematoxylin and eosin (H&E) (Leica,
   Germany). The images were captured using a fluorescence continuous
   scanning digital section microscope with a full slide scanning system
   (VS200, Olympus, Japan).

Immunohistochemistry and Immunofluorescence Staining

   Immunohistochemistry (IHC) staining was performed on 4 µm
   paraffin‐embedded sections of the ear and mucosal tissues. The sections
   underwent dewaxing and hydration steps, followed by heat‐mediated
   antigen retrieval using Tris–EDTA (C1038, Solarbio, China) in a
   pressure cooker. Time the pressure cooker once it reaches pressure and
   seal it after 100s. After gently washing the sections three times in
   PBS, 3% hydrogen peroxide was administered at 37 °C for 1 h. To block
   nonspecific antigens, 5% bovine serum albumin (BSA, FC0077, MP
   Biomedicals, USA) was used. Primary antibodies were incubated overnight
   at 4 °C, while fluorescent secondary antibodies were incubated for 1 h
   at room temperature. Sections were sealed with a mounting medium
   containing 4,’‐6‐diamidino‐2‐phenylindole (DAPI; ab104139, Abcam, UK)
   after being washed with PBS 3 times, and images were captured by
   fluorescence continuous scanning digital section microscope with full
   slide scanning system (VS200, Olympus, Japan) and analyzed by ImageJ
   software.

   Immunocytochemistry (ICC) staining was performed on CD8^+ T cells.
   CD8^+ T cells were prelabeled with PKH67 using PKH67 Green Fluorescent
   cell Linker Kit (MIDI67, Sigma‐Aldrich, USA) and cultured with PKH26
   (MIDI26, Sigma‐Aldrich, USA) prelabeled ApoVs (20 µg mL^−1) for 6 h.
   After Fluo‐8 AM or Mito‐track staining, CD8^+ T cells were fixed with
   2.5% glutaraldehyde (111‐30‐8, Macklin, China). Then, the primary
   antibodies were incubated overnight at 4 °C, while fluorescent
   secondary antibodies were incubated for 1 h at room temperature, and
   the nucleus was stained with Hoechst 33 342 (ab228551, Abcam, UK,
   diluted 1:50). Images were captured by Lattice doubling the resolution
   of structured illumination microscopy (SIM^2) (Elyra 7, ZEISS, Germany)
   and analyzed by ImageJ software.

   The antibodies used were: anti‐CD31 antibody (FAB3628G, R&D Systems,
   USA; diluted 1:100), anti‐TGF‐β antibody (3711S, Cell Signaling
   Technology, USA, diluted 1:200), anti‐TNF‐α antibody (11948s, Cell
   Signaling Technology, USA, diluted 1:200), mouse CD8 alpha Alexa Fluor
   488 MAb (FAB116G‐100, R&D system, USA, diluted 1:200), anti‐Ca[v]1.2
   antibody (ACC‐003, Alomone, Israel, diluted 1:200), anti‐Ca[v]β1
   antibody (PA5‐109285, Invitrogen, USA, diluted 1:200), anti‐BAX
   antibody(60267‐1‐Ig, Proteitech, China, diluted 1:200), Alexa Fluor 488
   Donkey anti‐Mouse secondary antibody (A21202, Invitrogen, USA, diluted
   1:200), Alexa Fluor 594 Donkey anti‐Mouse secondary antibody (A21203,
   Invitrogen, USA, diluted 1:200), Alexa Fluor 488 Donkey anti‐Rabbit
   secondary antibody (A21206, Invitrogen, USA, diluted 1:200) and Alexa
   Fluor 594 Donkey anti‐Rabbit secondary antibody (A21207, Invitrogen,
   USA, diluted 1:200).

PKH26 and PKH67 Staining

   To detect the behavior of ApoVs or CD8^+ T cells in vitro and in vivo,
   ApoVs or CD8^+ T cells was labelled with the PKH26 Red Fluorescent Cell
   Linker Kit (MIDI26, Sigma‐Aldrich, USA) and the PKH67 Green Fluorescent
   Cell Linker Kits (MIDI67, Sigma‐Aldrich, USA) following the
   manufacturer`s instructions. 200 ug ApoVs or 2 × 10^5 CD8^+ T cells
   were collected and washed once using medium without serum. Subject the
   ApoVs to centrifugation at 16 000 × g for a duration of 30 min, and the
   CD8^+ T cells to centrifugation at 400 × g for 5 min to form a loose
   pellet. Subsequently, carefully aspirate the supernatant, ensuring that
   no more than 25 µL remains. Adding 1 mL of Diluent C to the ApoVs
   pellet and resuspending with gentle pipetting to ensure complete
   dispersion. Then, 4 uL of fluorescent dye PKH26/PKH67 ethanolic dye
   solution was added and incubated at room temperature for 5 min. A
   volume of 2 mL of fetal bovine serum was added, followed by
   centrifugation at 16 000 × g for 30 min to isolate ApoVs, and at
   400 × g for 10 min for CD8^+ T cells, both conducted at 4 °C to remove
   the free PKH26/PKH67 dye. The resulting precipitates were subsequently
   resuspended in PBS for further analysis.

Adoptive Transfer Assessment

   Adoptive transfer of lymph node cells was performed as previously
   described with modifications.^[ [207]^12 ^] In brief, 6 to 8‐week‐old
   male wildtype mice were sensitized with OXA. Five days after
   sensitization, inguinal lymph node cells were isolated to prepare
   single‐cell suspensions. CD8^+ T cells were extracted and treated with
   ApoVs (10 µg mL^−1), PBS, or Verapamil (1 µM, 52‐53‐9, MedChemExpress,
   USA) for 6 h. Then, unsensitized mice were injected intravenously with
   5 × 10^7 CD8^+ T cells. Two hours later, baseline ear thickness was
   measured, and the mice were then challenged with 10 µL of 1.0% OXA on
   both sides of one ear. Ear thickness was measured 24 h later, and the
   increase in ear thickness above baseline was calculated.

CD8+ T Cells Isolation and Culture

   C57 mice (6‐8 weeks old, male, Air Force Military Medical University,
   Xi'an) were anesthetized with 1% pentobarbital sodium (50 mg kg^−1 body
   weight) and sacrificed by spinal cord dislocation. Spleens were removed
   and ground using a sterile plunger, and then the spleen cells were
   isolated through a 70 µm cell strainer. Centrifuge at 800 rpm for 5 min
   and discard the supernatant. Resuspend and lyse cells in red blood cell
   lysis buffer for 20 min at 4 °C. The samples were centrifuged at
   800 rpm for 5 min and resuspended in a solution prepared by diluting
   Magnetic‐Activated Cell Sorting (MACS) BSA Stock Solution at a ratio of
   1:20 with autoMACS Rinsing Solution. CD8^+T lymphocytes were
   magnetically labeled with CD8 (TIL) MicroBeads. Then cell suspension
   was then loaded onto a MACS Column, which was placed in the magnetic
   field of a MACS Separator. After removing the column from the magnetic
   field, the magnetically retained CD8^+ cells were eluted as the
   positively selected cell fraction. Total positively isolated CD8 ^+ T
   cells were stimulated using plate‐bound anti‐CD3 (2 µg mL^−1) plus
   soluble anti‐CD28 (1 µg mL^−1) monoclonal antibodies for two days and
   cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal
   bovine serum (FBS, Gibco, USA).

   Human CD8^+ T cells were isolated following the guidelines of the
   Ethical Committee of the School of Stomatology, Fourth Military Medical
   University (license number: KQ‐YJ‐2023‐164). Three healthy volunteers
   and three patients with oral lichen planus were informed of the
   objectives of this research, and written informed consent was obtained
   prior to conducting the study. In detail, mononuclear cells were
   obtained from the peripheral blood of healthy individuals and patients
   with oral lichen planus using Ficoll‐Paque PREMIUM (17 544 202, GE
   Life). Human CD8^+ T cells were magnetically labeled with CD8
   MicroBeads (130‐045‐201, Miltenyi Biotec) and isolated as mentioned
   above.

Apoptosis Assay

   The Annexin V‐FITC Apoptosis Detection Kit (BMS500FI‐100, ThermoFisher
   Scientific, USA) was utilized following the manufacturer's protocol.
   Annexin‐V fluorescein isothiocyanate (FITC) and propidium iodide (PI)
   combined working solution was prepared to examine the apoptotic CD8^+ T
   cells. Cells collected were incubated with the working solution for
   15 min at 4 °C in the dark. After staining, cells were analyzed using a
   flow cytometer, and the number of Annexin V‐positive cells was
   determined.

RNA Extraction and Sequencing

   The total RNA from the two groups of mice ears after 24 h of ApoVs
   treatment (20 mg mL^−1) was extracted following the instruction manual
   of the TRIzol Reagent (Life Technologies, California, USA). After
   measuring the RNA concentration and purity, 1 µg of RNA per sample was
   used as input material for the RNA sample preparations. Sequencing
   libraries were generated using the Hieff NGS Ultima Dual‐mode mRNA
   Library Prep Kit for Illumina (Yeasen Biotechnology) following the
   manufacturer's recommendations, and index codes were added to attribute
   sequences to each sample. The libraries were sequenced on an Illumina
   NovaSeq platform to generate 150 bp paired‐end reads, following the
   manufacturer's instructions. Differentially expressed genes (DEGs) were
   identified in the ApoVs injection group compared to the control group.
   These genes were defined as those with a fold change ≥1.5 and a false
   discovery rate (FDR) < 0.05. They were included for further analysis
   based on the Gene Ontology (GO) database. GO of biology process
   enrichment analysis of the up‐ and down‐regulated genes was implemented
   using the clusterProfiler package in RStudio software (version 4.3.1).
   Among the top 10 gene enrichment pathways, those with a p value < 0.05
   were exhibited.

   The total RNA from the CD8^+ T cells of mice (n = 3) was extracted
   after 6 h ApoVs treatment (10 ug mL^−1) and sequenced as mentioned
   above. DEGs were identified and defined as those with a fold change ≥2
   and a FDR < 0.05. A total of 322 upregulated genes were included for
   further analysis based on the KEGG and Reactome databases.
   ClusterProfiler package was used to analyze the upregulated genes in
   KEGG pathways using RStudio software (version 4.3.1). Reactome pathway
   enrichment analysis was performed using the Reactome pathway database
   ([208]https://reactome.org/). Top 10 KEGG gene enrichment pathways with
   FDR < 0.05 were visualized in bubble plots, and the top 12 Reactome
   gene enrichment pathways with FDR < 0.05 were also displayed.

Proteomic Analysis

   Proteins from SHED‐derived ApoVs and SHED‐derived EVs were extracted to
   obtain peptides, which were then analyzed using LC‐MS/MS analysis on a
   NanoElute UHPLC system (Bruker Daltonics, USA). The raw data was
   analyzed using the MaxQuant search engine (v.1.6.15.0). Differentially
   expressed proteins (DEPs) were defined as those with a fold change ≥2
   and FDR < 0.05 and were included for further analysis based on GO and
   Reactome Pathway databases. The top 10 enrichment pathways with an FDR
   < 0.05 were displayed in bubble and bar plots.

Western Blot Analysis

   Protein samples of ApoVs and CD8^+ T cells were analyzed as previously
   reported.^[ [209]^49 ^] The antibodies used were: anti‐CD9 antibody
   (#A19027, Abclonal, China, diluted 1:1000), anti‐CD63 antibody
   (#sc‐5275, Santa Cruz Biotechnology, USA, diluted 1:1000), anti‐TSG101
   antibody (#ET1701‐59, Huabio, China, diluted 1:1000), anti‐GM130
   antibody (#610 822, BD Pharmingen, USA, diluted 1:1000), anti‐Caspase9
   antibody (#9504, Cell Signaling Technology, USA, diluted 1:1000),
   anti‐Cleaved Caspase9 antibody (#9509, Cell Signaling Technology,
   USA, diluted 1:1000), anti‐Caspase3 antibody (#9662, Cell Signaling
   Technology, USA, diluted 1:1000), anti‐Cleaved Caspase3 antibody
   (#9664, Cell Signaling Technology, USA, diluted 1:1000), anti‐Ca[v]1.1
   antibody (ACC‐314, Alomone, Israel, diluted 1:1000), anti‐Ca[v]1.2
   antibody (ACC‐003, Alomone, Israel, diluted at 1:1000), anti‐Cavβ1
   antibody (PA5‐109285, Invitrogen, USA, diluted 1:1000), anti‐BAX
   antibody (60267‐1‐Ig, Proteitech, China, diluted 1:1000), anti‐GAPDH
   antibody (YM3029, Immunoway, USA, diluted 1:1000) and anti‐COX4
   antibody (#4850S, Cell Signaling Technology, USA, diluted 1:1000). Goat
   anti‐Mouse secondary antibody (115‐035‐003, Jacson, USA, diluted
   1:1000), Goat anti‐Rabbit secondary antibody (111‐035‐003, Jacson, USA,
   diluted 1:1000) and Rabbit anti‐Goat secondary antibody (305‐035‐003,
   Jacson, USA, diluted 1:1000).

Scanning Electron Microscopy (SEM)

   CD8^+ T cells were co‐cultured with SHED‐ApoVs (method as before) for
   1, 2, 3, 4, and 6 h separately. Then the cells were added onto the
   surface of coverslips which were coated with Poly‐D‐Lysine
   ([210]R30150,Yuan Ye, China) for 1 h. Then the cells were fixed with
   2.5% glutaraldehyde for 30 min, dehydrated with graded ethanol
   solutions (50%, 70%, 95%, 100%, 5 min each), and dried with
   hexamethyldisilane. The dried samples were coated with gold using a
   sputter coater and observed under SEM to evaluate the fusion of
   SHED‐ApoVs with the cell membranes of CD8^+ T cells.

Transmission Electron Microscopy (TEM)

   CD8^+ T cells from the spleen of mice were fixed with 3% glutaraldehyde
   at 4 °C for 5 min. Afterward, CD8^+ T cells were centrifuged at
   12 000 rpm for 10 min to remove the supernatant. Resuspend the deposit
   with 3% glutaraldehyde at 4 °C for further examination. The samples
   were applied to copper grids (BZ11022a, EMCN, China) and stained with
   2% phosphotungstic acid (GZ02536, Ruixin, China) using standard
   methods. Stained grids were examined using a transmission electron
   microscope (JEM‐1400FLASH, JEOL, Japan). Mitochondrial morphology in
   CD8^+ T cells was observed, and the abnormal mitochondria were defined
   as those with a loose cristae structure and a brighter appearance.

Mitochondrial Morphological Measurements

   The groups of CD8^+ T cells, with or without ApoVs treatment
   (10 ug mL^−1), were plated at 0.5 × 10^6 cells mL^−1 in 1.5 mL
   centrifuge tubes. Mitochondrial morphological measurements were
   conducted using MitoTracker Green FM (M7514, Invitrogen, USA) following
   the manufacturer's instructions and were observed using the Lattice
   doubling the resolution of structured illumination microscopy (SIM^2)
   (Elyra 7, ZEISS, Germany). The perimeter and area of individual
   mitochondrial branches were traced using ImageJ software, and the mean
   perimeter and area of mitochondria per cell were quantified.

Mitochondrial Permeability Measurement

   Cells from different groups were plated at 10^6 cells mL^−1 in 1.5 mL
   centrifuge tubes. Mitochondrial permeability was assessed using the
   MPTP Assay Kit (C2009S, Beyotime, China) following the manufacturer's
   instructions. Then cells were then plated at 10^4 cells mL^−1 in a
   confocal dish (1 mL dish^−1) that was treated with Poly‐D‐Lysine
   (Gibco, USA). After a 30‐minute treatment, cells were incubated with
   Hoechst 33342 (ab228551, Abcam, UK, diluted 1:50), and images were
   captured using Lattice SIM^2 (Elyra 7, ZEISS, Germany). The relative
   fluorescence intensity of Calcein AM was analyzed using the ImageJ
   software. Furthermore, the stained cells were analyzed using flow
   cytometry (BD Biosciences, USA), and the fluorescence intensity of
   Calcein AM was assessed with FlowJo software.

Intracellular Calcium Measurements

   Cytosolic Ca^2+ was measured using Fluo‐8AM (ab142773, abcam, UK). For
   instantaneous Ca^2+ measurement: After pretreatment with verapamil (1
   µM, 52‐53‐9, MedChemExpress, USA) or PBS for 30 min, CD8^+ T cells were
   incubated with 5 µM Fluo‐8‐containing buffer solution for 30 min at
   37 °C, then washed by Hanks Balanced Salt Solution with 20 mM HEPES
   (HHBBS) (2611S231799, maokangbio, China). Next, 5 × 10^4 cells per each
   well were seeded to 96‐well black plates in HHBS containing ApoVs
   (10 µg mL^−1) and 2 mM CaCl[2] (ST365, Beyotime, China). BioTek
   fluorescence microplate reader (Bioteck, USA) was applied to detect
   intracellular Ca^2+ for 6 min by exciting at 490 nm and detecting
   emission at 514 nm. For long‐term Ca^2+ measurement: The cells were
   cocultured with ApoVs (10µg mL^−1) and 2 mM CaCl[2] for 6 h and
   incubated with 5 µM Flou‐8‐containing buffer solution for 30 min at
   37 °C and washed by HHBBS. Then, 5 × 10^4 cells per well were seeded to
   96‐well black plates. Intracellular calcium fluorescence was detected
   by BioTek fluorescence microplate reader as above mentioned. Cytosolic
   Ca^2+ changes were presented as fluorescence intensity relative to
   baseline fluorescence over time: F/F0, in which F represents the
   fluorescence intensity measured and F0 represents the minimum
   fluorescence intensity.

Mitochondria Isolation

   Mitochondria isolation was performed using the Cell Mitochondria
   Isolation Kit (89 874, Thermo Scientific, USA) according to the
   manufacturer's instructions. The protein of isolated mitochondria was
   collected, COX4 were applied as mitochondrial reference protein and BAX
   expression level was analyzed by Western blot.

Membrane and Cytosol Protein Extraction

   Membrane and cytosol protein of CD8^+ T cells were extracted using
   Membrane and Cytosol Protein Extraction Kit (20127ES50, Yeasen, China)
   following the manufacturer's instructions. The expression levels of
   Ca[v]1.1, Ca[v]1.2, and Ca[v]β1 of membrane and cytosol were analyzed
   by Western blot respectively.

Enzyme‐Linked Immunosorbent Assay (ELISA)

   The following reagents were used according to the manufacturer's
   instructions for detection in serum and culture supernatants: Mouse
   IFN‐γ ELISA Kit (EMC101g.96, Neobioscience, China), Mouse TNF‐α ELISA
   Kit (EMC102a.96, Neobioscience, China), Mouse Perforin‐1 ELISA Kit
   (SEB317Mu, Cloud Clone, China), Mouse Granzyme B ELISA Kit (SEA600Mu,
   Cloud Clone, China), Human TNF‐α ELISA Kit (EHC103a.48, Neobioscience,
   China), Human IL‐1β ELISA Kit (EHC002b.96, Neobioscience, China), Human
   Perforin‐1 ELISA Kit (SEB317Hu, Cloud Clone, China), Human Granzyme B
   ELISA Kit (SEA600Hu, Cloud Clone, China).

JC‐1 Assay

   Cells were plated at 1 × 10^6 cells mL^−1 in black 96‐well plates
   (100 µL per well). Mitochondrial membrane potential was measured with
   JC‐1 kit (C2006, Beyotime, China), following the provided guidelines.
   The stained cells were analyzed by BioTek fluorescence microplate
   reader (Bioteck, USA).

Mitochondria ROS Measurement

   Cells were plated at 1 × 10^6 cells mL^−1 in black 96‐well plates
   (100 µL per well). Mitochondria ROS levels were measured with MitoSOX
   Red kit (HY‐D1055, MCE, USA), following the provided guidelines. The
   stained cells were analyzed by BioTek fluorescence microplate reader
   (Bioteck, USA).

Statistical Analysis

   GraphPad Prism (version 8.0) was utilized for statistical analysis. The
   two‐tailed Student's t‐test was applied to assess the differences in
   means between the two groups.

Conflict of Interest

   The authors declare no conflict of interest.

Supporting information

   Supporting Information
   [211]ADVS-12-2407446-s001.docx^ (21MB, docx)

Acknowledgements